Estereología

Practical Stereology
2nd Edition
John C. Russ
Materials Science and Engineering Department
North Carolina State University
Raleigh, NC
Robert T. Dehoff
Materials Science and Engineering Department
Florida State University
Gainesville, FL
Published by Plenum Press, New York, NY
ISBN 0-306-46476-4
Introduction
Since the first edition of this book was published more than a decade ago, a revolution in stereological techniques
has occurred. The new emphasis is on design-based techniques in which new types of probes are used to extract
specific kinds of information from structures, without the traditional requirements that they be isotropic, uniform
or random. The literature on new methods for sampling structures using oriented plane and line probes is
extensive and continuously expanding, and there is some danger of overlooking the utility of the traditional and
classical methods which retain important areas of applicability.
In rewriting the second edition of this book, we have tried to treat each of the three major classes of stereological
tools. Most general and easily applied are those that measure global microstructural parameters such as volume
fraction, mean intercept length, surface area and curvature, and line length. The classical model-based methods
that make geometric assumptions about the structures of interest are covered along with the simulation methods
that are used to generate the required models. And finally the new design-based techniques are introduced, using
vertical sections and cycloids, structured sampling strategies, and oriented surface probes. Some chapters
emphasize the procedures for applying stereological measurement techniques, while others emphasize the
derivation of the underlying relationships. The chapters are intended to be largely self-contained and so some
duplication of topics occurs.
All of these methods can be applied relatively simply by understanding what is being measured and practicing the
step-by-step methods of implementation. It is not required that the user be able to derive the techniques from basic
geometric probability (which is fortunate). The most important requirement for the user is to become comfortable
with what aspects of structure are important and how they can be measured. In most cases, the resulting
measurements are used to compare one structure to another or to correlate the geometric properties of the structure
with its history of development or its properties and performance. This must be done statistically, and so there is
also a brief but useful chapter on the statistical tools of primary importance. Again, these are introduced as
practical tools for usage and not derived.
The basic stereological tools are equally useful in a broad variety of applications. They are used in biological and
medical research, materials science, geology, food science, and other fields. There are some minor differences of
emphasis based on how sampling and sectioning can be accomplished with these different types of materials, and
some differences in terminology used to describe the results, but the underlying methods are the same.
Metallurgists may discuss grains where biologists see cells, but the idea of a space-filling tesselation of subunits is
the same. Although our own field is materials science, we have tried to use a mixture of terminologies throughout
in hopes of communicating the ideas to a broad spectrum of potential users of these methods.
We are indebted to Dr. Jeanette Norden, from the Department of Cell Biology at Vanderbilt University, who has
collaborated with us in teaching this material at a series of annual workshops at North Carolina State University
for more than fifteen years. These workshops consider in depth both computer-based image processing and
measurement, and the stereological interpretation of the measurements. It has been our experience that these two
topics are generally taught quite separately and individual researchers are often not familiar with both areas, which
is unfortunate. Our collaboration in teaching the workshops has led to the writing of this book, which we believe
combines our two somewhat different points of emphasis to provide a balanced treatment of the topics.
John C. Russ Robert T. Dehoff
Raleigh, NC Gainesville, FL
December 1999 December 1999
Table of Contents
Chap. 1 Introduction 1
Elements of microstucture 1
Geometric properties of features 4
Typical stereological procedures 6
Fundamental relationships 7
Intercept length and grain size 11
Curvature 12
Second order stereology 13
Stereology of single objects 15
Chap. 2 Basic Stereological Procedures 17
What stereology is 17
How stereology works 17
Why stereology works 22
Ground rules for applying stereology 25
Chap. 3 Geometry of microstructures 27
The qualitative microstructural state 29
The quantitative microstructural state 31
Metric properties 31
Topological properties 32
Ratios of global properties 34
The topographic microstructural state 35
Gradients: variation with position 35
Anisoptropies: variation with orientation 36
Associations 37
Chap. 4 Classical stereological measures 39
Two Dimensional Structures; Area Fraction from the Point Count 39
Volume Fraction from the Point Count 44
Two Dimensional Structures; Feature Perimeter from the Line Intercept Count 50
Three Dimensional Structures: Surface Area and the Line Intercept Count 57
Three Dimensional Microstructures; Line Length and the Area Point Count 63
Chap. 5 Less common stereological measures 71
Three Dimensional Features: Topological Properties and the Volume Tangent Count 71
Three Dimensional Features: the Mean Caliper Diameter 78
Mean Surface Curvature and Its Integral 80
The Sweeping Line Probe in Two Dimensions 89
Edges in Three Dimensional Microstructures 93
Chap. 6 Sample design in stereology 99
Population of point probes 99
Population of lines in two dimensions 102
Line probes in three dimensions 103
Planes in three-dimensional space 104
Disectors in three-dimensional space 105
Sampling strategies in 3D 106
Chap. 7 Procedures for IUR sampling 110
Volume fraction 112
Sampling planes 115
Isotropic planes 116
Isotropic line probes 120
Volume probes -the Disector 124
Networks 126
Chap. 8 Statistical interpretation of data 130
Sources of variability in experiments 130
Distributions of values 133
The mean, median and mode 135
The central limit theorem and the Gaussian distribution 136
Variance and standard deviation 138
Testing distributions for normality - skew and kurtosis 140
Some other common distributions 143
Comparing sets of measurements - the t test 145
Nonparametric comparisons 147
Linear regression 153
Nonlinear regression 157
Chap. 9 Computer-assisted methods 159
Getting the image to the computer 159
Display and storage 162
Image processing 166
Contrast manipulation 167
Correcting nonuniform brightmess 171
Reducing image noise 173
Rectifying image distortion 174
Enhancement 176
Overlaying grids onto images 179
Basic stereological calculations 183
Appendix: grid generation routines 185
Chap. 10 Computer measurement of images 193
Measurement using grids 193
Measuring area with pixels 198
Measurement parameters - size 205
Other feature measurements: shape and position 210
Image processing to enable thresholding and measurement 212
Image processing to extract measurable information 217
Combining multiple images 220
Summary 224
Chap. 11 Geometric modeling 225
Methods: analytic and sampling 225
Sphere intercepts 228
Intercept lengths in other bodies 229
Intercept lengths in three dimensions 233
Intersections of planes with objects 237
Bertrands paradox 240
The Buffon needle problem 241
Appendix: simulation routines 243
Chap. 12 Unfolding size distributions 246
Linear intercepts in spheres 246
Plane intersections 249
Other shapes 253
Simpler methods 256
Lamellae 258
Chap. 13 Anisotropy and Gradients 260
Grain structures in rolled metals 260
Boundary orientation 266
Gradients and neighbor relationships 268
Distances and irregular gradients 273
Alignment 275
Chap. 14 Finite section thickness 276
Projected images 276
Bias in stereological measurements 280
Measurements within sections 283
Chap. 15 Three-dimensional imaging 288
Limitations of stereology 288
Serial methods for acquiring 3D image data 288
Inversion to obtain 3D data 289
Stereoscopy as a 3D technique 292
Visualization 295
Processing 299
Measurement 301
References 304
Chapter 1 - Introduction
Elements of microstructure
Stereology is the science of the geometrical relationships between a structure that exists in three dimensions and
the images of that structure that are fundamentally two-dimensional (2D). These images may be obtained by a
variety of means, but fall into two basic categories: images of sections through the structure and projection images
viewed through it. The most intensive use of stereology has been in conjunction with microscope images, which
includes light microscopes (conventional and confocal), electron microscopes and other types. The basic methods
are however equally appropriate for studies at macroscopic and even larger scales (the study of the distribution of
stars in the visible universe led to one of the stereological rules). Most of the examples discussed here will use
examples from and the terminology of microscopy as used primarily in the biological and medical sciences, and in
materials science.
Image analysis in general is the process of performing various measurements on images. There are many
measurements that can be made, including size, shape, position and brightness (or color) of all features present in
the image as well as the total area covered by each phase, characterization of gradients present, and so on. Most of
these values are not very directly related to the three-dimensional (3D) structure that is present and represented in
the image, and those that are may not be meaningful unless they are averaged over many images that represent all
possible portions of the sample and perhaps many directions of view. Stereological relationships provide a set of
tools that can relate some of the measurements on the images to important parameters of the actual 3D structure. It
can be argued that only those parameters that can be calculated from the stereological relationships (using properly
measured, appropriate data) truly characterize the 3D structure.
Figure 1. Diagram of a volume (red), surface (blue) and linear structure (green) in a 3D space.
Chapter 1 - Basic Stereological Concepts Page # 1
What are the basic elements of a 3D structure or microstructure? Three-dimensional space is occupied by features
(Figure 1) that can be:
1. Three-dimensional objects that have a volume, such as particles, grains (the usual name for space-filling arrays
of polyhedra as occur in metals and ceramics), cells, pores or voids, fibers, and so forth.
2. Two-dimensional surfaces, which include the surfaces of the 3D objects, the interfaces and boundaries between
them, and objects such as membranes that are actually of finite thickness but (because they are much thinner
than their lateral extent) can often be considered as being essentially 2D.
3. One-dimensional features, which include curves in space formed by the intersection of surfaces, or the edges of
polyhedra. An example of a one-dimensional (1D) structure in a metal or ceramic grains structure is the network
of triple lines formed by the meeting of three grains or grain boundaries. This class also includes objects
whose lateral dimensions are so small compared to their length that they can be effectively treated as 1D.
Examples are dislocations, fibers, blood vessels, and even pore networks, depending on the magnification.
Features that may be treated as 3D objects at one magnification scale may become essentially 1D at a different
scale.
4. Zero-dimensional features, which are basically points in space. These may be ideal points such as the junctions
of the 1D structures (nodes in the network of triple lines in a grain structure, for example) or the intersection of
1D structures with surfaces, or simply features whose lateral dimensions are small at the magnification being
used so that they are effectively treated as points. An example of this is the presence of small precipitate
particles in metals.
Figure 2. Light microscope image of a metal (low carbon steel) showing the grain boundaries
(dark lines produced by chemical etching of the polished surface).
In the most common type of imaging used in microscopy, the image represents a section plane through the
structure. For an opaque specimen such as most materials (metals, ceramics, polymers, composites) viewed in the
light microscope this is a cut and polished surface that is essentially planar, perhaps with minor (and ignored)
relief produced by polishing and etching that reveals the structure (Figure 2).
For most biological specimens, the image is actually a projected image through a thin slice (e.g. cut by a
microtome). The same types of specimens (except that they are thinner) are used in transmission electron
microscopy (Figure 3). As long as the thickness of the section is much thinner than any characteristic dimension
of the structure being examined, it is convenient to treat these projected images as being ideal sections (i.e.,
infinitely thin) as well. When the sections become thick (comparable in dimension to any feature or structure
present) the analysis requires modification, as discussed in Chapter 14.
Figure 3. Transmission electron microscope image of rat liver. Contrast is produced by a combination
of natural density variations and chemical deposition by stains and fixatives.
When a section plane intersects features in the microstructure, the image shows traces of those features that are
reduced in dimension by one (Figure 4). That is, volumes (three-dimensional) are revealed by areas, surfaces
(two-dimensional) by lines, curves (one-dimensional) by points, and points are not seen because the section plane
does not hit them. The section plane is an example of a stereological probe that is passed through the structure.
There are other probes that are used as well - lines and points, and even volumes. These are discussed in detail
below and in the following chapters. But because of the way microscopes work we nearly always begin with a
section plane and a 2D image to interpret.
Figure 4. Sectioning features in a three dimensional space with a plane, showing the area intersection with a
volume (red), the line intersection witha surface (blue) and the point intersection with a linear feature (green).
Since the features in the 2D image arise from the intersection of the plane with the 3D structure, it is logical to
expect that measurements on the feature traces that are seen there (lower in dimension) can be utilized to obtain
information about the features that are present in 3D. Indeed, this is the basis of stereology. That is, stereology
represents the set of methods which allow 3D information about the structure to be obtained from 2D images. It is
helpful to set out the list of structural parameters that might be of interest and that can be obtained using
stereological methods.
Geometric properties of features
The features present in a 3D structure have geometric properties that fall into two broad categories: topological and
metric. Metric properties are generally the more familiar; these include volume, surface area, line length and
curvature. In most cases these are measured on a sample of the entire specimen and are expressed as per unit
volume of the structure. The notation used in stereology employs the letters V, S, L, and M for volume, surface
area, length, and curvature, respectively, and denotes the fact that they are measured with respect to volume using
a subscript, so that we get
V
V
the volume fraction (volume per unit volume, a dimensionless ratio) of a phase (the general stereological
term used for any identifiable region or class of objects, including voids)
S
V
the specific surface area (area per unit volume, with units of m
-1
) of a surface
L
V
the specific line length (length per unit volume, with units of m
-2
) of a curve or line structure
M
V
the specific curvature of surfaces (with units of m
-2
), which is discussed in detail later in Chapter 5.
Other subscripts are used to indicate the measurements that have been made. Typically the probes used for
measurement are areas, lines and points as will be illustrated below. For example, measurements on an image are
reported as per unit area and have a subscript A, so that we can have
A
A
the area fraction (dimensionless)
L
A
the length of lines per unit area (units of m
-1
)
P
A
the number of points per unit area (units of m
-2
)
Likewise if we measure the occurrence of events along a line the subscript L is used, giving
L
L
the length fraction (dimensionless)
P
L
or N
L
the number of points per unit length (units of m
-1
)
And if we place a grid of points on the image and count the number that fall on a structure of interest relative to the
total number of points, that would be reported as
P
P
the point fraction (dimensionless)
Volumes, areas and lengths are metric properties whose values can be determined by a variety of measurement
techniques. The basis for these measurements is developed in Chapters 2 through 4. Equally or even more
important in some applications are the topological properties of features. These represent the underlying structure
and geometry of the features. The two principle topological properties are number N
V
and connectivity C
V
, both
of which have dimensions of m
-3
(per unit volume). Number is a more familiar property than connectivity.
Connectivity is a property that applies primarily to network structures such as blood vessels or neurons in tissue,
dislocations in metals, or the porosity network in ceramics. One way to describe it is the number of redundant
connections between locations (imagine a road map and the number of possible routes from point A to point B). It
is discussed in more detail in Chapter 3.
The number of discrete objects per unit volume is a quantity that seems quite simple and is often desired, but is not
trivial to obtain. The number of objects seen per unit area N
A
(referring to the area of the image on a section plane)
has units of m
-2
rather than m
-3
. N
A
is an example of a quantity that is easily determined either manually or with
computer-based image analysis systems. But this quantity by itself has no useful stereological meaning. The
section plane is more likely to intercept large particles than small ones, and the intersections with particles that are
visible do not give the size of the features (which are not often cut at their maximum diameter). The relationship
between the desired N
V
parameter and the measured N
A
value is N
V
= N
A
/<D> where <D> is the mean particle
diameter in 3D. In some instances such as measurements on man-made composites in which the diameter of
particles is known, or of biological tissue in which the cells or organelles may have a known size, this calculation
can be made. In most cases it cannot, and indeed the idea of a mean diameter of irregular non-convex particles
with a range of sizes and shapes is not intuitively obvious.
Ratios of the various structural quantities listed above can be used to calculate mean values for particles or
features. For instance, the mean diameter value <D> introduced above (usually called the particle height) can in
fact be obtained as M
V
/(2N
V
). Likewise the mean surface area <S> can be calculated as S
V
/N
V
and the mean
particle volume <V> is V
V
/N
V
. These number averages and some other metric properties of structures are listed in
Table 1. The reasoning behind these relationships is shown in Chapter 4.
Table 1. Ratios of properties give useful averages
Property Symbol Relation
Volume <V> m
3
<V> = V
V
/N
V
Surface <S> m
2
<S> = S
V
/N
V
Height <D> m
1
<D> = M
V
/2N
V
Mean Lineal Intercept <> m
1
<> = 4V
V
/S
V
Mean Cross-Section <A> m
2
<A> = 2V
V
/M
V
Mean Surface Curvature <H> m
-1
<H> = M
V
/S
V
Typical Stereological Procedures
The 3D microstructure is measured by sampling it with probes. The most common stereological probes are points,
lines, surfaces and volumes. In fact, it is not generally practical to directly place probes such as lines or points into
the 3D volume and so they are all usually implemented using sectioning planes. There is a volume probe (called
the Disector) which consists of two parallel planes with a small separation, and is discussed in Chapters 5 and 7.
Plane probes are produced in the sectioning operation. Line probes are typically produced by drawing lines or
grids of lines onto the section image. Point probes are produced by marking points on the section image, usually
in arrays such as the intersections of a grid.
There probes interact with the features in the microstructure introduced above to produce events, as illustrated in
Figure 4. For instance, the interaction of a plane probe with a volume produces section areas. Table 2 summarizes
the types of interactions that are produced. Note that some of these require measurement but some can simply be
counted. The counting of events is very efficient, has statistical precision that is easily calculated, and is generally
a preferred method for conducting stereological experiments. The counting of points, intersections, etc., is done
by choosing the proper probe to use with particular types of features so that the events that measure the desired
parameter can be counted. Figure 5 shows the use of a grid to produce line and point probes for the features in
Figure 4.
Table 1.2. Interaction of probes with feature sets to produce events
3D Feature Probe Events Measurement
Volume Volume Ends Count
Volume Plane Cross-section Area
Volume Line Chord intercept Length
Volume Point Point intersection Count
Surface Plane Line trace Length
Surface Line Point intersection Count
Line Plane Intersection points Count
a
b
Figure 5. Sampling the section image from Figure 4 using a grid: a) a grid of lines produces line segments
on the areas that can be measured, and intersection points with the lines that can be counted; b) a grid of
points produces intersection points on the areas that can be counted.
With automatic image analysis equipment (see Chapter 10) some of the measurement values shown in Table 2 may
also be used such as the length of lines or the area or intersections. In principle, these alternate methods provide
the same information. However, in practice they may create difficulties because of biased sampling by the probes
(discussed in several chapters), and the precision and accuracy of such measurements are hard to estimate. For
example, measuring the true length of an irregular line in an image composed of discrete pixels is not very accurate
because the line is aliased by consisting of discrete pixel steps. As another example, area measurements in
computer based systems are performed simply by counting pixels. The pixels along the periphery of features are
determined by brightness thresholding and are the source of measurement errors. Features with the same area but
different shapes have different amounts of perimeter and so produce different measurement precision, and it is not
easy to estimate the overall precision in a series of measurements. In contrast, the precision of counting
experiments is well understood and is discussed in Chapter 8.
Fundamental relationships
The classical rules of stereology are a set of relationships that connect the various measures obtained with the
different probes with the structural parameters. The most fundamental (and the oldest) rule is that the volume
fraction of a phase within the structure is measured by the area fraction on the image, or V
V
= A
A
. Of course, this
does not imply that every image has exactly the same area fraction as the volume fraction of the entire sample. All
of the stereological relationships are based on the need to sample the structure to obtain a mean value. And the
sampling must be IUR - isotropic, uniform and random - so that all portions of the structure are equally
represented (uniform), there is no conscious or consistent placement of measurement regions with respect to the
structure itself to select what is to be measured (random), and all directions of measurement are equally
represented (isotropic).
It is easy to describe sampling strategies that are not IUR and have various types of bias, less easy to avoid such
problems. For instance, if a specimen has gradients of the amount or size of particles within it, such as more of a
phase of interest near the surface than in the interior, sampling only near the surface might be convenient but it
would be biased (nonuniform). If the measurement areas in cells were always taken to include the nucleus, the
results would not be representative (nonrandom). If the sections in a fiber composite were always taken parallel to
the lay (orientation) of the fibers, the results would not measure them properly (nonisotropic).
If the structure itself is perfectly IUR then any measurement performed any place will do, subject only to the
statistical requirement of obtaining enough measurements to get an adequate measurement precision. But few real-
world specimens are actually IUR, so sampling strategies must be devised to obtain representative data that do not
produce bias in the result. The basis for unbiased sampling is discussed in detail in Chapter 6, and some typical
implementations in Chapter 7.
The fundamental relationships of stereology are thus expected value theorems that relate the measurements that can
be made using the various probes to the structural parameters present in three dimensions. The phrase expected
value (denoted by < >) means that the equations apply to the average value of the population of probes in the 3D
space, and the actual sample of the possible infinity of probes that is actually used must be an unbiased sample in
order for the measurement result to give an unbiased estimate of the expected value. The basic relationships using
the parameters listed above are shown below in Table 3. These relationships are disarmingly simple yet very
powerful. They make no simplifying assumptions about the details of the geometry of the structure. Examples of
the use and interpretation of these relationships are shown below and throughout this text.
Table 3. Basic relationships for expected values
Measurement Relation Property
Point count = V
V
Volume fraction
Line intercept count = S
V
/2 Surface area density
Area point count = L
V
/2 Length density
Feature count <N
A
> = M
V
/2 = N
V
<D> Total curvature
Area tangent count <T
A
> = M
V
/ Total curvature
Disector count <N
V
> = N
V
Number density
Line fraction <L
V
> = V
V
Volume fraction
Area fraction <A
A
> = V
V
Volume fraction
Length per area <L
A
> = (/4)S
V
Surface area density
It should also be noted that there may be many sets of features in a microstructure. In biological tissue we may be
interested in making measurements at the level of organs, cells or organelles. In a metal or ceramic we may have
several different types of grains (e.g., of different chemical composition), as well as particles within the grains
and perhaps at the interfaces between them (Figure 6). In all cases there are several types of volume (3D) features
present, as well as the 2D surfaces that represent their shared boundaries, the space curves or linear features where
those boundaries intersect, and the points where the lines meet at nodes. In other structures there may be surfaces
such as membranes, linear features such as fibers or points such as crystallographic defects that exist as separate
features.
Figure 6. Example of a polyhedral metal grain (a) with faces, edges (triple lines where three faces from
adjacent grains meet) and vertices (quadruple points where triple lines meet and four adjacent grains
touch); (b) shows the appearance of a representative section through this structure. If particles form along
the triple lines in the structure (c) they appear in the section at the vertices of the grains (d). If particles
form on the faces of the grains (e) they appear in the section along the boundaries of the grains (f).
Faced with the great complexity of structures, it can be helpful to construct a feature list by writing down all of the
phases or features present (and identifying the ones of interest), and then listing all of the additional ones that
result from their interactions (contact surfaces between cells, intersections of fibers with surfaces, and so on).
Even for a comparatively simple structure such as the two-phase metal shown in Figure 7 the feature list is quite
extensive and it grows rapidly with the number of distinct phases or classes of features present. This is discussed
more fully in Chapter 3 as the qualitative microstructural state.

Figure 7. An example microstructure corresponding to a two-phase metal. Color-coding is shown to
mark a few of the features present: blue = phase, red = interface; green = triple points,
yellow = triple points.
Consider the common stereological measurements that can be performed by just counting events when an
appropriate probe is used to intersect these features. The triple points can be counted directly to obtain number per
unit area N
A
, which can be multiplied by 2 to obtain the total length of the corresponding triple lines per unit
volume L
V
. Note that the dimensionality is the same for N
A
(m
-2
) and L
V
(m/m
3
).
Figure 8. A grid (red) used to measure the image
from Figure 7. There are a total of 56 grid
intersections, of which 9 lie on the phase (blue
marks). This provides an estimate of the volume
fraction of 9/56 = 16% using the relationship P
P
=
V
V
. The total length of grid line is 1106 m, and
there are 72 intersections with the boundary
(green marks). This provides an estimate of the
surface area of that boundary of 272/1106 = 0.13
m
2
/m
3
using the relationship S
V
= 2P
L
. There
are 8 points representing triple points (yellow
marks) in the area of the image (5455 m
2
). This
provides an estimate the length of triple line of
28/5455 = 2.910
-3
m/m
3
using the relationship
L
V
= 2P
A
. Similar procedures can be used to
measure each of the feature types present in the
structure.
Other measurements are facilitated by using a grid. For example, a grid of points placed on the image can be used
to count the fraction of points that fall on a phase (Figure 8). The point fraction P
P
is given by the number of
events when points (the intersections of lines in the grid) coincide with the phase divided by the total number of
points. Averaged over many fields, the result is a measurement of the volume fraction of the phase V
V
.
Similarly, a line probe (the lines in the same grid) can be used to count events where the lines cross the
boundaries. As shown in Figure 8, the total number of intersections divided by the total length of the lines in the
grid is P
L
. The average value of P
L
(which has units of m
-1
) is one half of the specific surface area (S
V
, area per
unit volume, which has identical dimensionality of m
2
/m
3
= m
-1
).
Chapters 4 and 5 contain numerous specific worked examples showing how these and other stereological
parameters can be obtained by counting events produced by superimposing various kinds of grids on an image.
Chapter 9 illustrates the fact that in many cases the same grids and counting procedures can be automated using
computer software.
Intercept length and grain size
Most of the parameters introduced above are relatively familiar ones, such as volume, area, length and number.
Surfaces within real specimens can have very large amounts of area occupying a relatively small volume. The
mean linear intercept of a structure is often a useful measure of the scale of that structure, and as noted in the
definitions is related to the surface-to-volume ratio of the features, since = 4V
V
/S
V
. It follows that the mean
surface to volume ratio of particles (cells, grains, etc.) of any shape is <S/V> = 4/.
The mean free distance between particles is related to the measured intercept length of the region between particles,
with the relationship L = (V
V
/V
V
) where is the matrix and the particles. This can also be structurally
important, for example, in metals where the distance between precipitate particles controls dislocation pinning and
hence mechanical properties. To illustrate the fact that stereological rules and geometric relationships are not
specific to microscopy applications, Chandreshakar (1943) showed that for a random distribution of stars in space
the mean nearest neighbor distance is L = 0.554N
V
-1/3
where N
V
is the number of points (stars) per unit volume.
For small features on a 2D plane the similar relationship is L = 0.5N
A
-1/2
where N
A
is the number per unit area;
this will be used in Chapter 10 to test features for tendencies toward clustering or self-avoidance.
A typical grain structure in a metal consists of a space filling array of more-or-less polyhedral crystals. It has long
been known that a coarse structure consisting of a few large grains has very different properties (lower strength,
higher electrical conductivity, etc.) than one consisting of many small grains. The size of the grains varies within a
real microstructure, of course, and is not directly revealed on a section image. The mean intercept length seems to
offer a useful measure of the scale of the structure that can be efficiently measured and correlated with various
physical properties or with fabrication procedures.
Before there was any field known as stereology (the name was coined about 40 years ago) and before the
implications of the geometrical relationships were well understood, a particular parameter called the grain size
number was standardized by a committee of the American Society for Testing and Materials (ASTM). Although it
does not really measure a grain size as we normally use that word, the terminology has endured and ASTM
grain size is widely used. There are two accepted procedures for determining the ASTM grain size (Heyn, 1903;
Jeffries et al., 1916), which are discussed in detail in Chapter 9. One method for determining "grain size" actually
measures the amount of grain boundary surface S
V
, and the other method measures the total length of triple line
L
V
between the grains. The S
V
method is based on the intercept length, which as noted above gives the surface to
volume ratio of the grains.
Curvature
Curvature of surfaces is a less familiar parameter and requires some explanation. A fuller discussion of the role of
surface curvature and the effect of edges and corners is deferred to Chapter 5. The curvature of a surface in three
dimensions is described by two radii, corresponding to the largest and smallest circles that can be placed tangent to
the surface. When both circles lie inside the object, the surface is locally convex. If they are both outside the
object, the surface is concave. When one lies inside and the other outside, the surface is a saddle. If one circle is
infinite the surface is cylindrical and if both are infinite (zero curvature) the surface is locally flat. The mean
curvature is defined as 1/2 (1/R
1
+ 1/R
2
).
The Gaussian curvature of the surface is 1/(R
1
R
2
) which integrates to 4 over any convex surface. This is based
on the fact that there is an element of surface area somewhere on the feature (and only one) whose surface normal
points in each possible direction. As discussed in Chapter 5, this also generalizes to non-convex but simply
connected particles using the convention that the curvature of saddle surface is negative.
M
V
is the integral of the local mean curvature over the surface of a structure. For any convex particle M = 2D,
where D is the diameter . M
V
is then the product of 2<D> times N
V
, where <D> is the mean particle diameter
and N
V
is the number of particles present. The average surface curvature H = M
V
/S
V
, or the total curvature of the
surface divided by the surface area. This is a key geometrical property in systems that involve surface tension and
similar effects.
For convex polyhedra, as encountered in many materials grain structures, the faces are nearly flat and it might
seem as though there is no curvature. But in these cases the entire curvature of the object is contained in the edges,
where the surface normal vector rotates from one face normal to the next. The total curvature is the same 2D. If
the length of the triple line where grains meet (which corresponds to the edges between faces) is measured as
discussed above, then M
V
= (/2)L
V
. Likewise for surfaces (usually called muralia) in space, the total curvature
M
V
= (/2) L
V
where the length is that of the edge of the surface. For rods, fibers or other linear features the
total curvature is M
V
= L
V
; the difference from the triple line case is due to the fact that the fibers have surface
area around them on all sides.
Curvature is measured using a moving tangent line or plane, which is swept across the image or through the
volume while counting events when it is tangent to a line or surface. This is discussed more in Chapter 5 as it
applies to volumes. For a 2D image the tangent count is obtained simply by marking and counting points where a
line of any arbitrary orientation is tangent to the boundary. Positive tangent points (T
+
) are places where the local
curvature is convex and vice versa. The integral mean curvature is then calculated from the net tangent count as
M
V
= (T
+
T
-
)/A. Note that for purely convex shapes there will be two T
+
and no T
-
counts for each particle
and the total mean curvature H
V
is 2N
A
.
Second order stereology
Combinations of probes can also be used in structures, often called second-order stereology. Consider the case in
which a grid of points is placed onto a field of view and the particles which are hit by the points in the grid are
selected for measurement. This is called the method of point-sampled intercept lengths. The point sampling
method selects features for measurement in proportion to their volume (points are more likely to hit large than
small particles). For each particle that is thus selected, a line is drawn through the selection point to measure the
radius from that point to the boundary of the particle. If the section plane is isotropic in space, these radial lines are
drawn with uniformly sampled random orientations (Figure 9). If the section plane is a vertical section as
discussed in Chapters 6 and 7, then the lines should be drawn with sine-weighted orientations. If the structure is
itself isotropic, any direction is as good as another.
Figure 9. Point sampled linear intercepts. A grid (green) is used to locate points within features, from
which isotropic lines are drawn (red) to measure a radial distance to the boundary.
The volume of the particle v
i
= (4/3)<r
3
> where <> denotes the expected value of the average over many
measurements. This is independent of particle shape, except that for irregular particles the radius measured should
include all segments of the particle section which the line intersects. Averaging this measurement over a small
collection of particles produces a mean value for the volume <v>
V
= (4/3)<<r
3
>> where the subscript V
reminds us that this is the volume-weighted mean volume because of the way that the particles were selected for
measurement.
If the particles have a distribution of sizes, the conventional way to describe such a distribution is f
N
(V) dV where
f is the fraction of number of the particles whose size lies between V and V+dV. But we also note that the fraction
of the volume of particles in the structure with a volume in the same range if f
V
(V)dV. These are related to each
other by
f
V
dV = Vf
N
(V)dV (1.1
This means that the volume-weighted mean volume that was measured above is defined by
< v >
V
= V f
V
(V)dV
0
V
max
(1.2
and if we substitute equation (1.1) into (1.2) we obtain
< v >
V
= V
2
0
V
max

N
(V)dV=< v
2
>
N
(1.3
The consequence of this is that the variance
2
of the more familiar number weighted distribution can be computed
for particles of arbitrary shape, since for any distribution
2
=< v
2
>
N
< v >
2
N
(1.4
This is a useful result, since in many cases the standard deviation or variance of the particle size distribution is a
useful characterization of that distribution, useful for comparing different populations as discussed in Chapter 8.
Determining the volume-weighted mean volume with a point-sampled intercept method provides half of the
required information. The other needed value is the conventional or number-weighted mean volume. This can be
determined by dividing the total volume of the phase by the number of particles. We have already seen how to
determine the total volume using a grid count. The number of particles can be measured with the disector,
discussed in Chapter 7. So it is possible to obtain the variance of the distribution without actually measuring
individual particles to construct the distribution function.
There is in fact another way to determine the number-averaged mean volume of features <v>
N
without using the
disector. It applies only to cases in which each feature contains a single identifiable interior point (which does not,
however, have to be in the center of the feature), and the common instance in which it is used is when this is the
nucleus of a cell. The method (called the Nucleator) is similar to the determination of volume-weighted mean
volume above, except that instead of selecting features using points in a grid, the appearance in the section of the
selected natural interior points is used. Of course, many features will not show these points since the section plane
may not intersect them (in fact, if they were ideal points they would not be seen at all). When the interior point is
present, it is used to draw the radial line. As above, if the section is cut isotropically or if the structure is isotropic
than uniform random sampling of directions can be used, and if the surface is a vertical section then sine-weighted
sampling must be employed so that the directions are isotropic in 3D space as discussed in Chapters 6 and 7.
The radial line distances from the selected points to the boundary are used as before to calculate a mean volume
<v>
N
= (4/3)<<r
3
>> which is now the number-weighted mean. The technique is unbiased for feature shape.
The key to this technique is that the particles have been selected by the identifying points, of which there is one per
particle, rather than using the points in a grid (which are more likely to strike large features, and hence produce a
volume-weighted result).
Stereology of single objects
Most of the use of stereological measurements is to obtain representative measures of 3D structures from samples,
using a series of sections taken uniformly throughout a specimen, and the quantities are expressed on a per-unit-
volume basis. The geometric properties of entire objects can also be estimated using the same methods provided
that the grid (either a 2D array of lines and points or a full 3D array as used for the potato in Figure 7.4 of Chapter
7) entirely covers the object.
Figure 10. A leaf with a superimposed grid. The
grid spacing is 1/2 inch and 39 points fall on the
leaf, so the estimated area is 39(0.5)
2
= 9.75 in
2
.
This compares to a measured area of 9.42 in
2
using
a program that counts all of the pixels within the leaf
area.
In two dimensions this method can be used to measure (for example) the area of an irregular object such as a leaf
(Figure 10). The expected value of the point count in two dimensions is the area fraction of the object, or =
A
A
. For an (n n) grid of points this is just = /n
2
where is the number of points that lie on the
feature. The area fraction

A
A
=
A
n
2
l
2
where

l is the spacing of the grid. Setting the point fraction equal to the
area fraction gives

A = l
2
P . This means that the number of points that lie on the feature times the size of one
square of the grid estimates the area of the feature. Of course, as the grid size shrinks this is just the principle of
integration. It is equivalent to tracing the feature on graph paper and counting the squares within the feature, or of
acquiring a digitized image consisting of square pixels and counting the number of pixels within the feature.
When extended to three dimensions, the same method becomes one of counting the voxels (volume elements). If
the object is sectioned by a series of N parallel planes with a spacing of t, and a grid with spacing

l is used on
each plane, then the voxel size is

t l
2
. If the area in each section plane is measured as above then the volume is
the sum of the areas times the spacing t, or

V = t l
2
P
T
where is the total number of hits of grid points
on all N planar sections. This method, elaborated in Chapter 4, is sometimes called Cavalieris principle, but will
also be familiar as the basis for the integration of a volume as
V = A dz
.
Measurements of the total size of an object can be made whenever the sampling grid(s) used for an object
completely enclose it, regardless of the scale. The method can be used for a cell organelle or an entire organ. The
appropriate choice of a spacing and hence the number of points determines the precision; it is not necessary that
the plane spacing t be the same as the grid spacing

l .
Summary
Stereology is the study of geometric relationships between structures that exist in three-dimensional space but are
seen in two-dimensional images. The techniques summarized here provide methods for measuring volumes,
surfaces and lines. The most efficient methods are those which count the number of intersections that various
types of probes (such as grids of lines or points) make with the structure of interest. The following chapters will
establish a firm mathematical basis for the basic relationships, illustrate the step-by-step procedures for
implementing them, and deal with how to create the most appropriate sampling probes, how to automate the
measuring and counting procedures, and how to interpret the results.
Chapter 2 - Basic Stereological Procedures
What Stereology Is
As stated in Chapter 1, stereology is a collection of tools that make measurements of geometric properties of real
world microstructures practical.
In its typical application, information about the structure under study is available as a collection of images prepared
from that structure. Microscopes and other imaging devices usually present us with images that are two
dimensional. The visual information they contain is a biased sample of the three dimensional structure. The image
may be obtained from a section viewed in reflection, a slice viewed in transmission, or the projection of some
more or less rough external surface of the structure. The geometry of features in the image may be quantified by
measuring one or more geometric properties that may be defined for individual features, or for a set of features.
Although most uses of stereology seek geometric information about microstructures, its application is not limited
to microstructures. Stereology has been used to study the geometry of rock structures in mine roofs, in
astronomy, in geology, in agronomy, but the analysis of microstructures is its forte. Thus, every field of endeavor
that deals with microstructures has found stereology useful, including materials science, mineralogy, physiology,
botany, anatomy, pathology, histology, and a variety of other -ologies in the life sciences.
With modern image analysis software it is possible to define and measure several dozen such geometric properties
of features in a two dimensional image. Indeed, because a picture is worth a thousand words, the central
problem in image analysis is the reduction of a few megabytes of information that constitutes a grey scale or color
image to a few meaningful and useful numbers. Stereology provides one answer to the question, "Of all of these
dozens of numbers that can be defined and measured on an image, which are useful to me?" The specific answer
to that question depends explicitly upon the application under consideration, but stereology limits the numbers that
have useful meaning for the three dimensional microstructure sampled.
If your interest lies in obtaining quantitative information about the three dimensional structure that the image
samples then the answer to that question is limited. Only a small subset of the geometric properties that can be
measured in a two dimensional image have stereological meaning, that is, are unambiguously related to geometric
properties of the three dimensional structure which the image samples. Only those relatively few image properties
that are identified in the fundamental equations of stereology have potential for yielding quantitative three
dimensional information.
How Stereology Works
Insight into the geometry of a three dimensional microstructure is acquired by sampling the structure with probes.
Typically the structure is probed by an array of plane sections, or by a collection of line probes, point probes or
small thin volume elements called disector probes. Other probes have been devised for specific applications and
will be treated in later chapters, but these four probe types, points, lines, planes and volumes, are classical in
stereology. Operationally, these probes are generated by sectioning the structure with a plane and preparing the
revealed image for observation, as shown in Figure 1. In a given field of view a plane probe is delineated by
Chapter 2 - Basic Stereological Procedures Page # 17
framing an area for examination in the field of view. Thus, a field on a microstructural section is viewed as a
sample of the population of planes that can be constructed in the three dimensional space that the specimen
occupies. Line and point probes are obtained in such a field by superimposing a grid consisting of an arrangement
of lines and points, as shown in Figure 1. A variety of grids have been devised for these purposes, and examples
will be examined and recommendations made in later chapters.
Figure 1. Point, line and plane probes are constructed by superimposing a grid of lines
on a plane section through the microstructure.
The disector volume element probe, as shown in Figure 2, consists of a pair of planes spaced closely enough so
that unambiguous inferences may be made about how features appearing on the two planes are connected in the
volume between the planes.
These probes interact with features in the image to produce outcomes or events of stereological interest. Figure 1
shows a microstructure composed of two feature sets. Call the white areas features and the shaded areas
features.
1. A point on the grid may exhibit two outcomes in this structure: it may lie in the feature set, or in the
set
1
.
1
The third possibility, that the point lies on the boundary between the features, arises because in real microstructures the boundaries
and grid lines have finite widths. This possibility will be discussed in Chapter 4.
2. Lines in the grid interact with traces of the boundary (an interface surface in three dimensions) that
separates the two feature sets; here the event of interest is the intersection of the line probe with such a
trace.
3. The area of the plane delineated by the boundary of the grid interacts with and features in the structure;
the event of interest is the intersection of the plane probe with such three dimensional features.
4. A small volume of the structure called a disector is contained between matched fields delineated on two
plane sections a small known distance apart interacts with or features in the volume; the event of
interest is the appearance of the top of a particle within this volume.
Figure 2. A disector probe is constructed by comparing the microstructural features observed on two
closely spaced plane sections, to characterize the volume contained between them. In this example two
features continue through both planes, two features appear only on the upper plane, and three features
appear only in the lower plane, indicating that their tops must lie in the volume between the planes.
This brief list of probe/event combinations is by no means an exhaustive list of the interactions that are of interest
in stereology; others will be developed in appropriate chapters. However, these combinations are most frequently
applied and serve to illustrate how stereology works.
In a typical application the set of probes of a given class is scanned over a field and the events of interested are
noted and simply counted. For example:
1. The points in the grid are scanned and the number of points that lie in the phase is counted and recorded
for the field. (In Figure 2.1 four points lie in the phase.)
2. The lines in the grid are systematically scanned along their length and the number of intersections with
interface is counted and recorded (34 intersections in Figure 1);
3. The area of the field is scanned systematically and the number of features is counted (24 features in Figure
1)
2
.
4. Comparisons between features on the top (reference plane) and bottom (look-up plane) of the disector
shown in Figure 2 reveals the appearance of three new particle tops within the volume sampled.
Each of these simple counting experiments is repeated on a number of fields that appropriately sample the three
dimensional structure. What is meant by an appropriate sample is a central issue in stereology. Indeed, as will be
clearly shown in this development, the measurements of stereology are trivially simply; the fundamental
mathematical relationships that connect these measurements with three dimensional geometry are also trivially
simple. The design and collection of an appropriate sample of probes is the hard part of stereology.
Counts for a given probe/event measurement are usually reported as normalized counts by dividing the number of
counts by the total quantity of probe scanned in the field. For the examples cited above for Figure 1:
1. The count of points that lie in the phase is normalized by dividing by the total number of points in the grid
(25 points in Figure 1, to give a point fraction P
P
of 4/25 or 0.16;
2. The count of the number of intersections of lines is normalized by dividing by the total length of line probes
scanned in the field (34 intersection points on the 40 m of probe line length in Figure 1) to give a line
intercept count P
L
of 0.85 (counts per micrometer) or 0.85 x 10
4
= 8500 (counts per cm);
3. The count of features contained in the delineated area of the field is normalized by dividing by that area (24
features in the 160 m
2
of area probed in Figure 1) to give the feature count N
A
of 0.15 (counts per m
2
)
or 0.15 x 10
8
= 1.5 x 10
7
(counts per cm
2
).
4. Each new particle appearance noted in the disector volume identifies a point which is the upper bound of
that particle in the direction perpendicular to the planes of the disector. Since each particle has a single
upper bounding point, dividing this count by the volume contained within the disector provides an estimate
of N
V
, the number of features per unit volume in the structure. If in Figure 2 the area of the counting
frame outlined on the top frame is 130 m
2
and the distance between the planes is 6 m, the volume of the
disector is 780 m
3
. The corresponding estimate of number density is 3/780 = 0.0038 (particles/m
3
) =
3.8 x 10
9
particles per cm
3
.
For a given measurement, counts are recorded on a number of fields under the microscope, on photomicrographs,
or on a digitized image. The mean value and standard deviation of the counts from this sample of probes selected
for measurement are computed using standard statistical formulae (Chapter 8). The standard deviation is the basis
for estimating the precision of the estimate of the mean value and deciding whether a sufficient number of fields
have been be examined. The mean value of the counts in the sample is used to estimate the expected value for
2
Some of the features in the area intersect the boundaries of the counting frame. Adopting the rule that features that intersect two
adjacent edges of the frame boundary are counted (in the figure, the left and bottom edges) while those that intersect the other two edges
are excluded provides an unbiased feature count.
those counts for the entire population of probes. The central objective of sample design in stereology, (i.e., the
selection of fields to be included and how they are to be measured) is to devise a sample that provides an unbiased
estimate of this expected value or population mean for all such probes that can be constructed in three dimensional
space.
The connection between expected values of counts for a given probe/event combination and a geometric property
of the three dimensional structure is obtained by applying the appropriate fundamental relation of stereology.
These equations, elegant in their simplicity, have the status of expected value theorems. The simplest of these
relations,
= V
v
(2.1
where V
V
is the volume fraction occupied by the phase being counted and the brackets around signify the
expected value for this normalized count, in this case the point fraction. This equation may be read "The expected
value of the fraction of the population of points that exist in the volume of the structure under study that lie in the
phase of interest is equal to the fraction of the volume of the structure occupied by that phase." For the phase
sampled in Figure 1, the expected value of P
P
is V
V
=0.16. If the set of fields included in the count is

appropriately selected, the mean value of P
P
for this sample is an unbiased estimator of the expected value of P
P
for the population of points in the three dimensional space occupied by the specimen and thus of the volume
fraction of the phase in the three dimensional structure.
Other fundamental relations are equally simple. The expected value of the line intercept count for the population of
line probes in space is proportional to the surface area density of the interfaces being counted:
=
1
2
S
V
(2.2
where S
V
is the surface area of per unit volume of the set of interfacial features in the three dimensional structure
whose intersections were counted in the experiment. In Figure 1, the line intercept count estimates the surface area
per unit volume of the boundaries separating the and feature sets. Sv = 2 PL = 2 8500 = 17000
(cm
2
/cm
3
).
The feature count is related to the number density N
V
of features in the structure and their average caliper diameter
<D>:
< N
A
>= N
V
< D> (2.3
This latter relation is limited in its application to structures with features that are convex bodies, although it is
otherwise independent of their shape. For features of more general shape <N
A
> measures a property called the
integral mean curvature, discussed in Chapter 5.
Similarly, three dimensional features or particles that are convex have a single "upper bound point" in any
direction. The expected value of the number of points per unit volume of disector probes is a direct estimate of the
number of such points per unit volume in the structure:
< N
V
>= N
V
(2.4
The expected value relationships of stereology are powerful because the require no assumptions about the
geometry of the features being characterized. That point bears repeating. The expected value of counts made on
any of the probe/event combinations in an appropriately designed sample of probes provide an unbiased estimate
of the corresponding geometric property of the three dimensional feature set without geometric assumptions.
The typical stereological experiment begins by devising a procedure for selecting a set of fields to be examined that
provides an appropriate sample of the population of probes to be used in the measurement. Each field is examined
by overlaying an appropriate grid containing those probes. Interactions of the probes with features of interest in
the structure are noted and counted. The mean of these counts for the sample of probes is computed and
normalized. This sample mean is then used to estimate the population mean for the same count for the full set of
probes in three dimensions. The result is inserted in the appropriate fundamental relationship to yield an estimate
of the corresponding geometric property for the three dimensional structure. The standard deviation of the sample
measurements is used to compute the precision of this estimate.
Why Stereology Works
Why do simple counts of the number of probe/event occurrences on an appropriate sample of probes report
geometric properties of the three dimensional structure they sample? This question goes to the core of stereology,
which is contained in its collection of fundamental relationships. In this overview brief arguments are presented
with the intention of making these relationships plausible; more rigorous derivations of these relationships are
presented in later sections.
Keep in mind that the stereological relationships are expected value theorems. This means that the result applies
when the whole population of probes is included in the sample. The task is to design a sample of that population
which will yield an unbiased estimate of that expected value.
Consider the population of points in three dimensional space. In a two phase + structure this population of
points fills the volumes occupied by the and phases. If points are uniformly distributed in space the number of
points in the a part of the structure is proportional to the volume of the part. The number of points in the total
specimen is proportional to the total volume of the specimen. The volume fraction of the phase may be thought
of as the ratio of the number of points in the phase to the number of points in the structure. Equation (2.1)
follows. If a sample of points is drawn uniformly and randomly from this population, i.e., without bias, then the
fraction of points in in this sample will estimate the fraction of points in the population, which in equation (2.1)
is shown to be the volume fraction of the phase in the structure.
Figure 3. The set of vertical line probes (a) intersect an element of surface area dS if their positions lie
within the projected area dA on the horizontal plane (b). The relationship between dA and dS depends
on the angle between the line probe and the surface normal.
To make equation (2.2) plausible consider the population of vertical lines in three dimensional space, as shown in
Figure 3. An individual member of this set of lines may be located by its point of intersection P with a horizontal
plane. Now focus on an element (a small piece) of the surface dS somewhere in the three dimensional
structure. In the application of equation (1.2) the event of interest is the intersection of a line probe with such a
surface in space. In Figure 3, dA is the area of the projection of dS on the horizontal plane. The event of interest,
i.e., an intersection with dS, occurs for the subset of vertical line probes that pass through the area dA on the
horizontal plane. The number of lines that produces an intersection with dS is evidently reported by the number of
points in the horizontal plane that lie within its projected area on that plane, which is evidently determined by the
area dA. The projected area dA is simply related to the area of the surface element dS:
dA = dScos (2.5
where is the angle between the direction that is perpendicular to the surface element in the three dimensional
structure and the direction of the line probes. Averaging this result over the full population of lines in three
dimensional space averages the cosine of over the sphere of orientation. In Chapter 4 this average is shown to
be 1/2. Thus parallel line probes in space sense the projected area of surfaces (on a plane that is perpendicular to
the direction of the set of lines) they encounter in the structure. Including all orientations of lines in the probe set
reports (one half of) the true area of the surfaces in three dimensions.
Equation (2.3) derives from interactions of plane probes with features in the structure. To make this result
plausible focus on the subset of the population of plane probes that all share the same normal direction, (i.e., are
all parallel to each other). For example, the set of horizontal planes in space is illustrated in Figure 4. An
individual member of this set of planes may be uniquely identified by its point of intersection with the vertical
axis. The population of planes in this set is thus the population of points on the vertical axis. A feature in the
phase will be observed on a plane probe if the probe intersects the feature. A plane intersects the feature in Figure
2.4 if it lies within the vertical interval bounded by the two planes that are tangent to the top and bottom of the
feature. The number of planes that satisfy this condition is the same as the number of points that lie within the
interval on the vertical axis between the two tangent planes, since each plane is uniquely represented by its point
on this axis. Assuming the population of points along this line is uniformly distributed, the number of points in
this interval is proportional to the length of this interval. The length of the interval is equal to the tangent diameter
D (i.e., the distance between the two tangent planes) in the vertical direction. Thus the feature count on horizontal
planes senses tangent diameters in the vertical direction of the three dimensional features. If the sample of planes
includes the full set of orientations of plane probes then the tangent diameters will be averaged over the set of
orientations in space. Equation (2.3) follows.
Figure 2.4. The set of horizontal planes intersect a particle if their positions on the vertical axis lie between
the top and bottom tangent planes, i.e., within the caliper diameter D in the vertical direction.
The disector probe is a direct sample of the three dimensional volume of the structure. Equation (2.4) simply
recognizes that the expected value of the N
V
for the population of disectors is the same as the value for the
structure.
Point probes sample features according to their volume; line probes sense the area of surfaces and interfaces; plane
probes select features according to their tangent diameter; disector probes select features according to their
number. The demonstrations in this section show that these counting measurements sense directly their
corresponding geometric properties. That is the basis for the fundamental relationships in stereology.
Ground Rules for Applying Stereology
The fundamental relationships of stereology make no geometric assumptions. The interactions of probes with
features are free of such assumptions precisely because the probe/event combinations that are counted sense
specific geometric properties directly, regardless of how the features that display those properties are arrayed in
the structure. However the connecting relationships are expected value theorems relating the average value of
counts for the entire population of probes to a corresponding geometric property. In a given experiment some
subset of this population of probes is selected for inclusion in a sample. The mean of the sample is used to
estimate the mean of the probe population. In order to insure that this sample mean is an unbiased estimate of the
expected value it is essential to obey the ground rules for stereological sample design.
The ground rules for sample design center around the word uniform. The fundamental relationships assume that
the elements of the population of each class of probes, - points, lines, planes, disectors, etc., - are uniformly
distributed in space. Positions of probes are uniformly distributed in space in the derivation of these equations.
Probes that have directions, like lines and planes, have in addition orientations that are uniformly distributed over
the sphere of orientations in three dimensional space, i.e., are isotropically distributed.
If the structures being probed are uniformly distributed in space and isotropic in orientation, then any set of
sample probes can be used. However, it is difficult to know a priori whether this condition is met, and for many
real structures of interest it is not. A discussion of anisotropy and gradients is presented in Chapters 3, 6 and 13.
For statistical purposes it is also convenient that sample probes be selected randomly from these uniform
distributions because this simplifies the statistical interpretation of results. In some cases it has been shown that
relaxation of this requirement samples the structure more efficiently.
The typical design of the selection of fields, grids and probes for examination in a stereological experiment strives
to attain an Isotropic, Uniform, Random sample of the corresponding population of probes. In seeking an
unbiased estimate of the expected value of an event count the primary requirements are that the probe sample be
"IUR".
Summary
Stereology is the methodology that is required to obtain quantitative estimates of geometric properties of features
in three dimensional structures from measurements that are made on essentially two dimensional images.
The structure is sampled with point, line, plane, disector or other probes. Events that result from interactions of
these probes with features in the structure are counted. Normalized averages of these counts are used to estimate
the corresponding average count for the full population of probes. Fundamental relations of stereology connect
these expected values for the probe population to a geometric property of the structure being sampled.
Point probes sense the volume of three dimensional features. Line probes sense the area of interfaces in the
structure. Plane probes sense the average tangent diameter of features. Disector probes sense the number of
features in the structure.
The relationships of stereology are geometrically general because each kind of probe senses its corresponding
geometrical property directly. The result is thus independent of how the features that exhibit the property are
distributed in the structure.
Unbiased estimation of the stereological counting measurements requires that the selection of probes employed in
any experiment be chosen Isotropically, Uniformly and Randomly from the corresponding population of those
probes.
Chapter 3 - Geometry of Microstructures
The generic term that is used to describe the physical elements that make up a microstructure in this text applies the
concept of a phase borrowed from classical thermodynamics. Each phase in a microstructure is a set of three
dimensional features. To belong to the same phase the features must have the same internal physical properties.
Usually this means features of one phase have the same chemical makeup and the same atomic, molecular, crystal
or biological structure. The collection of parts of the structure that belong to the same phase is one example of a
feature set.
Microstructures are space filling, not-regular, not-random arrangements of the feature sets of phases in three
dimensional space. A microstructure may be a single phase tessellation
1
consisting of four feature sets, as shown
in Figure 1: polyhedral cells that have faces, edges and vertices arranged to fill the three dimensional space.
Alternatively a microstructure may consist of two phases - two distinguishable feature sets - labeled, for instance,
and , as shown in Figure 2, either or both of which may be cell structures, that fit together with precision to fill
up the space occupied by the structure. Microstructures frequently consist of several distinguishable phases each
contributing to the total collection of feature sets in the system, as shown in Figure 3. In some cases, voids or
porosity may be present and is also treated as a measurable phase.
Figure 3.1. Idealized illustration of a space filling tesselation with a cell removed to
show faces (red), edges (triple lines, blue) and vertices (quadruple points, green).
In the description of a microstructure it may be useful to focus on the properties of a particular feature set such as
the phase and its boundaries, edges and vertices if it has them. Each member of the feature set has its own
collection of geometric properties. For example, particles of the phase each have their own volume, surface area
and lineal dimensions such as tangent diameter. Properties of the whole collection of features in the set are called
global properties. Examples include the total volume, or boundary surface area, or number of particles in the set.
1
A tesselation is a subdivision of space into cells which then fill that space.
Chapter 3 - Geometry of Microstructure Page # 27
A more complete description of the structure might incorporate information about the spatial distribution of the
phase in the context of other feature sets in the structure.
Figure 2. Particles of a second phase may distribute themselves at vertices (a),
along triple lines (b), on the faces (c), or within the cell volume (not shown)
in a space filling structure. The white phase is , the colored phase is .
To facilitate the organization of an exercise in microstructural characterization, this chapter introduces three levels
of characterization of microstructures:
1. The qualitative microstructural state;
2. The quantitative microstructural state; and
3. The topographic microstructural state.
The first of these levels of description is a list of the classes of feature sets that exist in the structure. The second
level makes the description quantitative by assigning numerical values to geometric properties that are appropriate
to each feature set. The third level of description deals with nonuniformities in the spatial distribution that may
exist in the structure.
Figure 3.3. Microstructures may consist of a number of phases. In this example, based on a superalloy
metal, there are five: a matrix phase (white background), a precipitate (red) distributed through the matrix,
and three different phases (blue, yellow, and green) distributed along the undulating cell boundary.
The Qualitative Microstructural State
Microstructures consist, not only of the three dimensional features that make up the phases in the structure, but
also of two dimensional surfaces, one dimensional lines and zero dimensional points associated with these
features. The qualitative microstructural state is a list of all of the classes of feature sets that are found in the
structure. Surfaces, edges and points arise from the incidence of three dimensional particles or cells. For example
the incidence of two cells in space forms a surface. The kind of surface is made explicit by reporting the class of
the two cells that form it. If both belong to the feature set, the surface is an surface; if one is from the set
and the other is a member of the collection of cells, the interface is an interface. And so on. Triple lines are
formed by the incidence of three cells, (e.g., an triple line results from the incidence of an a cell with two
cells in space). Quadruple points require the incidence of four cells, and are so labeled, (e.g., ).
The qualitative microstructural state can be assessed or inferred by inspection of a sufficient number of fields to
represent the structure. In many cases, one field will be enough for this qualitative purpose. In making this
assessment keep in mind that the process of sectioning the structure reduces the dimensions of the features by one,
as shown in Figure 4. Sections through three dimensional cells of volume features appear as two dimensional
areas on the section. Sections through two dimensional surfaces appear as one dimensional lines or curves on the
section. Lineal features in space appear only as points of intersection with the sectioning plane; triple lines in a cell
structure intersect as triple points. Point feature sets in three dimensions, such as quadruple points, are in general
not observable on a sectioning plane.
Figure 4. The dimension of each feature is reduced by
one when a three dimensional structure is sectioned by
a plane. In the illustration, phases (an array of
transparent grains or cells) and (colored), surfaces
(grain or cell boundaries) and , and triple lines
and are sectioned so that volumes are
represented by areas, surfaces by lines, and lines by
points. Points in the original volume are not observable
in the plane.
Exhaustive lists of possible feature classes for single phase and two phase structures are given in Table 3.1. The
features that may exist in a three phase structure are listed in Table 3.2. Examples of each feature class are
presented on the sections shown in Figure 4.
Table 3.1. Feature classes that may exist in one and two phase microstructures.
Feature Class Single Phase () Two Phase (+)
Volumes ,
Surfaces , ,
Triple Lines , , ,
Quadruple Points , , , ,
Total Number of Classes 4 14
Table 3.2. Feature classes that may exist in a three phase microstructure.
Feature Class Three Phases (++)
Volumes ,,e
Surfaces , , , , .
Triple Lines , , , , , , , , ,
Quadruple Points , , , , , , , , ,
, , , , ,
Total Number of Classes 34
Characterization of any given microstructure should begin with an explicit list of the feature sets it contains. This
exercise is trivial for single phase structures. For systems with two or more phases this exercise has three
purposes:
1. To force you to think in terms of the three dimensional geometry of the structure;
2. To identify in the list of possible features those that are absent;
3. To provide an explicit basis for eliminating from characterization those feature sets that may not be of
interest.
For example, if the phase is porosity, then it contains no internal boundaries. This means that surface
features are absent, and any triple lines and quadruple points that contain two or more 's in their designation are
also absent. The list of feature sets contained in such a structure is: , ; , ; , ; , .
There are no features described by ; , ; , , .
The description of the qualitative microstructural state may be further expanded by ascribing qualitative aspects of
shape, scale, topology or topography. Shape is frequently conveyed qualitatively by comparing structural features
with familiar objects: alveoli in the lung are "bunches of grapes"; solidifying grains are "dendritic" or tree like, as
are neural cells; a structure may be equiaxed, plate-like or rod-like.
The Quantitative Microstructural State
Associated with each of the classes of feature sets listed in the last section is one or more geometric properties.
Use of stereology to estimate values for these properties constitutes specification of the quantitative microstructural
state. Properties that are stereologically accessible have the useful attribute that they have unambiguous meaning
for feature sets of arbitrary shape or complexity. These geometric properties may be associated with individual
features in the feature set, or as global properties of the whole feature set.
Metric Properties
As an example, consider a dispersion of particles of in an matrix, as shown in Figure 5. In three dimensions
individual particles of each have a value for their volume. Over the collection of all of the particles there is
some distribution of values of volume. Further, taken together, the entire collection of particles has a global
property which is its total volume. Stereological measures are generally related to these global properties of the full
set of features. They are usually reported in normalized units as the value of the property per unit volume of
structure. Thus, the total volume of the set of particles divided by the total volume of the structure that contains
them is reported as the volume fraction, written V
V
, of the phase. The volume fraction occupied by a feature set
may be quantitatively estimated through the point count, the most used relationship in stereology, see Chapters 2
and 4.
Figure 5. A simple two-phase microstructure provides a sample of the volumes
and surfaces that exist in the three-dimensional structure.
Surfaces that exist as two dimensional feature sets in the three dimensional microstructure possess the property
area. Each feature in a surface or interfacial feature set in the structure has a value of its area. The feature set as a
whole has a global value of its surface area. The concept of the area of a surface has unambiguous meaning for
feature sets of arbitrary size, shape, size distribution or complexity. Cell faces in a tessellation have an area. The
collection of a interfaces on the set of particles in an matrix has an area. The surface that separates particles
from the gas phase in a stack of powder has an area. The normalized global property measured stereologically is
the surface area per unit volume, written S
V
, sometimes called the surface area density of the two dimensional
feature set. The surface area may be estimated quantitatively with the line intercept count described in Chapter 2
and 4.
Lines or space curves that exist in the three dimensional microstructure, such as the or lines in the
structure in Figure 4, possess a length. Individual line segments, such as edges in a cell network, each have a
length. The full feature set has a global value of its length, reported as L
V
, the length per unit volume in
stereological measurements. Many feature sets, such as fibers in composite materials, axons in neurons, capillary
blood vessels, or plant roots, approximate lineal features. Length density of each of these feature sets may be
estimated stereologically by applying the area point count on plane probes through the structure as presented in
Chapters 2 and 4.
Topological Properties
Line length, surface area and volume are called metric properties because they depend explicitly on the dimensions
of the features under examination. Geometric properties that do not depend upon shape, size, or size distribution
are called the topological properties. Most familiar of these properties is the number of disconnected parts of a
feature set. The phase particles in Figure 6 may be counted in principle to report their number. The surfaces
bounding these particles also may be counted. In this case the number of disconnected parts in the collection of
surfaces is the same as the number of particles in the three dimensional feature set. (This will not be true if the
particles are hollow spheres, for example. Then each particle is bounded by two surfaces and the number of
surfaces is twice the number of particles.) The number of edges in the network of triple lines in a tessellation is
countable. The normalized value of this generic topological property is the number density, N
V
.
A less familiar topological property is the connectivity of a feature set. Connectivity reports the number of extra
connections that features in the set have with themselves. To visualize the connectivity of a three dimensional
feature determine how many times the feature could be sliced with a knife without dividing it into two parts, as
shown in Figure 6. The sphere, the potato or the blob in the top row in Figure 6 all have connectivity zero, since
any cut will divide any of these features into two parts. Features that may be deformed into a sphere without
tearing or making new joints are topologically equivalent to a sphere and are said to be simply connected.
The features in the second row in Figure 6 can all be cut once without separating the feature into two parts. These
features all have connectivity equal to one and are topologically equivalent to a torus. Connectivities of the
remaining features are given on the figure. A microstructure that consists of a network, such as a powder stack, or
the capillaries in an organ, may have a very large value of connectivity. The normalized property is the
connectivity per unit volume, C
V
.
Figure 3.6. Connectivity, C, is the maximum number of cuts that can be made
through a three-dimensional feature without separating it into two parts.
A third topological property of a feature set, called the Euler (pronounced oiler) characteristic, , is the
difference between the number of connected parts and the global connectivity of the feature set:
= N C (3.1
This combination is a useful measure of the topological properties that combines both rudimentary concepts. It is
more directly accessible stereologically, as will be shown in Chapter 5. Measurements of the topological
properties require a sample of the three dimensional volume of the structure either by serial sectioning and
reconstructing the three dimensional feature set or by applying the disector, see Chapters 2 and 5.
Table 3.3 reviews the primary geometric properties of three dimensional microstructures that can be estimated with
stereology. Values for each may be estimated and appropriately assigned among the list of feature sets contained in
the qualitative microstructural state to provide the quantitative microstructural state for the microstructure.
Some additional geometric properties may be defined that involve the concept of the local curvature at a point on a
surface, or at a point on a line. These concepts require more detailed development. They are unfamiliar and not as
widely used as those listed in Table 3.3. For the interested reader they are developed in detail in Chapter 5, along
with the stereological methods that permit their estimation.
Table 3.3. Geometric Properties of Feature Sets
Features Geometric Property Symbol/Units
Topological Properties
, ... Number Density N
V
m
-3
, ... Connectivity Density C
V
m
-3
Metric Properties
, ... Volume Fraction V
V
m
3
/m
3
, a... Surface Area Density S
V
m
2
/m
3
, ... Length Density L
V
m/m
3
Ratios of Global Properties
Ratios of selected global properties also provide some useful measures of microstructural geometry. A
representative measure of the scale (i.e., size) of the features in the feature set is the mean lineal intercept, <>.
This property is the average length of lines that intersect the features in the set. It is the mean surface - to - surface
distance through the three dimensional features in the structure. It is given by
< >=
4V
S
=
4V
V
S
V
(3.2
This property must be applied knowledgeably in interpreting its meaning. For a collection of same size spheres it
is equal to 2/3 the sphere diameter.
=
4V
S
=
4
4
3
R
3
( )
4 R
2
=
4
3
R (3.3
If the spheres have a size distribution, this property is related to a ratio of the third to the second moment of the
distribution function; it is not the mean particle radius (Underwood, 1970a). For a collection of rod shapes <>
reports the diameter and provides no information about their length (<> = 3/2 D). For plates, it is primarily
determined by the plate thickness (<> = 2 ) and provides no information about dimensions in the plane of the
plate.
Other accessible ratios of global properties report the mean cross sectional area of particles, <A>, and the average
mean surface curvature, <H>, which may be a useful quantity when capillarity or surface tension effects play a
role in shaping the microstructure. A more detailed presentation of the measurement and meaning of these ratios is
given in Chapter 5
The Topographic Microstructural State
Many if not most microstructures exhibit nonuniformities in the distribution and disposition of the feature sets that
make them up. Quantitative measures of these nonuniformities lead to the specification of the topographical
microstructural state.
Gradients: Variation with Position
If a metal rod is heated and then dropped into a water bath the outside quenches while the inside cools more
slowly. The change in microstructure that accompanies this heat treatment will yield a structure that varies from the
surface to the axis of the rod because different cooling rates produce different microstructures. Figure 7 shows an
axial section through a cylindrical sample which has a radial gradient of the second phase with the amount
decreasing from the surface toward the axis of the cylinder. Other properties, like the number density or surface
density will also vary along the radius of the rod. Variations of geometric properties with position in the structure
are called gradients.
Figure 7. An axial section through a cylindrical sample with a gradient in the
amount of the phase decreasing radially from the surface to the center.
Such spatial variations are common in biological structures because the function of an organ usually requires the
structure to vary from the surface to the core. If the positional variations may be captured in a plane, then global
values of the properties may be obtained on a single properly chosen sample plane which averages these variations
appropriately. If the spatial variation is more complex, estimation of global properties requires a carefully designed
sample of sections and fields for observation, see Chapter 6. As an alternative you may wish to characterize the
gradient, i.e., to quantify the variation of, say, the volume fraction of the phase with distance from the surface.
In this case the sample design requires estimation of V
V
at each of a selected set of positions along the radius by
viewing a number of fields that lie along a plane that is parallel to the surface. In designing such a sampling
strategy it is necessary that you have a clear concept of the geometry of the spatial distribution of the feature set.
Anisotropies: Variations with Orientation
Muscle fibers tend to be aligned along the macroscopic axis of the muscle. Similarly, if a polycrystalline metal bar
is deformed directionally as in rolling or extruding, the grains are stretched out in the deformation direction and
flattened normal to that direction, as shown in Figure 8. Wool fibers in felt tend to lie in the same plane, although
they are (ideally) uniformly distributed in orientation within that plane. This tendency for features to be more or
less aligned with one or more preferred directions in space is given the generic term of anisotropy.
Figure 8. Grains in a polycrystal that has been directionally deformed
are elongated in the direction of deformation.
The anisotropy of a collection of particles resides in the surfaces that bound the particles. To visualize this attribute
of structures in three dimensions imagine that the surface bounding the particles is made up of a large number of
small patches of equal area. The direction associated with a patch is described by the vector that is perpendicular to
the tangent plane at the patch, i.e., locally perpendicular to the surface, called the local surface normal. If these
vectors are distributed uniformly over the sphere that describes all possible orientations in three dimensional space
then the feature set is said to be isotropic. If the distribution of vectors is not uniform on the sphere, but tends to
cluster about certain directions, the feature set exhibits anisotropy. This nonuniformity with respect to orientation
in space can be quantified using appropriate sampling strategies that involve making line intercept counts on
oriented line probes, as discussed in Chapter 4. Such nonuniformities may also be appropriately averaged by
using a sampling strategy based upon the method of vertical sections (Baddeley et al., 1986), also discussed in
Chapter 4.
Collections of space curves or other linear features in space may also exhibit anisotropy. This anisotropy may also
be quantified by making area point counts on oriented plane probes (Gokhale, 1992), see Chapter 4.
Associations
In a truly uniform structure the spatial distribution of any one feature set is not influenced by other feature sets that
may exist in the system. In real microstructures there is always some tendency for relations between the various
constituents in the system. There are associations among elements of different feature sets. Mitochondria may tend
to be in contact with cell walls. Particles of the phase may tend to reside at grain boundaries in the matrix, or at
grain edges or corners, as shown in Figures 2 and 9a. In a three phase structure particles (nucleoli) may be
completely contained within particles (nuclei) and have no contact with the matrix, as shown in Figure 9b.
Figure 9. Illustration of association tendencies in microstructures: (a) all of the particles (orange)
are on triple points, and thus on the grain edges in three dimensions (Figure 2b);
(b) all of the blue nucleoli () are contained within the yellow nuclei ().
Quantitative measures of tendencies for positive or negative associations of elements in a microstructure are based
on comparisons of measured values of properties that report the interactions with values computed from a model
that assumes the distributions are in fact independent. For example, the particles in Figure 9a form triple lines of
the type when they lie on the grain boundary. An area point count (P
A
) measures the total length of these
triple lines per unit volume of structure. A line intercept count (P
L
) reports the area of grain boundary and,
separately, the area of boundary. The length of triple line that would result if the interactions between the
two interfacial feature sets were random can be computed. Comparison of the measured length of triple line
with that expected for the random structure gives a quantitative measure of the tendency for particles to be
associated with grain boundaries.
Inclusions or other small particles in the structure may appear as clustered, random or ordered (uniformly
distributed). A variety of measures of this tendency have been proposed; most are based on comparisons between
the observed properties and those of some model spatial distribution of points that can be easily visualized.
Summary
There are at three levels of characterization in the description of the geometric state of a microstructure.
The qualitative microstructural state is simply a list of the three, two, one and zero dimensional features that exist
in the structure at hand.
Each of the feature sets in the list has geometric properties that have unambiguous meaning for features of
arbitrary complexity. These properties may be visualized for individual features in the microstructure or as global
properties for the whole feature set. There are two classes of geometric properties associated with these sets:
Topological properties including number density N
V
, connectivity density C
V
, and Euler characteristic
V
;
Metric properties including volume fraction V
V
, surface area density S
V
, length density L
V
, and curvature
measures to be developed in Chapter 5.
Evaluation of one or more of these geometric properties constitutes a step in the assessment of the quantitative
microstructural state.
Real microstructures exhibit variations of some properties with macroscopic position in the structure. Some
feature sets may display variation with orientation in the macroscopic specimen. Proper sample design may
provide appropriate averages of global properties. Alternatively these gradients and anisotropies may be assessed
quantitatively. Comparison of appropriate combinations of these properties with predictions from random or
uniform models for the structure provide measures of tendencies for feature sets to be positively or negatively
associated with each other.
Chapter 4 - Classical Stereological Measures
This chapter reviews the most commonly used stereological measurements. More detailed discussions and
derivations may be found in the collection of traditional texts in the field (Saltykov, 1958; DeHoff and Rhines,
1968; Underwood, 1970; Weibel, 1978; Kurzydlowski and Ralph, 1995; Howard and Reed, 1998). Each section
of the chapter focuses on a manual stereological measurement. In each case, the procedure is illustrated with a
microstructure and a superimposed grid. Each figure lists:
1. The probe population that is required in the measurement;
2. The specific probe that is used in the illustration;
3. The event that results when this probe interacts with the microstructure;
4. The measurement to be made and its specific result;
5. The stereological relationship that connects the measurement to the geometric property of the structure;
6. The calculation of the appropriately normalized version of the measured result;
7. The calculation of the geometric property of the microstructure that is estimated from this measurement.
A discussion of the corresponding procedure used in computer-based image analysis follows the descriptions of
the manual measurements.
The chapter begins with the measurement of the area of features in a two dimensional structure since this is easily
visualized. Measurement of volume fraction, surface area density and line length are then reviewed.
Two Dimensional Structures; Area Fraction from the Point Count
Figure 1 shows a two dimensional structure consisting of two feature sets. Label the white background and the
colored regions . The fraction of the area of the structure occupied by the phase is A
A
. This area fraction is

measured using point probes. The population of points in this two dimensional world is sampled by
superimposing a grid of points on the structure. An outline of the measurement process is given in the caption of
Figure 1.
The square 5x5 grid in Figure 1 constitutes a sample of 25 points (the intersections in the grid) out of this
population of all of the points that could be identified in the area of the specimen. The event of interest that results
from the interaction of the probe sample with the structure is, "the point hits the phase". The actual measurement
is simply a count of these events, i.e., the number of points in the 5x5 grid in Figure 1 that lie within the areas.
In Figure 1, this count gives 8 points in the phase; that count is the result for this placement of the probe sample
in this field of the microstructure. This point count is related to the area fraction of the phase in this two
dimensional structure by the fundamental stereological relationship,

= A
A
(4.1
The left side of this equation, , is the "expected value" of the fraction of points that hit the phase in a
sample set of grid placements; the right hand side is the area fraction of in the microstructure.
Chapter 4 - Classical Stereological Measures Page # 39
Figure 1. Measurement of the area fraction of the dark phase A
A
Probe population: Points in two dimensional space
This sample: 25 points in the grid
Event: Points lie in the phase
Measurement: Count the points in the phase
This count: 8 points in the phase
Relationship: =A
A
Normalized count: P
P
=8/25=0.32
Geometric property: A
A
=0.32
In the example of Figure 1, the point fraction is 8/25 = 0.32. In practice the grid will be placed on a number of
fields, each producing a particular point count for beta hits. This distribution of number of hits has a mean value
P , and a corresponding sample mean value for the point fraction, P
P
=
P
P
T
, where P
T
is the total number of
points in the grid, 25 in this example. Following usual statistical practice, this sample mean value, P
P
, is use to
estimate the expected value of the point fraction in the phase for the population of points in the structure,
and, through equation (3.1), the area fraction of the phase. The area fraction of the phase in this structure may
be evaluated by counting hits in , or simply by subtracting A
A
from 1.
Figure 2 shows a three phase structure composed of , and areas. In this example, a 5x5 grid is placed on the
structure. The procedure described in the preceding section may be applied separately to hits in , hits in and
hits in the phase. This placement of the grid gives 10 hits on the phase, 8 on the particles and 7 hits on .
The corresponding point fractions of the three phases are obtained by dividing by P
T
= 25. Estimates for the three
area fractions for this single placement of the grid are A
A

= 0.40; A
A
= 0.32; A
A
= 0.28. Statistically valid

results will require replication of these counts on a number of appropriately chosen fields.
Figure 2. Measurement of the area fractions in a three phase structure A
A
, A
A
, A
A

Event: Points lie in each phase, , ,
Measurement: Count the points in each phase
This count: 10 points in , 8 points in , 7 points in
Relationship: =A
A
Normalized count: P
P
=10/25=0.40; P
P
=8/20.32; P
P
=7/25=0.28
A
=0.40; A
A
=0.32; A
A
=0.28
Area fractions of constituents can be measured directly in computer assisted image analysis. This is a
straightforward application of the point count applied with a very high density of points. In this case, each pixel in
the image is a point probe. The phase of interest in the count is segmented from the rest of the image on the basis
of its grey shade range or its color, perhaps along with some other geometric characteristics of the phase, such as
size or shape. Numerical gray shades or color values for those pixels that satisfy the conditions set up to identify
the phase are set to black, and the remaining pixels are set to white. The "detected image" is thus a "binary image".
The computer then simply counts black pixels in this binary image. The point fraction is the ratio of this count to
the total number of pixels in the image. These counts can be made essentially in real time.
It might seem that the pixel count in an image analysis system would provide a much greater precision in the
estimate of A
A
than a manual count of a comparatively very limited number of points. However, analysis has
shown that the manual count may give about the same precision, as reflected in the standard deviation of the
counts for a collection of fields. This is partly because much of the variation in A
A
derives from differences from
one field to another. Also, there is a great deal of redundant information in the pixel count, since many pixels lie in
any given individual feature.
The point count method is most efficient when the grid spacing is such that adjacent points rarely fall within the
same feature, cell or region in the image (they are then said to be independent samples of the structure). An
advantage of this method is that when the hits produced by the point grid are all independent, the number of hits
can be used directly to estimate the measurement precision, as discussed in Chapter 8.
The most difficult step in image analysis is detection or segmentation, i.e., making the computer "see" the phase to
be analyzed as the human operator sees it. There are always pixels included in the discriminated phase that the
operator can "see" are not part of the phase, and pixels not included that "should be". Detection difficulties
increase with the complexity of the microstructure. Such problems frequently can be minimized with more careful
sample preparation and additional image processing and more steps in the analysis. In any experimental situation it
is necessary to balance the effort required to develop an acceptable sample preparation strategy and discrimination
algorithm against the inconvenience of the manual measurement which incorporates the most sophisticated of
discrimination systems, the experienced human operator with independent knowledge about the sample and its
preparation.
In these procedures for estimating area fraction of a phase it is not necessary to know the actual dimensions of the
grid, since the measurement involves only ratios of counts of points. The scale of the grid relative to the
microstructure, and the number of points it contains, do not influence the expected value relationship, equation
(4.1). These choices do influence the precision of the estimate obtained, through the spread in the number of
counts made from one grid placement to the next, but not the estimated expected value. For example, as a limiting
case, if the grid were very small in comparison with the features in the structure then most of the time the entire
grid would either lie within a given phase, or be outside of it. For a 5 x 5 grid, counts for most placements of the
grid would give a count of 25 or zero. The mean number of counts would still be the area fraction for a large
number of observations, but the standard deviation of these counts would be comparatively large. A
correspondingly large number of fields would have to be viewed to obtain a useful confidence interval on the
estimate of the mean.
The point count can also be used to estimate the total area of a single feature in a two dimensional structure. The
solution to this problem is an example of a sampling strategy that is pervasive in the design of stereological
experiments called the "systematic random sample". Figure 3 shows a single particle of the phase. Design a grid
of points with outside dimensions large enough to include the whole particle. In order to estimate the area of the
feature with a point count it is necessary to calibrate the dimensions of the grid at the actual magnification of the
image. A stage micrometer may be used to measure the overall dimensions of the grid and compute the spacing
between grid points. Let the spacing thus measured be

l
0
, and the total number of points in the grid be P
T
. For
the 6 x 8 point grid in Figure 3, there are 48 grid points. Calibration of the magnification gives the grid spacing as

l
0
= 5.7 m. The population of points that the grid samples is contained in the area A
T
given by P
T

l
0
2
( = 48
(5.7)
2
= 1560 m
2
in Figure 3) .
Figure 3. Area of a feature in two dimensions
This sample: 6 8 = 48 points in the grid
Calibration

l
0
= 5.7 m
Event: Points lie in the feature
Measurement: Count the points in the feature
This count: 20 points in the feature
Relationship:

A = l
0
2
P
A
=(5.7m)
2
20 = 650 m
2
Focus on the square box in the upper left corner of the area in Figure 3. A specific placement of the grid may be
specified by locating the upper left grid point Q at any point (x,y) within this box. A given choice of (x,y)
specifies the location of the remaining 48 probe points in the grid. Imagine that the point (x,y) is moved to survey
all of the points in the corner box. Then the remaining grid points systematically sample all of the points in the area
A
T
, i.e., the population of points of interest.
The number of points P that lie within the feature is counted for a given placement of the grid (20 points hit the
particle in Figure 3). The point fraction P
P
for that placement of the grid may be computed as P/P
T
. If the point Q
at (x,y) that locates the grid is chosen uniformly from the points in the corner box then the point fraction for this
sample value is an unbiased estimate of the expected value for the population and equation (4.1) applies.
Statistically speaking, the point fraction in this experiment is an unbiased estimate of the fraction of the area A
T
occupied by the particle. The expected value relation is

= A
A
=
A
A
T
=
A
P
T
l
0
2
(4.2
The number of points P
T
in the grid in this experiment is fixed. The number of points that hit the area of the
figure, P, varies from trial to trial. Thus
=

P
T
(4.3
where is the expected value of the number of points observed within the feature on each trial. Insert this
result into equation (4.2) and solve for A

A = (P
T
l
0
2
) = (P
T
l
0
2
)

P
T
= l
0
2
 (4.4
This simple result is the statistical equivalent of tracing the figure on graph paper and counting the number of
squares that lie in the figure. The area of the feature is seen to be the area associated with a given point (

l
0
2
)
times the number of points that hit the figure. This is approximately true for any single placement of the grid. The
argument that leads to this perhaps obvious result demonstrates that the area is exactly equal to the area of a grid
square times the expected value of the number of hits on the feature. This transforms the simple geometric
approximation into a statistical result, with the attendant potential for replication of the experiment, evaluation of
standard deviation and estimate of the precision of the result. For the feature in Figure 3, the 20 hits for the
placement of the grid shown estimates the area of the feature to be 650 m
2
.
Volume Fraction from the Point Count
The most commonly measured property of three dimensional feature sets is their volume, usually reported as the
volume fraction, V
V
, of the phase. This property may be estimated using either plane, line or point probes; the
simplest and most commonly used measurement relies on point probes. The population of points to be sampled by
these probes is the set of points contained within the volume of the specimen in three dimensional space. Point
probes are normally generated by first sectioning the sample with a plane, and generating a grid of points on the
plane section. As in the two dimensional structure described in the last section, the event of interest is "point hits
the phase" where " phase" is taken to mean the set of features at the focus of the analysis. These points are
simply counted. The stereological relation that connects this point count with the volume of the structure is

= V
V
(4.5
Visualize a specimen composed of two phases and . This structure is revealed by sectioning it with planes and
examining fields on these sections, as shown in Figure 4. The population of points in the three dimensional
specimen is probed by the 5 x 5 grid of points superimposed on this structure. For this sample, 5 points lie within
the phase. This count is replicated on a series of fields on the set of sectioning planes. The mean P and
standard deviation
P
of these counts are computed. The mean point fraction, P / P
T
, is used to estimate the
expected value for the population of points, , and, through equation (4.5), the volume fraction occupied by
the phase. For the field shown in Figure 4, this estimate is 5/25 = 0.25.
Figure 4. Measurement of the volume fraction V
V
of a phase in three dimensions
Probe population: Points in three dimensional space
Event: Points lie in the phase
Measurement: Count the points in the phase
This count: 5 points in the phase
Relationship: =V
V
Normalized count: P
P
=5/25=0.25
Geometric property: V
V
=0.25
In order to obtain an estimate of the volume fraction of in the three dimensional structure represented by the
plane section in Figure 4 it is necessary to repeat this measurement on a number of fields that are chosen to
represent the population of points in three dimensions and average the result. Table 4.1 provides an example of
results that might be derived from a set of observations of 20 such fields. The mean number of counts on these
fields is 7.60 and the standard deviation of the set of 20 observations is found to be 1.5.
The standard deviatoin of the population mean in this experiment is given by
P
=

P
n
where n = 20, the
number of readings in this sample. P 2
P
is the 95% confidence interval associated with this set of readings.
The result in Table 4.1 may be interpreted to mean that the probability is 0.95 that the expected value of P for the
populationof points lies within the interval 7.60 0.68. Since each field was sampled with 25 points, the
normalized point count and its confidence interval is obtained by dividing both numbers by 25. There is thus an
0.95 probability that the expected value of the point fraction lies in the interval 0.277 to 0.231. Since the expected
value of the point fraction estimates the volume fraction by equation (4.5), this range is also the confidence
interval for the estimate of the volume fraction.
Table 4.1. Point Counts from the Structure in Figure 4.
P
P

P
P 2
P
P
P
2
P
P
V
V
2
V
V
7.60 1.51 0.34 7.60 0.68 0.304 0.027 0.304 0.027
Figure 5 shows a structure consisting of three phases, , and . In this structure the small particles lie within
the phase; there are no particles in the matrix. This is a very common structure in life science applications
where an organelle () is a part of the cells. The phase is not of interest in the experiment; the fraction of the
volume of the cells that are occupied by organelles is the object of this example. There are 70 points in the
grid. For the field shown, 30 points hit the phase and 8 hit ; the remaining 32 points are in . These counts are
replicated on a number of fields. The mean number of hits in each phase, P
and P
, are computed, along with

their standard deviations. Corresponding point fractions are obtained by dividing by P
T
= 70 for this case. The
resulting point fractions are used to estimate their corresponding expected values and hence the volume fractions,
V
V
, V
V
, and, by difference, V
V
by applying equation (4.5).

The volume fraction of the structure occupied by the cells including the organelles contained within them is the
sum V
V
+ V
V
. To find the fraction of that volume occupied by organelles, take the ratio, V
V
/ (V
V
+ V
V
).
For the single field shown the estimates are: V
V
= 8/70 = 0.11; V
V
= 30/70 = 0.43 and V

V
0.46. The fraction

of the volume of the regions occupied by is 0.11/(0.43+0.11) = 0.20.
In this analysis it is important to obtain valid estimates of the volume fractions of the three phases separately, and
then combine the results to obtain the desired comparison. As an alternate (incorrect) procedure, imagine taking
counts of and for each field, P
and P
. Add the and counts, (P
+ P
). Next, take the ratio [P

/ (P
+
P
)]. Average this ratio over a number of fields to estimate the fraction of cells occupied by organelles. This
procedure does not give the same result as that described in the previous paragraph because the average of a sum
of ratios is not the same as the ratio of the averages. To obtain valid estimates it is important to measure the
volume fractions of the various phases with respect to the structure as a whole, and subsequently manipulate these
results to provide measures of relative volumes among the phases.
Figure 5. Small particles of the phase (blue) are located within features (green) in this
three phase structure. There are no particles in the phase (white). A 70 point grid is
used to estimate the fraction of the volume of occupied by by separately estimating
the volume fractions of the and phases in the structure.
Event: Points lie in , or phase
Measurement: Count the points in each phase
This count: 30 points in , 8 points in
Relationship: =V
V
Normalized count: P
P
=30/70=0.43; P
P
=8/70=0.11
V
=0.43; V
V
=0.11;
V
V
,
=0.11/(0.43+0.11)=0.20
Figure 6 illustrates the use of the point count to estimate the total volume of a single feature in a three dimensional
microstructure. A three dimensional array of points is used to sample the feature. Visualize a box large enough to
contain the feature. N equally spaced planes are sliced through the feature; let h be the distance between planes. N
= 5 and h = 11.5 m in Figure 6. Each plane has an (m x n) grid of points with a calibrated grid spacing

l
0
imposed on it. Grids with (5 x 5) points with spacing of 5.2 m are shown in Figure 6). In this way a three
dimensional grid of points with (N x m x n) points {(5 x 5 x 5) = 125 in Figure 6} is constructed which includes
the entire feature. The box associated with each grid point has dimensions (v
0
=

l
0

l
0
h), {(5.2 5.2 11.5
= 311 m
3
in the figure}. Counts are made of the number of points that hit the feature on each of the five
sectioning planes. The sum of these five counts is the total number of points in the three dimensional grid that hit
the particle. In Figure 6 the total number of hits is (1 + 3 + 2 + 3 + 1 = 10 hits).
Figure 6. The Cavalieri principle is used to estimate the volume of a single feature by
counting points on a series of sectioning planes. The spacing between the planes is h.
This sample: 5 x 5 x 5 = 125 points in the grid
Calibration: h = 11.5 m,

l
0
=5.2 m

v
0
= l
0
l
0
h =5.2 5.2 11.5 = 311 m
3
Event: Points lie in the feature
Measurement: Count the points in the feature
This count: 1+3+2+3+1=10 points in the feature
Relationship: V
=P
T
v
0

=v
0

=311 m
3
10 = 3110 m
3
Visualize a small box with the dimensions v
0
=

l
0

l
0
h at the upper left rear corner of the large box that
contains the feature. The upper, left, back corner of the three dimensional grid will be located at some point Q =
(x,y,z) within this small box. For any given choice of Q, the positions of the remaining (N x m x n) points in the
grid is determined. They provide a systematic sample of the population points within the containing box. As Q is
moved to all of the points in the small corner box, the grid of points samples all of the population of points within
the containing box. Thus, a random choice for the position of Q from its uniform distribution of possible points in
the small box produces a systematic random sample of the population of points in the containing box. The fraction
of points in the three dimensional grid thus provides an unbiased estimate of the expected value for the population
of points in the containing box and equation (4.5) may be applied:

= V
V
=
V
V
T
=
V
P
T
v
0
(4.6
The number of points that hit the feature varies from trial to trial. Since the total number of points P
T
is the same in
all placements of the three dimensional grid, the expected value of this point count estimates the point fraction

=

P
T
(4.7
Insert this result into equation (4.6) and solve for V
:
V
= P
T
v
0

= P
T
v
0

P
T
= v
0

(4.8
Thus, the volume of the feature is the volume associated with an individual point (

l
0

l
0
h) times the
expected value of the number of hits that points in the grid make with the feature. For a single position of the grid,
v
0
P
provides an estimate of the volume of the feature. For the example illustrated in Figure 6, a total of 10 hits
are noted. The volume associated with a grid point was computed earlier to be 311 m
3
. Thus the volume of the
feature is estimated at (10)(311) = 3,110 m
3
.
The structured random sample is a much more efficient procedure than random sampling in which grid points are
independently placed in the volume. In the latter case, points will inevitably cluster in some areas producing
oversampling, while being sparse in others producing undersampling. It will take on the average nearly three
times as many independent random points to reach the same level of precision as with the use of the structured
approach, but the same answer will still result, namely that the point fraction that hit the phase or structure
measures the volume.
The point counting procedure to estimate the volume of a three dimensional object is an example of the oldest of
the stereological procedures based upon the "Cavelieri Principle" (Howard & Reed, 1998a). The object to be
quantified is sliced into a collection of slabs of known thickness. Some method is used to measure the area of each
slab, such as the point count described in an earlier section. An alternate procedure might use a planimeter, an area
measuring mechanical instrument used by cartographers before the advent of the computer, to measure the
individual cross section areas. If the slabs are of uniform thickness, and uniform content, weighing each slab,
together with a calibration of the weight per unit area, could be used. The underlying principle takes the volume of
each slab to be its cross sectional area times its thickness, so that the volume of the object is the sum of the
volumes of the slabs
V = A
i
h
i=1
N
=< A > ( Nh) (4.9

where <A> is the average cross section area of the slabs and (N h) is the total height of the object.
Two Dimensional Structures; Feature Perimeter from the Line Intercept Count
Figure 7. Use of the line intercept count to estimate the total length
of the boundary lines of a two dimensional feature set.
Probe population: Lines in two dimensional space
This sample: Four horizontal lines in the grid
Calibration: L
0
=17.7m, total probe length = 4 17.7 = 70.8 m
Event: Line intersects the feature boundary line
Measurement: Count the intersections
This count: 17 intersections
Relationship: =(/2)L
A
Normalized count: P
L
=17 counts / 70.8 m = 0.24 counts/m
Geometric property: L
A
= (/2) P
L
= (/2)0.24 = 0.38 (m/m
2
)
Each of the collection of features in the two dimensional structure shown in Figure 7 has a boundary, and each
boundary has a length commonly referred to as its perimeter. The normalized parameter, L
A
, is the ratio of the
total length of boundaries of all of the features in the specimen divided by the area that the specimen it occupies.
This perimeter length per unit area can be estimated by probing the structure with a set of line probes represented
in Figure 7 by the four horizontal lines in the superimposed grid. The event of interest in the measurement is "line
intersects boundary". A simple count of these events forms the basis for estimating L
A
through the fundamental
stereological formula (Underwood, 1970a):
=
2
L
A
(4.10
P
L
in this equation is called the "line intercept count"; it is the ratio of the number of intersections counted to the
total length of line probe sampled (only the horizontal lines in the grid were used in this example). Each of the
lines in Figure 7 is found by calibration to be 17.7 micrometers (m) long. The total length of line probe sampled
in this placement of the grid is 4 x 17.7 = 70.8 m. Since there are 17 intersections marked and noted in Figure 7,
for this example P
L
is 17/70.8 = 0.24 counts / m probed. The left hand side of equation (4.10) is the expected
value of this measurement for the population of lines that can be constructed in two dimensional space. Inverting
equation (4.10) gives the estimate of the perimeter length per unit area for the features shown in Figure 7:
L
A
=

2
P
L
=

2
0.24 = 0.38
m
m
2

(4.11
The area within the grid shown in Figure 7 is (17.7)
2
= 313 m
2
. A rough estimate of the boundary length of the
features contained in the grid area is thus 0.38 (m/m
2
) 313 m
2
= 119 m. There are 8.5 particles in the area
of the grid. (Particles that lie across the boundary are counted as 1/2.) A rough estimate of the average perimeter
of particles may be estimated by dividing the total boundary length in the area by the number of particles: 119 m /
8.5 = 14 m. Inspection of the features in Figure 7 indicates that this result is plausible.
Each member of the population of lines in two dimensional space has two attributes: position, and orientation. The
set of lines in the grid used for a probe in Figure 7 represent a few different positions in the population of lines,
but only a single orientation. In order for a sample of line probes to provide an unbiased estimate of a value for the
population of probes it is necessary that the probe lines uniformly sample all orientations of the circle. A
representative sample of the population of orientations of lines could be obtained by rotating the stage by uniform
increments between measurements. A much more direct strategy, which guarantees uniform sampling of line
orientations, uses test line probes in the shape of circles to make the P
L
measurement.
Figure 8 shows a microstructure with phase boundaries that tend to be aligned in the horizontal direction. In
Figure 8a and 8b a square grid of points is superimposed on this structure. Calibration shows that the grid is
square 23.7 m on a side. The set of five horizontal lines are used to sample the population of lines in two
dimensional space in Figure 8a. The total length of lines probed in the horizontal direction is (5 23.7 = 118.5
m). The events, line intersects boundary, are marked with red markers in Figure 8a. There are 8 intersections,
giving a line intercept count in the horizontal direction of {8/118.5 = 0.068 (counts/m)}. If the set of vertical
lines in the grid are used as a line probe (Figure 8b), the resulting count is 62; for this direction, P
L
= 62/118.5 =
0.52 counts/m. It is clear that the line intercept count is different in different directions in this aligned structure,
and that this reflects the anisotropy of the structure.
Indeed, counts in different directions may be used to characterize the anisotropy quantitatively. Replications on a
collection of fields produce mean and standard deviations of the counts made in each direction. A polar plot of P
L
as a function of , the angle the line direction makes with the horizontal direction, as shown in Figure 9, called the
"rose of the number of intersections" [Saltykov, 1958; Underwood, 1970b] provides a graphical representation of
this anisotropy in a two dimensional structure.
Figure 8a. Use of horizontal line probes to measure the total projected length of
boundaries of two dimensional features on the vertical axis L
A,proj
(vert)
Probe population: Horizontal lines in two dimensional space
This sample: Five horizontal lines in the grid
Calibration: L
0
Relationship: (horz) = L
A,proj
(vert)
Normalized count: P
L
=8 counts / 118.5 m = 0.068 counts/m
A,proj
(vert) = P
L
(horz) = 0.068 (m/m
2
)
Figure 8b. Use of vertical line probes to measure the total projected length of
boundaries of two dimensional features on the horizontal axis L
A,proj
(horx)
Probe population: Vertical lines in two dimensional space
This sample: Five vertical lines in the grid
Calibration: L
0
Relationship: (horz) = L
A,proj
(vert)
Normalized count: P
L
=62 counts / 118.5 m = 0.52 counts/m
A,proj
(vert) = P
L
(horz) = 0.52 (m/m
2
)
The true length of boundaries in this anisotropic structure may be estimated by applying test lines that are made up
of circular arcs, as shown in Figure 8c. The population of orientations in this set of circular line probes provides a
uniform sample of the population of line orientations in two dimensional space. This line grid is equivalent to eight
circles, each with a calibrated diameter of 23.7/4 = 5.92 m. The total length of this collection of line probes is
thus 8 [ (5.92m)] = 148.9 m. The number of intersections with these line probes marked in Figure 8c is 55,
giving a line intercept count P
L
= 55 / 148.9 = 0.37 (counts / m). Note that this result is within the range
between the result for horizontal lines (0.068) and the vertical lines (0.52). Equation (4.10) gives the
corresponding estimate for the true perimeter length: L
A
= (/2) 0.37 = 0.58 m / m
2
.
Figure 8c. Use of circular line probes to measure the true total length of
boundaries in two dimensional features, L
A
Probe population: Lines in two dimensional space
This sample: Set of lines equivalent to 8 circles
Calibration: d=23.7/4=5.9m, total probe length = 8 d = 148.9 m
Relationship: = (/2) L
A
Normalized count: P
L
=55 counts / 148.9 m = 0.37 counts/m
A
= (/2) P
L
= 0.58 (m/m
2
)
The number of features per unit area in Figure 8 is counted to be 21 / (23.7)
2
= 0.037 (1/m
2
). A rough estimate
of the average perimeter of features in this structure is L
A
/ N
A
= 0.58 / 0.037 = 16 m. Inspection of Figure 8
shows that this is not an unreasonable estimate of the average particle perimeter.
Figure 9. Rose-of-the-number-of-intersections for a two-dimensional anisotropic structure
Most image analysis programs provide a measurement of the perimeter of individual features directly on the
digitized image. Boundary pixels are identified in the detected binary image. The boundary line is approximated as
a broken line connecting corresponding points in adjacent boundary pixels. The perimeter of the feature is then
computed as the sum of the lengths of the line segments that make up this broken line. The attendant
approximation may produce significant errors. The magnitude of these errors depends upon how many different
directions are used to construct the broken line boundary.
Figure 10. Illustration of the bias in measuring perimeter of a circle in a digitized image: a) four directions
gives the perimeter of a square; b) eight directions gives that of an octagon.
Consider the circle shown in Figure 10. Its digitized image is represented by the blue plus red pixels.
Identification of which pixels lie on the boundary may vary with the choice of the number of neighbors that are
required to be "non-". The broken line used to compute the perimeter is illustrated for two different cases. In
Figure 10a only four directions are used in constructing the perimeter. It is easy to see that the line segments
connecting neighboring pixels can be combined to make the square that encloses the circle. If the diameter of the
circle is d, then its actual perimeter is ( d). The reassembled broken lines make a square with edge length d; its
perimeter is (4 d). This overestimates the perimeter of the circle by a factor of 4/ = 1.27. That is, use of this
algorithm gives a built in bias for every particle of about + 27%. In Figure 10b the segments assemble into an
octagon; the ratio of perimeter of the broken line to the circle is computed to be 1.05. Thus, even for this more
sophisticated algorithm, evaluation of the perimeter of simple particles has a bias of about +5%.
Constructing an n-sided polygon with sides running in 16 possible directions (a 32-gon) further improves the
accuracy of the perimeter. For example, measuring circles varying from 5 to 150 pixels in diameter using this
technique gives results that are within 1% as shown in Table 4.2. However, the bias is always positive (the
measured value is longer than the true value). Another new method for perimeter measurement is based on
smoothing the feature outline and interpolating a super-resolution polygon using real number values (fractional
pixels) for the vertices (Neal et. al., 1998). This method has even better accuracy and less bias, but is not yet
widely implemented. The presence of any bias in measurements is anathema to stereologists and so they generally
prefer to use the count of intercepts made by a line grid with the outlines.
Table 4.2 - Perimeter measurements using a 32-sided polygon
Diamet er ( pixels) Act ual Perimet er Measured Perimet er Er r or ( %)
5 15.7078 15.6568 -0.326
10 31.4159 31.8885 +1.504
15 47.1239 47.2022 +0.166
20 62.8319 63.4338 +0.958
30 95.2478 95.4562 +0.219
40 125.6637 126.6030 +0.747
50 157.0796 158.1100 +0.656
75 235.6194 236.6480 +0.437
100 314.1593 314.6880 +0.168
125 392.6991 393.1660 +0.119
150 471.2389 471.8700 +0.134
A simple experiment will permit the assessment of a given software in its measurement of perimeter. Generate, by
hand or computer graphics, a collection of black circles or different sizes. Acquire the image in the computer.
Detect the collection of circles. Instruct the software to measure the area A and perimeter P of each feature and
compute some measure of the "circularity" of the features, such as the formfactor 4A/P
2
. This parameter has a
value of 1 for a perfect circle. Measured values may be consistently smaller than 1.00, and may vary with size of
the circle in pixels. Use of the manual line intercept count to estimate perimeter does not suffer from this bias.
Three Dimensional Structures: Surface Area and the Line Intercept Count
Figure 11. A three-dimensional specimen with internal surfaces.
Figure 11 shows a specimen that contains internal interfaces. Usually interfaces of interest in microstructures are
part of the boundaries of particles, e.g., the interface in a two phase structure, but this is not a requirement for
estimating their surface area, reported as the surface area of interface per unit volume of structure. This property,
S
V
, is sometimes referred to as the surface density of the two dimensional feature set imbedded in a three
dimensional structure. Line probes are used to sense the area of surfaces. A sample of the population of lines in
three dimensional space is usually constructed by sectioning the structure with a plane to reveal its geometry, then
superimposing a grid of lines on selected fields in that plane. The events to be counted are the intersections of the
probe lines with traces of the surfaces revealed on the plane section. The line intercept count, P
L
, described in the
preceding section in the context of analyzing a two dimensional structure, i.e., the ratio of the number of
intersection points to the total length of probe lines in the sample, is made for the grid in each field examined. The
governing stereological relationship is
=
1
2
S
V
(4.12
In the preceding section the structure under analysis was viewed as a two dimensional microstructure, and the
properties evaluated were two dimensional (perimeter, area). If indeed the microstructures shown are plane probe
samples of some three dimensional microstructure, then the features observed have the significance of sections
through three dimensional features. To obtain an unbiased estimate of the expected value of PL for the population
of lines in three dimensional space, the probes in the sample must be chosen uniformly from the population of
positions and orientations of lines in three dimensional space, and the geometric properties estimated are those of
the three dimensional microstructure sampled.
In this context, the line intercept count obtained from Figure 7, P
L
= 0.24 (counts/m) takes on new meaning. If
the field chosen and the line probes are a properly representative sample of the population of lines in three
dimensions, then this value is an unbiased estimate of the expected value for that population. This requires a
carefully thought out experimental design that prepares and selects fields and line directions to accomplish this
task. For the single sectioning plane represented in Figure 7, this will only be valid if the surfaces bounding the
features in the figure are themselves distributed uniformly and isotropically. Then inversion of equation (4.12)
then gives S
V
= 2 (0.24) = 0.48 m
2
/ m
3
for the surface density of the interface in this structure.
It is difficult to visualize the meaning of this value of surface density for the particles in Figure 7. As an aid in
seeing whether this result is plausible, estimate the mean lineal intercept for the particles in the structure by
applying equation (3.2) in Chapter 3. The points in the grid may be used to estimate the volume fraction of the
phase by applying equation (4.5). For the placement shown, 3 points hit the phase, giving a rough estimate of
V
V
= 3/16 = 0.19. This result may be combined with the surface density estimate to calculate a rough value of the
mean lineal intercept of the particles:
< >
=
4V
V
S
V
=
4 0.19
0.48
= 1.6m (4.13
The grid points in Figure 7 are about 6 m apart. The mean lineal intercept averages longest dimensions with
many small intercepts near the surface of the particles. Thus, this very rough estimate appears to be reasonable.
The structure in Figure 8 may also be viewed as a section through a three dimensional anisotropic microstructure.
This structure is squeezed vertically and elongated horizontally. However, a single orientation of sectioning plane
is insufficient to establish the nature of this anisotropy in three dimensions. Indeed, out of the population of line
probes in three dimensional space only those that lie in the section plane of Figure 8 are sampled. The observation
that P
L
is different in different directions takes on a three dimensional character, as does the anisotropy that this
difference measures. The concept of the rose of the number of intersections as a quantitative description of the
anisotropy in the microstructure extends to three dimensional microstructure. The three dimensional rose is a plot
of the line intercept count as a function of longitude and latitude on the sphere of orientation.
Figure 12. Orientation in three-dimensional space is specified by a point on a sphere represented by
spherical coordinates (,). The population of line orientations is sampled without bias if the line probes
used are cycloids on vertical sections (see text). The dimensions of the cycloid can be expressed in terms
of the height, h, of the circumscribed rectangle (sometimes referred to as the minor axis).
An understanding of the meaning of such a construction may be aided by visualizing the meaning of the directed
line intercept count. Consider the subpopulation of lines in three dimensional space that all share the same
orientation, specified by the longitude, , and the co-latitude (angle between the direction and the north pole), ,
as shown in Figure 12. The line intercept count may be performed on a set of directed line probes taken from this
subpopulation. This directed count, P
L
(, ) measures the total projected area, A(, ) of surface elements in the
structure on the plane that is perpendicular to the direction of the test lines. Thus, in Figure 8, the low value for
counts for horizontal line reflects the fact that the features have a small footprint when projected on the plane
perpendicular to this direction. The large number of counts for vertical line probes derives from the large projected
area of these particles on the horizontal plane. The governing stereological relationship is (Underwood, 1970b):
= S
V
< cos > (4.14
where <cos > is the average value of the angle between the direction of the test probes and the directions normal
(perpendicular) to all of the surface elements in the structure.
The circular test lines used for the two dimensional analysis of the structure in Figure 8 provide an ingenious and
economical mechanism for automatically averaging over the population of line orientations in two dimensional
space. However in three dimensional space line probe orientations are uniformly distributed over the sphere of
orientation. Averaging over the circle of orientations in a given plane does not provide an unbiased sample of the
population of line orientations in three dimensions, as is demonstrated by the shaded regions in Figure 12a.
Circular test probes in a plane sample the set of orientations in a great circle on the sphere, such as the circle
containing the pole in Figure 12a. This subset of line orientations in a great circle is uniformly sampled in all
directions by a circular line probe in the plane. This implies that the length of line probes with orientations within
five degrees of the pole (green cap) is the same as then length sampled within five degrees of the equator (red
stripe). The green cap is clearly a very much smaller fraction of the area of the sphere of orientation than is the red
stripe. The green cap represents a much smaller fraction of the set of line orientations in three dimensions than
does the red stripe. But the circular test probe samples these regions with the same length of line probe. Thus,
such a probe design over-samples orientations near the pole and under-samples them near the equator. This design
does not provide a uniform sample of the population of line orientations in three dimensions, and will produce a
biased estimate of expected values for the line intercept count.
An unbiased sample of this population is provided by a sample design known as the method of vertical sections
(Baddeley et al., 1986), illustrated in Figure 13. A reference direction is chosen in the macroscopic specimen,
shown in Figure 13a. In the remainder of the analysis, this is called the "vertical direction". Sectioning planes are
prepared which contain (are parallel to) this reference direction, as shown in Figure 13b. These sections are
chosen so that they represent the variations in the structure with position, and with orientation in "longitude", i.e.,
around the vertical direction.
In a typical experiment, a longitude direction is chosen at random and a section is cut that contains this direction
and the vertical direction. Two other vertical sections, 120 away from the first, are also prepared. This provides a
random systematic sample of the longitude orientations. Fields are chosen for measurement in these sections that
are uniformly distributed with respect to position on the vertical plane. The vertical direction is contained in each
field, as shown in Figure 13c, and is known.
Figure 13. The stereological experimental design based on vertical sections gives
an unbiased sample of the population of line orientations in three-dimensional space.
In order to provide an unbiased sample of the population of line orientations on the sphere, a grid is constructed of
line probes that have the shape of a cycloid curve
1
, shown in Figure 13d. This curve has the property that the
fraction of its length pointing in a given direction decreases as the tangent rotates from the vertical direction in a
manner that is proportional to the sine of the angle from the vertical. The need for this sine-weighting is indicated
in Figure 12b. Two equal intervals in the range of values are shown, one at the pole (green) and the other at the
equator (red). The cycloid test line exactly compensates for the sine weighting required of the direction with
longer length of lie probe near the equator (red segment) and shorter near the pole (green segment). This sine
weighting of the line length produces an unbiased sample of the population of orientations in three dimensional
space. A grid consisting of cycloid test lines with known dimensions, so that the total length of lines probed is
known, forms the basis for an unbiased P
L
count in three dimensional space.
1
The cycloid curve is generated by a point on the rim of a wheel as the wheel rolls along a horizontal road. Cycloid segments used in
the method of vertical sections as shown in Figure 4.13 correspond to the curve traced out by the first half turn of the wheel.
Figure 14. Line intercept count made on a presumed vertical section using a cycloid
test line grid gives an unbiased estimate of the surface area per unit volume, S
V
.
Arrow indicates the vertical direction in the specimen.
Probe population: Lines in three dimensional space
This sample: Vertical section with cycloid line probes; 12 cycloid units
joined together in three lines
Calibration: L
0
=28.8 m. Each unit has height h=28.8/6 = 4.8 m
length of each cycloid =

l = 2h = 9.6 m
total probe length = 12

l = 115.1 m
Event: Cycloid probe intersects the feature boundary line
Relationship: = (1/2) S
V
Normalized count: P
L
=24 counts / 115.1 m = 0.21 counts/m
Geometric property: S
V
= 2 P
L
= 0.42 (m
2
/m
3
)
Figure 14 reproduces the anisotropic structure in Figure 8 assuming it is a representative vertical section through a
three dimensional structure. The height of the box containing the grid is calibrated at 28.8 m. The grid is made
up of 12 cycloid segments joined together in rows of four. The height of each cycloid segment is (28.8/6) = 4.8
m. The length of a cycloid segment is twice its height, or 9.6 m. (Figure 12 has the dimensions of a cycloid
segment.) Thus, the total length of lines in the grid is (12 segments x 9.6) = 115.1 m. A total of 24 intersections
with a boundaries are marked and counted. The line intercept count, P
L
= 24/ 115.1 = 0.21 (counts/m). This is
an unbiased estimate of the expected value for lines in three dimensional space. It may thus be used to
provide an estimate of the surface area density through the equation:
S
V
= 2 
= 2 0.21
1
m ( )
= 0.42
m
2
m
3

(4.15
In the analysis of Figure 8 as a two dimensional structure, line intercept counts were made in the vertical and
horizontal directions (Figure 8). Results of this analysis gave P
L,h
= 0.059 (1/m) and P
L,v
= 0.45 (1/m).
Interpret these counts as directed probes in the three dimensional population of lines. According to equation
(4.14), these counts measure the total projected area of particles on a plane perpendicular to the probe direction,
and are related to the average of the cosine of the angle that surface elements make with the probe direction.
Measurement with the cycloid test line grid provides an estimate of the unbiased value for S
V
. These results may
be combined to provide a first estimate of the average of the cosines of surface element normals with the horizontal
direction and the vertical direction in Figure 8:
< cos >
h
=
P
L,h
S
V
=
0.059
0.42
= 0.14
< cos >
v
=
P
L, v
S
V
=
0.45
0.42
= 1.09
(4.16
The maximum value of the cosine function is of course 1.00. The estimate for the vertical projection violates this
limting value. This impossible result arises because counts were obtained from a single placement of the grids
involved, and these counts are only estimates of their expected values. Further, the variance of the ratio of two
statistics, like P
L,v
/ S
V
is the sum of the variances of the individual statistics; the precision of the estimate from a
single field is low. Nonetheless, it is clear from these results (as from a casual inspection of the structures, that
most of the surface elements in this structure have normal directions near the vertical where is near zero and cos
is near 1.0.
In order to help visualize the geometric significance of the surface area value extimated above it is useful to
compute the mean lineal intercept of the features in the structure. This requires an estimate of the volume fraction
of the phase. Apply the point count to the grid in Figure 8: there are three hits on particles. Thus, a very rough
estimate of V
V
is 3/25 = 0.12. Insert this result into the expression for the mean lineal intercept given in equation
(3.2), Chapter 3.
< >=
4 0.12
0.42
= 1.2m (4.17
For plate-like structures such as those shown in Figure 8, <> reports the thickness of the plate. This value is
plausible for the average plate thickness in this structure.
Three Dimensional Microstructures; Line Length and the Area Point Count
Figure 15. Illustration of the use of a plane probe to intersect linear structures in a volume.
Lineal features in a three-dimensional specimen reveal themselves on the plane probe
as a collection of points. Counting the number of intersections provides a tool to
measure the total length of the structures as shown in Figure 16.
Figure 15 shows a specimen with some lineal features as part of its microstructure. The triple lines in a cell
structure or grain structure described in Chapter 3 provide a common example of such lineal structural features.
Dislocations in crystals observed in the transmission electron microscope provide another example. Edges of
surfaces are also lineal features. Tubular structures, such as capillaries, or plant roots, may be approximated as
collections of space curves. The total length of a one dimensional feature set may be estimated from observations
on a set of plane probes. Line features intersect plane probes in a collection of points, as shown in Figure 16.
These points are counted over a field of known area. The area point count P
A
is the ratio of the number of points
of emergence of these line features in the area, normalized by dividing by the area. If the collection of fields
included in the measurement provides a uniform sample of the population of positions and orientation of planes in
three dimensional space, then this count provides an unbiased estimate of L
V
, the length of line features in unit
volume of structure:
=
1
2
L
V
(4.18
Construction of a representative sample of the population of planes in three dimensional space is a challenge, since
sectioning the sample to form a plane probe divides the sample. For the special situation in which the lineal
features are contained in a transparent medium and can be viewed in projection a procedure similar to the method
of vertical sections is available (Gokhale, 1992).
Figure 16. Point count made on a section through a structure containing
linear features (shown here with finite cross-sectional area for clarity).
Probe population: Planes in three dimensional space
This sample: Square area contained within the measurement frame
Calibration: L
0
=40.0 m; probe area A
0
= (40)
2
= 1600 m
2
Event: Plane intersects the linear features (observed as a small feature)
Relationship: = (1/2) L
V
Normalized count: P
A
=13 counts / 1600 m
2
= 0.0081 counts/m
2
V
= 2 P
A
= 2 0.0081 = 0.016 (m/m
3
) = 16 (km/cm
3
)
Figure 17 shows a single phase cell structure (e.g., grains in a polycrystal, or botanical cells). The triple points on
this section result from the intersection of the plane probe with the triple lines in the three dimensional structure.
Calibration shows that the square area to be sampled is 31.1 m on a side; the area thus probed in this field is
(31.1
2
= 967 m
2
). The triple points in this area are marked and counted. The grid facilitates counting the triple
points by dividing the area into sixteen small squares, which are counted systematically. There are 37 triple points
in the area. Thus, P
A
= 37/ 967 = 0.038 (counts/m
2
). Assuming this field is an unbiased sample of the
population of planes in the specimen, equation (4.18) may be used to estimate the length of triple lines in this cell
structure:
L
V
= 2 = 2 0.038 = 0.076
m
m
3
(4.19
In more familiar units, this length corresponds to 76 (km/cm
3
). This at first surprisingly large result is not unusual
for line lengths in microstructures.
Figure 17. Section through a cell structure; a count of the triple points measures
the length of triple lines in the three-dimensional structure, L
V
.
This sample: Square area contained within the grid
Calibration: L
0
=31.1 m; probe area A
0
= (31.1)
2
= 967 m
2
Event: Plane intersects the triple line (observed as triple point)
Relationship: = (1/2) L
V
Normalized count: P
A
=37 counts / 967 m
2
= 0.038 counts/m
2
V
= 2 P
A
= 2 0.038 = 0.076 (m/m
3
) = 76 (km/cm
3
)
Figure 4.18. A two phase microstructure in which the a phase is a cell structure. The length per unit
volume, L
V
, of three kinds of triple lines may be characterized: , , and (occupied)
This sample: Square area contained within the grid
Calibration: A
0
=182.6 m
2
Event: Plane intersects each class of triple line
This count: =19, =39, (occ)=15
Relationship: = (1/2) L
V
Normalized count: P
A
=19 / 182.6 m
2
= 0.10 counts/m
2
P
A
=39 / 182.6 m
2
= 0.21 counts/m
2
P
A
(occ)=15 / 182.6 m
2
= 0.08 counts/m
2
V
= 2 P
A
= 2 0.10 = 0.20 (m/m

3
)
L
V
= 2 P
A
= 2 0.21 = 0.42 (m/m

3
)
L
V
(occ) = 2 P
A
(occ) = 2 0.08 = 0.16 (m/m

3
)
Figure 18 shows a two phase structure ( + ) with particles of the phase distributed along the interfaces
and triple lines. This is inferred from the qualitative observation that the particles are all situated at the
former triple lines. One can visualize three different types of triple lines in this structure through their
corresponding triple points: , , and triple points that are not present because they are occupied by
particles (). These three classes of triple points are marked with respectively red, green and blue markers in
the field be analyzed in Figure 18. These results are tabulated in Table 4.3.
Table 4.3. Triple point counts in Figure 18. Calibrated probe area is 182.6 m
2
.
Category Mark Counts P
A
(1/m
2
) L
V
(m / m
3
)
red 19 0.10 0.20
green 39 0.21 0.42
(occ) blue 15 0.08 0.16
The total length of triple line, occupied plus unoccupied, is 0.36 (m / m
3
). The fraction of the triple
line occupied by particles is 0.16 / 0.36 = 0.44. Since the volume fraction of is small (probably only a few
percent, since there are no hits on for the points in the grid in Figure 18) this observation that almost half of the
cell edge network in three dimensional space is occupied by the phase measures the evidently strong tendency of
to be associated with the cell edge network. Further, although the phase is sparse, the triple line length in the
category is about the same as the total length as that of the cell edge network.
Invoking equation (4.18) in these last two examples presumes that the fields chosen are unbiased, though
extremely limited, samples of the population of plane probes in three dimensional space. The realization of this
condition borders upon intractable, particularly with respect to acquiring planes whose normals sample the
population of orientations on the sphere. The method of vertical sections devised for line probes has the advantage
that each vertical plane section contains all of the line orientations from vertical to the equator. A plane probe
through an opaque specimen has a single orientation; each orientation contained in the sample requires a different
sectioning plane, and each section cuts the sample into two parts. Preparation of a series of plane probes with
normals that are distributed in some sine weighted fashion with respect to an overarching vertical direction in the
specimen may be possible if a large volume of specimen material is available (the Orientator, shown in Chapter 7),
but it requires a significant effort. A simpler sample design that may accomplish this is shown in Chapter 6.
If the sample is transparent, so that the lineal features may be viewed as a projected image, then a strategy similar
to the method of vertical sections for lines may be applied. Figure 19 shows the projected image of a thick slab of
a microstructure with internal lineal features. A probe line on the projection plane corresponds to a probe plane,
defined by that line and the projection direction, that passes through the transparent structure. Intersection points
between the probe line and the lines in the projected image have a one-to-one correspondence to intersections of
the lineal features in the structure with the associated probe plane. The area probed is L
0
t, where L
0
is the length
of the line probe on the image and t is the specimen thickness. The P
A
count is the number of intersections with
the probe line, divided by this area.
If the lineal structure is not isotropic, P
A
will be different for different directions of the probe line in the projection
plane. Vertical and horizontal lines give different counts in the structure shown in Figure 19. The statistical test
compares the number of counts using the square root of the number as the standard deviation. In the example, 30
vertical counts is clearly greater than 12 horizontal counts (3030 or 305.5 compared to 1212 or 123.5). In
order to obtain an unbiased sample of the population of orientations of planes in space, choose a vertical direction
for the specimen. Prepare slices of known thickness that contain this vertical direction and uniformly sample the
equatorial circle of longitudes; these are called "vertical slices". To obtain a uniform sampling of the latitude angle
(angle from the pole) it is again necessary to sample more planes with orientations near the equator than near the
pole. The orientation distribution of plane normals must be sine weighted.
Figure 19. A microstructure consisting of linear features viewed as a projection
through a section of thickness t = 2 m.
This sample: Planes represented by their edges which appear as lines on the grid
(vertical and horizontal lines represent two sets of planes)
Calibration: L
0
=20 m, A
0
= L
0
T 5 planes = 20 2 5 = 200 m
2
Event: Linear features intersect the horizontal or vertical planes
Measurement: Count the intersections with the horizontal and vertical grid lines
This count: Vertical (red arrows) = 30
Horizontal (green arrows) = 12
Relationship: = (1/2) L
V
Normalized Count: P
A
= (30+12)/200 m
2
= 0.21 (counts/m
2
)
Geometric Property: L
V
= 2 P
A
= 0.42 m / m
3
This can be accomplished by using cycloid shaped line probes on the projected image, as shown in Figure 20. In
this application, the cycloids in the grid must be rotated 90 from the direction used in the line probe sample design
because the normals to the cycloid curve (representing the plane normals in the structure) must be sine weighted.
Each cycloid curve, when combined with the projection direction, generates a cycloid shaped surface with normals
that provide a sine weighting of the area of the surface as a function of colatitude orientation. If P
L
is the line
intercept made on these cycloid line probes in the projected image, then the length of lineal features in unit volume
of the structure is given by
L
V
= 2 =
2
t
 (4.20
Figure 20. A line probe on a projected image represents a surface probe in the volume
being projected (a). A cycloid-shaped line probe (b) produces a cycloidal surface probe,
which uniformly samples the latitudinal angle of the probe. Note that the cycloid is
rotated 90 degrees with respect to the orientation used in Figure 13.
This result also forms the basis for estimating the total length of a single lineal object suspended in three
dimensional space, like a the whole root structure of a plant, a wire frame object or the set of capillaries of an
organ. In this application it must be possible to view the whole lineal structure from any direction. Choose a
vertical direction, then a vertical viewing plane. The plane of observation must have an area A
0
large enough to
view the entire object. Visualize a box with the area of the projection plane and depth t large enought to contain the
whole object. The volume of the box is A
0
t. Construct a grid of cycloid test lines with major axis parallel to the
vertical direction on the projection plane. The total length L
T
of these probes is calibrated and known. Count
intersections of the cycloid line probes with the projected lineal features. If P
L
is the number of intersections per
unit length of line probes, then manipulation of equation (4.20) gives
L
V
=
L
A
0
t
=
2
t
=
2
t

L
T
L =
A
0
L
T

(4.21
where is the expected value of the number of intersections that form between the cycloid grid and the
projected image of the structure. Thus, a simple count of the number of intersections between the cycloid grid and
lines in the projected image, replicated and averaged over a number of orientations for the vertical projection plane,
provides an unbiased estimate of the actual length of the feature in three dimensions.
Summary
Normalized geometric properties, volume fraction V
V
, surface area density S
V
and length density L
V
are
accessible through simple manual counting measurements applied to interactions between grids that act as
geometric probes and corresponding features in the three dimensional microstructure. Corresponding
measurements performed with image analysis software may automate these measurements if the features to be
analyzed can be detected satisfactorily. Geometric properties of single features can also be estimated if the
observations can encompass the entire feature.
The connecting relationships make no assumptions about the geometry of the microstructure. It may be simple or
complex, and exhibit anisotropies and gradients. Each of the relationships involves the expected value of counts of
some event that result from the interaction of probe and microstructure. The typical stereological experiment is
designed to yield an unbiased estimate of the corresponding expected value. This requires a sample design that
guarantees that the population of probes, encompassing all possible positions and, for lines and planes, all
orientations on the sphere, will be uniformly sampled.
Uniform sampling of all positions requires selection of fields that accomplish that goal. Uniform sampling of
orientations can be accomplished with appropriately configured test probes. In two dimensional structures,
circular test lines automatically sample all orientations uniformly. The method of vertical sections uses cycloids to
guarantee an unbiased sample of orientations of line probes in three dimensional space. A similar procedure, with
the cycloids rotated ninety degrees, and applied to projected images, gives isotropic sampling of the population of
orientations of planes in three dimensions.
Chapter 5 - Less Common Stereological Measures
This chapter deals with manual stereological measurements that provide access to geometric properties that are
related to the curvature of the objects of analysis. It begins with the concept of the spherical image of surfaces and
its relation to the number and connectivity of the features the surfaces enclose. The probe for this analysis is a
plane that is visualized to sweep through the three dimensional structure. This probe is implemented through the
disector, or more generally by serial sectioning. The concept of integral mean curvature is then developed with
some insight into the meaning of this rather abstract notion. Plane probes yield a feature count in the plane which
measures integral mean curvature. The same information may be obtained in a more general way through the area
tangent count. Polyhedral features have edges and corners which contribute to the spherical image of the feature
and its integral mean curvature. The latter property provides access to the mean dihedral angle at edges through a
combination of counting measurements.
Three Dimensional Features: Topological Properties and the Volume Tangent Count
The topological properties, number N
V
and connectivity C
V
, provide rudimentary information about three
dimensional feature sets. A fundamental property of surfaces that is related to these topological properties derives
from the concept of the spherical image, of the surface.
Figure 1. The spherical image of a point P on a surface (a) is a point P on the unit sphere (b).
The spherical image of a patch of surface dS (c) is a patch d on the unit sphere (d).
If a surface is smoothly curved, every point P on it has a tangent plane, as shown in Figure 1a. (If the surface is
not smoothly curved, i.e., has edges and corners, then they also contribute to the spherical image.) A tangent
plane at P also has a vector, pointing outward from the surface, called the local surface normal. This vector
represents the local orientation of the patch of surface at P. This orientation can be mapped in longitude and
colatitude as a point P on the sphere of orientation, as shown in Figure 1b. The point P on the unit sphere is
Chapter 5 - Less Common Stereological Measures Page # 71
called the spherical image of the point P on the surface. It represents the local direction of the surface. For a small
patch on a curved surface the collection of its normal directions map as a small patch on the unit sphere, as shown
in Figure 1c,d. This is the spherical image of the patch.
Figure 2. Convex bodies (a) are composed of convex surface elements. Non-convex (b,c) bodies have
convex and saddle surfaces, and may also have concave surface elements. Holes in multiply connected
closed surfaces (d) contribute a net of -4 spherical image for each hole.
Now think about a convex surface (no bumps, dimples or saddles), as shown in Figure 2a. The spherical images
of the collection of normal directions for such a surface cover the sphere of orientation exactly once. For every
point that represents a direction on the sphere there is a point on the surface that has a normal in that direction. No
normal direction is represented more than once. Thus, the spherical image of any convex body covers the unit
sphere exactly once, as shown in Figure 2a, and may be evaluated as the area of the unit sphere, which is 4
steradians
1
. This result is the same for every convex body, no matter what its shape or size. The spherical image
thus has the character of a topological property, at least for convex bodies, i.e., it has a value that is independent
of the metric properties of the body. For a collection of N convex bodies, the spherical image is 4N.
1
A steradian is a unit of solid angle represented by an area on the sphere of orientations analogous to the unit, radian, which applies to
angles in a plane. The circle in a plane subtends 2 radians (the circumference of a circle with unit radius). The sphere in three
dimensions subtends a solid angle of 4 steradians.
Particles with smooth bounding surfaces that are not convex, as shown in Figure 2b and c, may have three
different kinds of surface patches: convex, concave, and saddle surface, as shown in the figure. At any point on a
convex patch of surface, the tangent plane is outside the surface. At points on a concave patch, the tangent plane is
inside the surface. At any point on a saddle surface patch the tangent plane lies partly inside the surface, partly
outside. Saddle surface patches (curved outward in one direction and inward in another like a potato chip) are
required to smoothly transition between convex and concave elements, or to surround holes in the particle, as in
the inside of a doughnut, as shown in Figure 2c. Patches of each kind of surface have their own collection of
normal directions which may be mapped on the unit sphere.
It can be shown , and this appears remarkable at first sight, that if the parts of area of the unit sphere that are
covered by the convex and concave patch images are counted as positive and the parts covered by saddle images
are counted as negative, then the result, called the net spherical image,
net
=
convex
+
concave

saddle
= 4(1 C) (5.1
where C is the connectivity of the particle. For simply connected features (no holes), C = 0, and
net
= 4. This
is a generalization of the result for convex bodies described above. Where the shape departs from a simple convex
closed surface, the extra convex and concave spherical image is exactly balanced by the (negative) spherical image
of saddle surfaces on the feature, so that the unit sphere again is covered (algebraically speaking) exactly once. If
the feature has holes in it, every hole requires a net of (- 4) steradians of saddle surface, which thus gives rise to
equation (5.1). For a collection of N particles in a structure, with a collective connectivity of C, the net spherical
image of the collection of features is
net
= 4(N C) (5.2
The difference (N - C) is called the Euler characteristic of the particles in the structure.
The probe used to measure these properties is the sweeping tangent plane. Figure 3 shows a set of particles in a
specimen. Imagine that the plane shown at the top of the figure is swept through the structure from top to bottom.
Such a probe can be realized in practice by confocal microscopy in which a plane of focus is translated through a
transparent three dimensional specimen. For opaque specimens where this is not possible, the sweeping plane
probe may be visualized by producing a set of closely spaced plane sections (serial sections) through the structure
and examining the changes that occur between successive sections. The disector probe (Sterio, 1984) is the
minimum serial sectioning experiment, employing information from two adjacent planes.
The events of interest to the measurement of spherical image are the formation of tangents with the particle
surfaces as the plane sweeps through the particles in the structure. Three kinds of tangent events may occur: at a
convex surface element (++); at a concave element (--), or at an element of saddle surface (+-). These three
situations are pictured in Figure 3. These events are marked and counted separately. The result is normalized by
dividing by the volume swept through by the sweeping probe. The spherical image per unit volume of each class
of surface in the structure is given by
V
++
= 2T
V
++
= 2T
V
V
+
= 2T
V
+
(5.3
Combining equations (5.1) , (5.2) and (5.3) leads to a simple relation between the Euler characteristic of a
collection of particles and the volume tangent counts
N
V
C
V
=
1
4

V
++
+
V

V
+ ( )
=
1
4
2T
V
++
+ 2T
V
2T
V
+ ( )
(5.4
which further simplifies to
N
V
C
V
=
1
2
T
V
net
=
1
2
T
V
++
+ T
V
T
V
+ ( )
(5.5
Figure 3. The sweeping plane probe forms tangents with convex (++), concave ()
and saddle (+) surface elements.
Probe population: Sweeping plane in three dimensional space
This sample: The specimen
Calibration: None required
Event: Plane forms tangents with convex, concave and
saddle surface
Measurement: Count each class of tangent formed
This count: T
++
= 16; T
= 1; T
+
= 11
Relationship: N C = (1/2) T
net
= (1/2) (T
++
+ T
T
+
)
Geometric property: Actual count: N C = (6 3) = 3
Tangent count: N C = (1/2)(16+111) = 6/2 = 3
For a collection of convex bodies C
V
= 0, as are T
V
-- and T
V
+-. Every particle has exactly two convex tangents,
and counting tangents is evidently equivalent to counting particles. More generally, for a collection of simply
connected (not necessarily convex) particles, no matter what their shape, C
V
= 0, and the net tangent count gives
the number of particles in the structure. At the other extreme, for a fully connected network (one particle) N
V
<<
C
V
, and the net tangent count (which will be mostly saddle tangents) gives (minus) the connectivity. The case
where both number and connectivity are about the same order of magnitude is fortunately rare in microstructures.
In this case the separation of number and connectivity is a challenge. A sufficient volume of the sample must be
examined, either in a confocal microscope or by serial sections, to encompass many whole particles so that
beginnings and ends of specific particles may be identified and counted to determine N
V
. This information may
then be combined with the measurement of the Euler characteristic to estimate the connectivity.
Figure 4. The simple disector analysis measures the number density, N
V,
of convex features in three dimensional space.
Probe population: Set of disector planes in three dimensional space
Calibration: A
0
=64 m
2
; h = 0.5 m, V
0
= 32 m
3
Event: Feature in counted unbiased frame on the reference plane
does not appear on the look-up plane
Measurement: Count these events
This count: N=6
Relationship: N
V
= N / V
0
Geometric property: N
V
= 6 / 32 m
3
= 0.19 m
3
= 1.910
11
per cm
3
The disector is a minimum serial sectioning experiment composed of two closely spaced planes. The distance
between the planes, h, must be measured so that, together with area of the field analyzed on the plane, the volume
contained in the disector sample is known. The spacing h must be small enough so that tangent events that occur
in the volume between the planes can be inferred unambiguously. Usually this means that the spacing must be a
fraction (perhaps 1/4 or 1/5) of the size of the smallest features being analyzed. Figure 4 shows the two planes of
the disector viewed side by side for a simple structure composed of convex bodies. An unbiased counting frame
2
with area, A
0
, is delineated on the first plane, called the reference plane. The second plane is called the look-up
plane. Each feature on the reference plane is viewed in conjunction with the corresponding region on the look up
plane. If the particle still persists on the look-up plane it is not marked. If there is no particle section observed in
the region then it is inferred that the particle came to an end between the planes. The particle is marked on the
reference plane to indicate that a convex tangent has occurred within the volume of the disector. Counting the
marks is equivalent to counting particle ends. Since in this simple case of convex particles each particle has only
one end, this is equivalent to counting particles. If N particle ends are counted, then N/(h A
0
) is an estimate of
N
V
. If the disector is chosen to sample the population of disector volumes uniformly then this provides an
unbiased estimate of N
V
.
Figure 5 a,b (continued...)
2
A potential bias in counting particles in a plane probe frame arises because some particles will intersect the boundary of the frame.
Further, large particles are more likely to intersect the frame boundary than small ones. The bias can be eliminated by assigning a
count of 1/2 to particles that cross the frame. A more general strategy involving the concept of an unbiased frame is described in detail
in a later section of this chapter.
c
Figure 5. The sweeping tangent analysis analysis using the disector measures the Euler characteristic of
features in three dimensional space. The events are most easily seen when the two images are overlaid
(Figure 5c), and are marked with arrows (red = T++, green = T, blue = T+). Note that features
are not counted if any part of them touches that touch the exclusion line in either image.
Probe population: Set of disector planes in three dimensional space
Calibration: A
0
= (18 m)
2
= 324 m
2
; h = 0.5 m, V
0
= 162 m
3
Event: Features in counted unbiased frame on the each plane
indicate various tangent events in the volume
Measurement: Count these events
This count: T++ = 3 (red)
T = 3 (green)
T+ = 5 (blue)
Relationship: N
V
C
V
= 1/2 (T
V
++
+ T
V
T
V
+
)
Geometric property: N
V
C
V
= 1/2 (3 + 3 5) / 162 m
3
= 0.0031 m
3

= 3.110
9
cm
3
If the look-up and reference planes are now reversed, particles on the second plane that do not appear on the first
imply that a convex tangent has formed within the volume of the disector. On the average, the number of counts in
the second case will be equal to that in the first, but this will not be true for individual disector fields.
Figure 5 shows a more complex structure with particles that have concave as well as convex boundary segments
on a section. The presence of concave boundary elements on the plane probes results from sections through saddle
surface elements and/or concave elements in the three dimensional structure. The sweeping plane probe is
expected to form tangents of all three types of surfaces. A tangent is formed with a convex surface element (T
++
)
if an isolated particle on the reference plane does not appear on the look-up plane, and also if an isolated particle
on the look-up plane does not appear on the reference plane. A tangent with a concave surface element (T
) is
indicated if a hole on the reference plane disappears on the look-up plane, and also if a hole seen on the look-up
plane disappears on the reference plane. If one feature on the reference plane becomes two on the look-up plane,
or two on the reference plane becomes one on the look-up plane, then a tangent with an element of saddle surface
(T
+
) is inferred. The three classes of tangent planes inferred from the disector sample in Figure 5 are indicated
with red (++), green () and blue (+) markers. The analysis of the disector is presented in the figure caption.
The Euler characteristic estimated from this single observation is 0.0031 m
3
. If it is assumed that these particles
are simply connected (C
V
=0), the number of particles can be estimated to be 0.0031 m
3
. Relative amounts of
each of the three classes of spherical image may also be estimated from the tangent counts by applying equations
(5.3).
Three Dimensional Features: the Mean Caliper Diameter
The notion of the diameter has a straightforward meaning for a sphere. For more general convex bodies, the
"diameter" of a particle is different in different directions. Figure 6 illustrates the concept of the caliper diameter,
D, of a convex particle. Choose a direction and visualize the sweeping plane probe moving along that direction.
The probe will form two tangents with the particle. The perpendicular distance between these two tangent planes is
the caliper diameter of the particle in that direction. This diameter has a value for each direction in the population of
orientations. If the value of the diameter is averaged over the hemisphere of orientation (the other hemisphere
gives redundant information), the mean caliper diameter of the particle is obtained. This concept may be applied to
convex bodies of any shape. It provides one measure of the particle size of features in the structure.
Figure 6. The mean caliper diameter, D, is the distance between parallel tangent planes in any direction
on a convex particle. This distance is sensed by plane probes in three-dimensional space.
An estimate of the mean caliper diameter may be obtained using a plane probe through the three dimensional
structure. Consider the subpopulation of plane probes that all have the same normal direction. Individual planes in
this subpopulation are located by their position along the direction vector. In order for a plane probe to produce an
intersection with a particle in the structure, its position must lie between the planes that are tangent to the ends of
the particle in that orientation. The event of interest is that the plane probe intersects the particle. This event will
produce a two dimensional feature, a section through the particle, observed on the plane probe. The number of
these events, i.e., the number of these two dimensional features that appear on the probe, is counted. The count is
normalized by dividing by the area of the probe sample to give N
A
, the number of features per unit area of probe
scanned. The governing stereological relationship is,
N
A
= N
V
D (5.6
The number of particles per unit volume can be estimated separately using the disector probe as described in the
last section. With this information, the mean caliper diameter of particles in the structure can be evaluated from the
expected value of a feature count on plane probes. This result is limited to convex bodies, but the shapes of the
particles are not otherwise constrained. In order to obtain the mean caliper of particles averaged over orientation it
will be necessary to sample the population of plane orientations in three dimensions. The difficulties in obtaining
an unbiased sample of orientations of plane probes have already been discussed.
For a single orientation of plane probes, this count provides a measure of the mean caliper diameter of particles in
the direction perpendicular to the probe planes. This result can be visualized as an application of equation (4.14) in
Chapter 4, which is the stereological basis for measuring the length of lineal features in a three dimensional
structure. Visualize a subpopulation of parallel planes that share some normal direction given by coordinates (, )
on the orientation sphere. Replace each convex particle with a stick that is the same length as the caliper diameter
in the direction perpendicular to those planes D(, ). Then each particle section on the plane probe may be
visualized as resulting from an intersection of a plane probe with one of these sticks. N
A
for the particle sections is
the same as P
A
for the collection of sticks for this structure. The feature count thus measures the collective length
of these sticks, or more precisely, the collective lengths of the caliper diameters of the particles in the structure, in
the direction that is perpendicular to the plane probes.
N
A
, ( ) = N
V
D , ( ) (5.7
For a collection of particles that show a tendency to be elongated or flattened in a particular direction in space
measurement of N
A
on planes with different orientations provides one measure of the anisotropy of the particle
shapes. Ratios of N
A
counts on planes oriented in different directions directly yield ratios of the mean caliper
diameters in those directions:
N
A

1
,
1
( )
N
A

2
,
2
( )
=
N
V
D
1
,
1
( )
N
V
D
2
,
2
( )
=
D
1
,
1
( )
D
2
,
2
( )
(5.8
Note that these caliper diameters are measured in the direction that is parallel to the normal to the plane probe.
Thus a feature count on a section, when combined with a disector measurement of N
V
gives access to the mean
caliper diameter of convex particles, one convenient measure of particle "size". If the particle shapes are
anisotropically arranged in space, then a feature count on an oriented plane probe provides a measure of the mean
particle diameter in the direction perpendicular to the probe.
Mean Surface Curvature and Its Integral
This section introduces curvature related geometric properties that are significantly more abstract than the
straightforward geometric properties defined and measured in previous sections. These hard-to-visualize
properties are introduced here because they have geometric meaning that is general for structures of arbitrary
geometry, and they can be measured (they are what is measured) by the feature count when the features are not
limited to being convex, as they were in the previous section. For some special classes of features (plates or
muralia, or rods or tubules) these geometric properties have a meaning that can be visualized and put to conceptual
use. For more general kinds of three dimensional features, although the meaning is abstract, at the very least the
measurement provides an additional descriptor of the geometry of the system.
The central concept, the mean curvature at a point on a smooth surface, is the geometric property of surfaces
through which capillarity effects operate. If surface energy or surface tension plays a role in the problem being
investigated, access to a measure of the mean curvature provides geometric information that has direct
thermodynamic meaning (DeHoff, 1993).
Figure 7. Curvature at a point P on a curve in two dimensional space is the reciprocal of
the radius of a circle that passes through three adjacent points on the curve at P.
In order to present the concept of curvature at a point on a surface, it is first useful to recall the concept of
curvature of a plane curve in two dimensional space. Figure 7 shows a curve in the xy plane. To define the
curvature at a point P place two other points, A and B, on the curve. Construct a circle through these three points.
This circle has a center, O, and a radius r. Now let A and B approach P. The point O moves and the radius
changes. In the limit as A and B arrive at P, the center approaches the center of curvature for the point P, and the
radius becomes the radius of curvature. This limiting circle, which just kisses the curve, is called the osculating
circle. The curvature of the curve at P is the reciprocal of this radius. It can also be shown that the curvature is the
rate of rotation of the tangent to the curve as the point P moves along the arc length, s:
k =
1
r
=
d
ds
(5.9
This two dimensional concept is the basis for defining the curvature at a point on a surface in three dimensional
space.
Figure 8. Curvature at a point P on a surface is reported by two principle normal curvatures
1
and
2
measured on two orthogonal normal planes through P. Of all the possible planes through P that
contain the normal direction (a), the two with the minimum and maximum radii (b) give
the principle normal curvatures.
Figure 8a shows a smooth surface. Focus on the point P on this surface. There is a tangent plane at P, and a local
surface normal, perpendicular to that plane. Imagine a plane that contains this normal direction and intersects this
surface at P. The intersection of this "normal plane" with the surface is a plane curve that passes through P. The
radius of curvature, r, and the curvature, k, of that curve may be defined at P using the concepts developed in the
previous paragraph.
Next visualize another normal plane that makes some arbitrary angle with the first plane. The curve of intersection
of this plane with the surface also has a value of curvature at P. If the piece of surface is a piece of a sphere, these
two curvatures will be the same. However, for an arbitrarily shaped surface, the curvature values on two different
normal planes will be different. As the normal plane is rotated, the curvature of the intersecting curve at P changes
smoothly. As the normal plane is rotated through half a circle, there will be some direction for which the curvature
has a maximum value, and another direction for which its value is a minimum. In differential geometry, which
deals with the geometry of entities in the vicinity of a point on the entity, it is shown that the directions for which
the maximum and minimum values occur are 90 apart. These two directions in the tangent plane are called the
principle directions. The curvatures in these two directions,
1
and
2
, are called the principle normal curvatures
at the point P, corresponding to radii r
1
and r
2
. In differential geometry it is shown that these two curvature values
completely describe the local geometry near a point on a smooth surface. The local configuration for an element of
surface around a point P is shown in Figure 8b.
For a spherical surface with radius R,
1
=
2
= 1/R at every point on the surface. For a right circular cylinder of
radius r, the maximum curvature occurs on a plane perpendicular to the axis:
1
= 1/r. The minimum curvature
occurs on a plane containing the cylinder axis, which intersects the surface in a straight line so that
2
= 0. The
values of
1
= 1/r and
2
= 0 are the same at all points on the cylindrical surface. For smooth surfaces with
arbitrary geometry,
1
and
2
vary smoothly from point to point and have some distribution of values over the
surface area.
For a surface that encloses a three dimensional volume, i.e., for a closed surface, these curvatures may be
assigned a sign. If the curvature vector points toward the inside of the surface it is defined to be positive; if it
points outward, it is negative. The possible combinations of signs of the curvature give rise to the three classes of
surface elements described earlier in this chapter:
Convex surface (++) - if both curvatures point inward and are thus positive;
Concave surface () - if both point outward and are thus negative;
Saddle surface (+) - if one points inward and the other points outward.
These three classes of surfaces were illustrated in Figure 2. In general, if a surface has any departure from a
simple convex shape, extra convex lumps, or concave dimples, it must possess patches of saddle surface that
connect these smoothly. At the boundaries of saddle surface one of the curvatures changes sign (passes from {+
} to {++}, or from {+} to {}), so that one of the principle curvatures passes through zero. Such points occur
only along the lines bounding saddle patches (or on the surface of a cylinder), and are called parabolic points.
Two combinations of the principle normal curvatures find widespread application in geometry, topology, physics
and stereology:
The mean curvature, defined by H =
1
2
(
1
+
2
), the algebraic average of the two curvatures, and
The Gaussian curvature, defined by =
1

2
.
Both have a value at each point on a surface and thus vary smoothly over the surface.
The mean curvature, H, is important for two distinct reasons. Capillarity effects play a crucial role in the formation
and performance of most microstructures. These effects arise from the physical action of curved surfaces on their
surroundings. The geometric property of the surfaces that determines the nature of these capillarity phenomena is
shown in thermodynamics to be the local mean curvature, H. Thus an important component of the behavior of
surfaces involves the local mean curvature, its distribution and its average value over the surfaces involved.
The second compelling reason for interest in this geometric property has to do with its integrated value for the
collection of surfaces in a microstructure, called the integral mean curvature. This property has a single value for a
closed surface. It may also be evaluated for a collection of surfaces that make up a microstructure. Visualization of
its meaning is elusive. Consider an element (a small patch) of surface with incremental area dS; suppose H is the
value of mean curvature at that element. Compute the product HdS for the element. Then add these values together
for all of the surface elements that bound the particle. The resulting quantity, defined mathematically by
M HdS
S
(5.10
has units of length. H has units of length
-1
and dS has units of length
2
. (The double integral signs signifies that
the local incremental value HdS is summed over the whole area of the surface, S.) This abstract geometric
property has geometric meaning that can be visualized for particular classes of three dimensional features.
As a simple example, apply the concept to a spherical particle of radius R. The principle curvatures are both equal
to (1/R), and have the same value at every point on the sphere. Thus, for every point on a sphere the mean
curvature is simply 1/R:
H =
1
2
1
r
1
+
1
r
2

=
1
2
1
R
+
1
R

=
1
R
(5.11
Put this value into the definition of M:
M
sphere
= HdS
S
=
1
R
dS =
S
1
R
dS =
1
R
S
sphere
=
1
R
4R
2
= 4R
S
(5.12
This result generalizes through the Minkowski formula; for general convex bodies,
M
convex
= 2D (5.13
where D is the mean caliper diameter defined in the previous section.
We have seen that for a collection of convex bodies the feature count on a plane probe, N
A
, reports the product of
the number density and the mean caliper diameter. For non-convex bodies, i.e., for the general case, a
generalization of the feature count reports the integral mean curvature, M. In the discussions up to now, the
classes of features considered produced sections that are free of holes on the plane probe. However, in the general
case of arbitrarily shaped particles in three dimensional space, some plane probes will produce features with holes
in them. In this most general kind of structure, the feature count generalizes to the Euler characteristic of the
particle sections, which is defined to be the number of particles minus the number of holes (N - C). It is useful to
think of this as a count of the net number of closed loops bounding particles in the structure, where loops
enclosing particles are counted as positive and those enclosing holes are negative. The fundamental stereological
relationship is
N
A
net
= N
A
C
A
=
1
2
M
V
(5.14
where M
V
is the integral mean curvature of the surfaces bounding the particles in the three dimensional structure
divided by the volume of the structure. Thus, no matter how complex the features in three dimensions are, the net
feature count on a plane probe provides an unbiased estimate of this abstract property, the integral mean curvature.
It is the three dimensional property that the feature count measures.
Applying Minkowskis formula for convex bodies, equation (5.13), to equation (5.14):
N
A
convex
=
1
2
M
V
convex
=
1
2
N
V
M
V
convex
=
1
2
N
V
2D = N
V
D (5.15
recovers equation (5.6), now seen to be a special case of equation (5.14).
Figure 9 reproduces the microstructure in the left side of the disector in Figure 4 so that a feature count may be
made and illustrated. In this case, the value of the mean curvature M
V
estimated to be 4.45 m/m
3
can be used to
estimate the mean caliper diameter. The value of N
V
obtained from the disector analysis in Figure 4 is 0.19 m
3
.
Apply the Minkowski formula, equation (5.13), to compute:
D =
M
V
2N
V
=
4.45
2 0.19
= 3.72m (5.16
Figure 10 reproduces the microstructure on the left side of the disector in Figure 5. A net area loop count estimates
the integral mean curvature for this structure of general geometry.
The integral mean curvature is a key geometric property of a collection of plate shaped particles, or, more
generally muralia, i.e., "thick surfaces", features that are small in one dimension and extensive in the other two, as
shown in Figure 11a. For such features it can be shown that M measures the length of the perimeter of the edge of
the the plate or muralia, L (DeHoff, 1977):
M
muralia
=

2
L (5.17
At the other extreme of feature shapes, the integral mean curvature reports visualizable information for rod or more
generally for tubule features. A feature satisfies the definition of a tubule if it is small in two of its dimensions and
extensive in the third, as shown in Figure 11b. For this class of features,
M
tubules
= L (5.18
where L is the length of the tubule.
Figure 9. A feature count measures the integral mean curvature, M
V
, of the bounding
surfaces in three dimensional space.
This sample: The unbiased frame in the field
Calibration: L
0
= 6.9 m; A
0
= (6.9 m)
2
= 47.9 m
2
Event: Feature lies within the unbiased frame
Measurement: Count the features
This count: N=34
Relationship: <N
A
> = (1/2) M
V
Normalized count: N
A
= 34 counts/47.9m
2
= 0.71 counts/m
2
Geometric property: M
V
= (2) 0.71 = 4.45 m/m
3
Figure 12 shows a plane probe section through a microstructure consisting of muralia. The vast majority of
sections will appear as curved strips in the microstructure. Orientations and positions of the probe that produce fat
sections are not very likely. For muralia equation (5.17) yields,
N
A
muralia
=
1
4
L
V
muralia
(5.19
where L
V muralia
is the total perimeter of plates or length of edges of muralia in unit volume of structure. If it is
assumed that Figure 12 is an unbiased sample of the population of positions and orientations of plane probes in
the structure, the feature count, N
A
= 0.074 (1/m
2
) gives, for the total length of feature edges in the three
dimensional structure = 0.295 (m/m
3
).
Figure 10. Net feature count for non-convex features in the area measures the integral mean curvature,
M
V
, of the surfaces bounding the features in the three dimensional microstructure.
This sample: The unbiased frame in the field
Calibration: L
0
= 17.7 m; A
0
= (17.7 m)
2
= 313 m
2
Event: Feature (particle or hole) lies within the unbiased frame
This count: 8 particles; 2 holes
Relationship: <N
A
>
net
= <N
A
>
+
<N
A
>
= (1/2) M
V
Normalized count: N
A

net
= 8 - 2 counts/313m
2
= 0.019 counts/m
2
V
= (2) 0.019 = 0.120 m/m
3
Most sections through tubules are small and equiaxed, as shown in Figure 13. Orientations and positions which
produce a very long section only occur for planes that are nearly parallel to the local axis of the tubule, and are
close to it in position. Such sections will be relatively rare, particularly if the radius is small. From equation (5.18)
the expected value of the feature count gives
N
A
tubules
=
1
2
L
V
tubules
(5.20
where L
V tubules
is the collective length of the rods or tubules in unit volume. For the example shown in Figure
13, NA = 0.29 (1/m
2
) which estimates a total tubule length of 0.58 (m/m
3
)
Figure 11. Muralia (a) are thick surfaces; a flat muralium is a plate. Tubules (b) are thick space curves
A plane probe intersects particles in three dimensional space to produce a collection of two dimensional features on
the sectioning plane. The number of such features, normalized by dividing by the area of the probe, is
proportional to the integral mean curvature of the surfaces bounding the three dimensional particles in the structure
divided by the volume of the specimen (equation 5.14). Integral mean curvature has simple geometric meaning for
convex bodies, for which it reports the mean caliper diameter, platelets or muralia, for which it reports the total
length of edge, and tubules, for which it reports the total tubule length. These results assume the set of plane
probes in the sample are drawn uniformly from the population of positions and orientations of plane probes in
three dimensional space. Oriented plane probes give information about these lengths in the direction that is
perpendicular to the probes.
Figure 12. A feature count on a section through muralia measures the integral mean curvature, M
V
, or the
muralia surfaces in three dimensional space. In this case, M
V
is simply related to the total length of edges
of the muralia, L
V muralia
This sample: Area within the unbiased frame
Calibration: A
0
= 122 m
2
Event: Feature lies within the unbiased frame
This count: N=9
Relationship: <N
A>
= (1/2) M
V muralia
= 1/4 L
V muralia
Normalized count: N
A
= 9 counts/122 m
2
= 0.074 counts/m
2
V muralia
= 4 N
A
= 4 0.074 = 0.295 m/m
3
Figure 13. A feature count is used to estimate M
V
, the integral mean curvature. For tubules M
V
is
proportional to the L
V tubules
length of the tubules.
This sample: Area within the unbiased frame
Calibration: L
0
= 6.9 m; A
0
= (6.9 m)
2
= 47.9 m
2
Event: Feature lies within the unbiased frame (indicated by color)
This count: N=14
Relationship: <N
A>
= (1/2) M
V tubules
= 1/2 L
V tubules
Normalized count: N
A
= 14 counts/47.9 m
2
= 0.29 counts/m
2
V tubules
= 2N
A
= 1.84 m/m
3
L
V muralia
= 2N
A
= 0.58 m/m
3
The Sweeping Line Probe in Two Dimensions
Figure 14 shows a feature in two dimensional space. Any point P on the boundary has a tangent direction and,
perpendicular to the tangent and pointing outward, a normal direction. Mapping these directions onto a unit circle
creates the circular image of the points, and a small arc of the boundary ds has a small range of normal directions
that map as a segment of arc on the unit circle, d, as shown in Figure 14a. The circular image of a convex feature
maps point for point on the unit circle exactly once. Thus, no matter what the shape of the particle boundary, so
long as it is convex, its circular image is exactly 2 radians, the circumference of the unit circle.
Figure 14. The circular image of a convex feature in a plane is equivalent to the unit circle. Every point on
the feature periphery has a normal direction that corresponds to one point on the circle, as shown by the
colored vectors in Figure 14a. A segment dS on the feature maps to a segment of arc d on the circle. A
non-convex feature has regions of negative curvature as shown in Figure 14b. The angular range which
these cover (shown in green) exactly cancels the extra positive curvature (shown in blue and orange),
again leaving a net circular image of 2.
If the boundary shape departs from convex, as shown in Figure 14b, then part of it will consist of convex arc
segments and part will be made up of concave arc segments. Mapping the rotations of the boundary normal on the
unit circle as the point P moves aroung the perimeter of the particle will produce ranges of overlap as shown in
Figure 14d. However, if concave segments are defined to contribute a negative circular image, then the net
rotation of the normal vector around the perimeter remains exactly 2 radians because the point chosen to begin
and end the map is the same point. Thus, like the spherical image of a surface discussed earlier, the circular image
of the boundary of a two dimensional feature also has the character of a topological property. It is equal to 2
radians, no matter what is the size and shape of the particle enclosed by the boundary.
In a two dimensional structure particles of the phase are said to be "multiply connected" if they have holes in
them. The connectivity of a two dimensional particle, C, is equal to the number of holes in the particle. The
boundary of each of these holes, no matter what their size or shape, contributes a net circular image of (-2)
because overall, a hole contributes a net of 2 of concave arc. Thus, the net circular image of a two dimensional
particle with C holes in it is
net
=
+

= 2(1 C) (5.21
For a collection of N features with a total of C holes in them,
net
= 2(N C) (5.22
The difference in the topological properties (N - C) is called the Euler characteristic of the two dimensional
collection of particles. These concepts for a two dimensional structure mirror those presented earlier for the
spherical image and the topological properties of three dimensional closed surfaces.
It was shown earlier that a volume tangent count, based on a sweeping plane probe through a three dimensional
structure, measured the spherical image of the surface bounding particles in the volume. An analogous probe and
measurement applies to the boundaries of two dimensional features in two dimensions. Figure 15 illustrates the
concept of the sweeping line probe in two dimensions. The line in the figure is swept across the field. In the
process the moving line forms tangents with elements of the boundary. Tangents formed with elements of the
boundary that are convex (T
+
) with respect to the phase and elements that are concave (T
) may be marked
separately and subsequently counted. Dividing these counts by the area of the field included in the count gives the
area tangent count. This is the two dimensional analogue of the volume tangent count described in a previous
section.
The area tangent count applied to boundary elements of a collection of features measures the circular image of
those features:
A+
= T
A+
;
A
= T
A
A
net
= T
A+
T
A
( ) = T
A
net
(5.23
Combine this result with equation (5.22) to show that the Euler characteristic of a collection of two dimensional
features with holes is simply related to the net area tangent count:
N
A
C
A
=
1
2
T
A
net
=
1
2
T
A+
T
A
( ) (5.24
For a collection of convex features C
A
= 0 (there are no holes) and T
A

= 0, so that N
A
= 1/2 T
A

+
; the result
that every particle has two tangents is self evident in this case. If the features are simply connected, i.e., have no
holes, then every bounding loop has two terminal convex tangents and, for each concave tangent there is a
balancing extra convex tangent. The net tangent count still gives two per particle. If there are holes, equation 5.24
gives the Euler characteristic of the collection of features.
Figure 15. An area tangent count is used to estimate the Euler characteristic and integral mean curvature.
A horizontal line is swept down across the image and the locations of tangents with convex
and concave boundaries are counted.
Probe population: Sweeping line in two dimensional space
This sample: Area within the frame
Calibration: L
0
= 20 m; A
0
= (20 m)
2
= 400 m
2
Event: Line forms tangents with convex and concave boundaries
Measurement: Count each class of tangent formed
This count: T
+
= 45 (red)
T
= 19 (green)
Relationship: <T
Anet
>= (1/2) (T
+
T
) = (1/) M
V
Normalized count: N
A
C
A
= (1/2) (45 - 19) /400 m
2
= 0.0325 counts/m
2
V
= 0.204 m/m
3
If the two dimensional structure in this discussion results from probing a three dimensional microstructure with a
plane then the expected value of the Euler characteristic on the section is an estimator of the integral mean
curvature of the corresponding collection of a surfaces in the volume, according to equation (5.14). Thus, the net
tangent count provides an unbiased estimate of the integral mean curvature:
T
Anet
= 2 N
A
net
=
1
M
V
(5.25
It may appear that the tangent count merely provides the same information that a feature and hole count could
provide, since in a two dimensional structure the features are visible in the field and can be separately marked and
counted. The tangent count would appear to give redundant information. However, there are some valid
arguments for considering replacing the feature count with the tangent count:
1. Tangents occur at a point; a point is either inside the boundary of the field, or outside it. Bias due to
particles intersecting the boundary of the field is not a factor in the tangent count;
2. In some structures, e.g., lamellar structures, or structures in which both phases occupy about the same area
fraction, features of, say, the phase may wander in and out of the boundary of the field several times so
that it is not possible to make the feature count.
3. In a three phase structure ( + + ) part of the boundary of particles is boundary, and part may be
boundary. In this case it is possible make separate area tangent counts of these two kinds of interface and
assess the circular image (and the integral mean curvature) of each.
4. If the boundary on a section has vertices, separate application of the tangent count to smooth segments
versus vertices provides a measure of the dihedral angle at edges in the three dimensional structure the
section samples. This is described in more detail in an example below.
5. Since separate counts are obtained for convex and concave elements of boundary the circular images of
convex and concave arc in the plane probe structure may be separately estimated. While this information
does not have a simple relation to the geometry of the parent three dimensional microstructure (DeHoff,
1978) it may be useful information in some applications.
Thus, the net tangent provides an unbiased estimator of M
V
in all of its applications.
Edges in Three Dimensional Microstructures
Because microstructures are space filling, triple line structures, such as those described in the qualitative
microstructural state discussion in Chapter 3, are common. A triple line results when three "cells", some or all of
which may be the same phase, are incident in three dimensional space. Three surfaces also meet at a triple line,
namely the three pairwise incidences of the cells involved. From the viewpoint of any particular cell, the triple
lines it touches are edges of that polyhedral shaped body. More generally an edge is a geometric feature which
results from the intersection of two surfaces that bound a feature to form a space curve, as shown in Figure 16. In
addition to the properties of space curves (length, curvature and torsion) an element of edge has a dihedral angle.
The two surfaces that meet at an element of edge each have a local normal vector. The dihedral angle, in Figure
16, is the angle of rotation between these two surface normals
3
. In general this angle varies from point to point
along the edge.
The dihedral angle also plays an important role in the physical behavior of structures in which surface tension
plays a role. In the thermodynamics of surfaces that meet at triple lines it is shown that the dihedral angles at the
three edges are determined by the relative surface energies of the three surfaces that meet there. For example, the
3
Like surfaces, edges may be convex (ridge with a maximum); concave (valley with a minimum) or saddle (ridge with a minimum or
valley with a maximum) in character. Valley edges by definition have a negative value of .
tendency of one phase to spread over a surface between two other phases, i.e., to "wet" the surface, is measured
by this dihedral angle. This property plays an important role in soldering, adhesion, liquid phase penetration,
welding, powder processing and the shape distribution of phases at interfaces.
Figure 16. An edge is formed by two surfaces meeting to form a space curve. The angle between the
surface normals at a point P on an edge, , is called the dihedral angle.
An element of length of an edge may be thought of as a limiting case of an element of surface for which one of the
radii of curvature goes to zero, i.e., sharpens to an angle. With this point of view it can be shown that for a
polyhedral particle the edges make their own contribution to the integral mean curvature of the boundary of a
particle:
M
edges
=
1
2
dL
L
(5.26
where the integration is over the length of edge in the structure. To lend some credence to this assertion that this
property is in fact the contribution to M due to edges it may be shown that this result may be used in conjunction
with Minkowskis formula for convex bodies, equation (5.13), to compute the mean caliper diameter of
polyhedral shapes. Consider the cube with edge length e shown in Figure 17a. The dihedral angle at all points on
the twelve edges is /2. The total length of edge is 12e. The surfaces (faces) all have zero curvature, so that the
contribution of the surfaces to M is zero. Combine equation 5.26 with the Minkowski formula:
M
cube
= M
faces
+ M
edges
= 0 +
1
2
2
L =

4
12e = 3e = 2D
cube
D
cube
=
3
2
e
(5.27
Figure 17. The integral mean curvature for features with edges contains a contribution from edges. For the
cube (or any flat faced polyhedron), all of the integral mean curvature resides at the edges. A feature with
curved faces and edges has both contributions.
This is the same result that is obtained by evaluating the caliper diameter of a cube as a function of orientation and
averaging over orientation.
Similar analysis of other shapes provides an efficient way to calculate D averaged over orientation. This is
generally far easier than the modelling approach using Monte-Carlo sampling introduced in Chapter 11, but the
latter method can also produce the distribution of intercept lengths or areas which are needed to unfold particle size
distributions.
Figure 17b shows a cylinder with radius r and length l. In this case there are contributions to M from both the
curved surface and the edges. On the curved face of the cylinder the curvature in the axial direction is zero, and the
mean curvature everywhere has the value (1/2r). The dihedral angle at every point on the edges is /2. Evaluate M
for a cylinder:
M
cyl
= M
faces
+ M
edges
= H dS +
1
2
S
dL
L
=
1
2r
S
dS +
1
2
2
L
dL =
1
2r
2rl +

4
2 2r
M
cyl
= l +
2
r = 2D
cyl
D
cyl
=
1
2
l +

2
r
(5.28
Again, this is the same result that is obtained if D is computed as a function of orientation and averaged over the
hemisphere. Note that for the limiting cases of a plate (r >> l), D =

2
r, while for a rod (l >> r), D =
1
2
l .
Figure 18. The mean dihedral angle at edges, <> on the (colored) phase of this structure can be
measured with a triple point count combined with a tangent count.
Probe population: Planes in three dimensional space and sweeping line
probes in two dimensions
Calibration: L
0
= 14.7 m; A
0
= (14.7 m)
2
= 215 m
2
Event: Plane intersects triple line;
Sweeping line forms tangent at triple line
Measurement: Count the triple points; count the tangents
This count: 49 triple points, 32 tangents
Relationship: <> = TA / PA
Normalized count: P
A
= 45 counts/215 m
2
= 0.23 counts/m
2
T
A
= 32 counts/215 m
2
= 0.15 counts/m
2
Geometric property: <> = 0.15 / 0.23 = 2.05 radians = 117 degrees
Edges can also be treated as a limiting case of a curved surface in the sense that the integral mean curvature of an
edge can be measured by the tangent count, equation (5.24):
T
A
edges
=
1
M
V
edges
=
1
1
2
dL
L
V
(5.29
where is the dihedral angle measured at any point along the edge. An average dihedral angle may be defined,
=
dL
L
V
dL
L
V
=
2M
V
edges
L
V
(5.30
M
V edges
can be measured with the tangent count at edges, using equation (5.29), and L
V
can be measured by
counting points of intersection of the edge line with the plane probe. Thus, the average dihedral angle for any
specific type of edge can be estimated from the ratio
=
2 T
A
edges
2P
A
=
T
A
edges
P
A
(5.31
Figure 18 shows a two phase structure with triple lines. A sweeping line probe is visualized to move from
the top to the bottom of the field. Tangents with the edge are marked and counted. The total number of
triple points is also counted. With the usual assumptions about the sample plane, these properties are normalized
and used to estimate M
V

edge
and L
V
. These computations are indicated in the caption to Figure 18. The ratio
is then used to estimate the average dihedral angle on edges of the particles. The result, 2.05 radians or 117
degrees, is the average value of the angle between the surface normals at the edge. This result may be used, for
example, in the assessment of the relative surface energies of the and interfaces in this system.
Summary
The disector probe is required to obtain information about the topological properties, number (N
V
) and
connectivity (C
V
) of surfaces bounding three dimensional features. This is achieved by comparing the structures
on a reference plane and a look-up plane in order to infer the occurrence of tangents with the surfaces formed by a
plane that is visualized to sweep the volume between the planes of the disector. Counts of the three kinds of
tangents measure the spherical image of convex, concave and saddle surfaces in the structure. The Euler
characteristic, (N
V
- C
V
), may be estimated from these tangent counts in the disector probe
N
V
C
V
=
1
2
T
V
net
=
1
2
T
V
++
+ T
V
T
V
+
[ ]
For most structures the connectivity is small in comparison with the number of disconnected parts of the
structure, and the disector probe provides the primary strategy for estimating number density in microstructures.
If the three dimensional particles are convex, then a feature count on a plane probe estimates the product of the
number density and mean caliper diameter:
N
A
= N
V
D
Feature counts on oriented plane probes measure the caliper diameter in the direction of the plane probe normal.
N
A
, ( ) = N
V
D , ( )
For more general structures, the (net) feature count (or equivalently, the net area tangent count) reports the integral
mean curvature of the surface in the structure:
N
A
net
= N
A
C
A
=
1
2
T
A
net
=
1
2
T
A
+
T
A
=
1
2
M
Particles of arbitrary shape in the three dimensional structure may, for some positions and orientations of a plane
probe, produce sections with holes in them. In this general case, the feature count is interpreted as the number of
features in the section minus the number of holes. This net number of features can also be determined from the
area tangent count, which visualizes a line that sweeps across the field of view and forms tangents with convex
and concave elements of the boundaries of particle sections. In the general case, either of these counts reports the
value of integral of the mean surface curvature of the boundaries of particles in the three dimensional
microstructure. For convex bodies, M is proportional to the mean caliper diameter. For muralia (plates) it reports
the length of perimeter of the features in three dimensions. For tubules (rods), M reports their total length in the
volume.
If the features in the three dimensional collection of particles have edges, then these edges contribute to the integral
mean curvature of the boundaries of the particles so that
M
V
total
= M
V
surfaces
+ M
V
edges
= H dS
S
V
+
1
2
dL
L
V
where is the dihedral angle between the surface normals at the each point on the edge. This result may be used to
estimate the average dihedral angle along a collection of edges in the structure.
=
T
A
edges
P
A
Chapter 6 - Sample Design in Stereology
The fundamental relations in stereology are expected value theorems that relate measurements that are made on
geometric probes passed through a three dimensional microstructure and corresponding geometric properties of
the three dimensional structure that the probes sample. Implementing any of these relations requires that a sample
of the population of appropriate probes be produced and the appropriate measurements be made upon the set of
probes in the sample. The mean value of this set of sample measurements is computed. This mean value is then
taken as an estimate of the expected value of the measurement on the population of probes from which the sample
set is drawn.
It is highly desirable that this estimate be unbiased. If this is not guaranteed by the choice of the set of probes
included in the sample set then a great deal of effort may be expended to produce an estimate of a geometric
property which may be highly precise, but wrong.
In order for the set of probes in the sample to provide an unbiased estimate of the expected value for the
population it is required that the sample set be drawn uniformly from the population of probes. This is the central
rule in the design of the probe set included in a stereological experiment. This condition must be satisfied because
it is the central assumption made in the derivation of each of the fundamental relations that lie at the heart of the
methodology of stereology. In order to design a sample of probes that is selected uniformly from the population of
those probes it is necessary to understand what the population is, for each class of probes used in stereological
measurements.
This chapter presents the population for each of the most important probes that are used in stereology: points,
lines, planes and volumes. Once the population can be visualized for a particular probe class, the ground rules for
obtaining a sample of probes drawn uniformly from each population will become evident. Schemes and strategies
for obtaining a uniformly drawn sample set will then be discussed for each probe class.
Population of Point Probes
A point on a one dimensional line is specified by assigning a value to a variable x that reports the distance from
some conveniently defined origin on the line. The population of points on a line is the set of values that this
variable may be assigned. For some interval of length L
0
, which may be viewed as the "specimen" in this one
dimensional world, the population of points in the specimen is the set of values of x that correspond to points
within L
0
. Since the possible numerical values for x are considered to be distributed uniformly, the set of points is
distributed uniformly within L
0
.
This concept may be extended to a two dimensional "specimen" which encompasses the total area A
0
of interest in
an experiment. A point within the specimen is defined by constructing a convenient Cartesian coordinate system
with x and y axes and assigning particular values to x and y. The population of points in the specimen is the set of
all possible combinations of values for x and y that lie within A
0
. Again, since the numerical values of x and y are
distributed uniformly, the set of points in the population are uniformly distributed over the area of the specimen.
Chapter 6 - Sample Design in Stereology Page # 99
In order to specify a point in three dimensional space it is necessary to assign a value to three variables, e.g., the
Cartesian coordinates (x, y, z). Each point in the volume V
0
of the macroscopic specimen is given by its
corresponding three coordinates. The population of points that lie within the specimen is the collection of all
possible values of the triplet (x, y, z) that describe points contained within its boundaries. These points are
uniformly distributed in the volume of the specimen.
Since the points in these Cartesian spaces represent a continuum there is a conceptual problem in counting how
many points are contained in a given domain in any of these that spaces. Even the smallest finite domain contains
an infinite number of points. Indeed domains of different sizes and shapes each contain an infinite number of
points. There is an infinite number of points in the interval from x = 0 to x = 1 on the x-axis, but there is also,
conceptually, an infinite number of points in the interval from 0 to 1/2. Intuitively it seems that there should be
"twice as many" points between 0 and 1 as there are between 0 and 1/2.
a b
c
Figure 1. Measures of line length (a), area (b) and
angle (c) as discussed in the text.
This problem is solved by introducing the concept of the "measure of the set of points" in a domain in any space.
The measure of the set of points in an interval on a line is the length of the interval assuming that the density of
points is uniform along the line. Thus, the measure of the set of points contained in a one dimensional "specimen"
of length L
0
is equal to L
0
. The measure of the set of points in some short segment of length contained within
the specimen, as shown in Figure 1a, is equal to the length . With these concepts, the fraction of points in the
specimen that lie within the segment is the ratio of these measures: f
= /L.
In two dimensional space the measure of the set of points in any domain is equal to the area A of that domain. The
population of points in the two dimensional specimen in Figure 1b has a measure equal to A
0
. The set of points
that lie within the phase particles in this figure has measure A
, the total area of this collection of particles. The

fraction of points that lie within the phase is the ratio of these measures: A
/A
0
= A
A
, the area fraction occupied

by the phase. Thus for the entire population of points in the specimen the point fraction equals the area fraction,
which is the basis for equation (4.2) in Chapter 4.
In three dimensional space the measure of the set of points in a domain is the volume V of the domain. For a
specimen this measure is the volume V
0
of the specimen. The measure of the set of points within a set of
features contained in the specimen is their volume V
. The fraction of points in the specimen that lie within the set
of features is the ratio of these measures, V
/V
0
. This is the basis for the classic volume fraction relation in
Chapter 4, equation (4.5)
In a stereological experiment a sample is drawn from the population of points in the specimen. The total number of
points in the sample is P
T
and P
is the number of these points that lie within the feature set. The ratio of these
two counts is then used to estimate the expected value for this ratio of these measures for the populations of points
in the specimen. This estimate will be unbiased if and only if the points in the sample are drawn uniformly from
the population of points in the volume of the specimen, V
0
.
As example, suppose there were a gradient in the volume of the phase so that the volume fraction of increased
as the specimen was traversed from top to bottom. If in the experimental sample of point probes was confined to a
single plane near the top of the specimen then the mean of this sample will underestimate V
V
since there is less
phase near the top than the bottom. This sample provides a biased estimate of the expected value of P
P
. If it were
known a priori that the gradient in was one dimensional and in the vertical direction, then a plane in the vertical
direction would capture the gradient and sample it uniformly. Armed with the a priori information in this case,
points drawn in a single appropriately chosen plane could produce an unbiased estimate of P
P
.
If the particles are themselves uniformly positioned over the area of the specimen then the sampling problem is
simplified. For example, all of the points in the sample could come from one region in the specimen and the
resulting mean value would be an unbiased estimate of the population expected value. Uniformity (or the absence
of gradients) may be characteristic of some man-made materials, but most specimens will contain gradients in
structure. The assumption of uniformity must be verified before it is used in an implementation that simplifies the
stereological design.
Thus, in the general case the positional variation of the volume of the feature set is either unknown, or of such a
nature that it cant be captured in a single plane. In the absence of a clear knowledge that the volume of the features
in the sample is uniformly distributed, or has a simple, one dimensional spatial variation, the sample of point
probes must be distributed uniformly throughout the volume of the specimen. In this general case an unbiased
estimate for P
P
is obtained only if the set of points chosen for inspection in the sample is uniformly distributed
over the volume V
0
of the macroscopic specimen.
A specimen exhibiting gradients in V
V
does not necessarily require a great deal more effort to estimate P
P
than
does a specimen in which the phase is uniformly distributed. It is only necessary to plan ahead (design) in
cutting up the specimen for preparation and inspection. For example, the specimen may be diced into a number of
chunks. Pieces selected randomly and uniformly from these chunks (applying the fractionator {Howard & Reed,
1998b} principle described below) may all be mounted in a single mount. A single plane sectioned through this
aggregate would provide fields of view that uniformly sample the volume V
0
of the specimen. Grids imposed on
fields selected for measurement would provide an unbiased sample of the population of points in V
0
.
This sampling strategy, which inspects points that are uniformly drawn from the population of points in the
specimen, gives an unbiased estimate of the expected value for the structure no matter how the features are
distributed.
The Population of Lines in Two Dimensions
The only attribute that points have is position {x, (x,y), or (x,y,z)}. A line drawn in a plane has both position, p,
and orientation, . In two dimensions an orientation of a line is represented by a point on a unit circle of
orientations, as shown in Figure 1c. The population of orientations is the set of points on the unit circle. The
measure of the set of orientations in two dimensional space given by the measure of the set of points on the
circumference of the unit circle: 2. The measure of the set of orientations contained in some angular range , as
shown in the figure, is the length of the arc on the unit circle that contains the set of points that correspond to the
directions encompassed by . The fraction of orientations that lie in is the ratio of these two measures, /2.
For a line probe, in which adding 180 degrees to the angle of the line produces the same result, the orientations are
measured over half the unit circle and the fraction of orientations contained within some angular range is /.

Figure 2. An array of lines with a given orientation in a plane (a),
and the measure of a range of angles (b) as discussed in the text.
Consider now the subset of the population of lines in two dimensional space that all have the same direction, as
shown in Figure 2a. To aid in describing the population of positions of lines, construct a convenient Cartesian
coordinate system with origin O and x and y axes. Construct a line OA through the origin in a direction
perpendicular to that of the subset of oriented lines. An individual line in this subset of lines is located uniquely by
a position on the line OA. The measure of the set of lines that have the orientation shown and are contained in
some interval BC on OA is the measure of the set of points in this interval, and is therefore the length BC.
Thus, a given line in two dimensional space is given by a pair of numbers: (p, ). The population of lines is
represented by the values of from 0 to and, for a given value of , a range of p values that encompass the
specimen. The population of orientations may be guaranteed to be uniformly sampled by simply using circles as
line probes as illustrated in Chapter 4, Figure 8.c. If the set of circles contained in the sample is distributed
uniformly with respect to position over the specimen area then the population of lines is guaranteed to be sampled
uniformly in both position and orientation. Observations obtained from such a sample will provide an unbiased
estimate of the expected value of some measure derived from the population of lines in two dimensional space.
Line Probes in Three Dimensions
As in two dimensions, a line in three dimensional space has both orientation and position. An orientation may be
described by a point on the unit sphere. The coordinates of such a point are given by two angles, as they are on a
globe that maps the earth, as shown in Figure 2b (see also Chapter 4, Figure 12). The longitude, , is represented
by a polar circle that passes through a point on the circle of the equator. The angle is measured relative to a
reference direction defined to be 0 longitude (through Greenwich on the globe). The latitude may be specified by
the angle between the point (, ) on the sphere and the north pole. (By convention on the globe latitudes are
specified by the compliment of , i.e. by the angle along a longitude at from the equator to the point (, ). In
this text latitude will be specified by the angle from the pole, sometimes called the colatitude.)
A continuous range of orientations in space maps as some domain on the unit sphere called its spherical image, as
discussed in Chapter 5. The measure of the set of orientations represented by this domain is the measure of the set
of points on the unit sphere contained in its spherical image, which is an area on the unit sphere, as shown in
Chapter 5, Figure 1. In terms of the variables (, ) a small patch of this area is given by sin d d . The sin
f factor arises because the arcs bounding this element in latitude are arcs of a small circle (as opposed to a great
circle), which has a radius of sin where =1 is the radius of the sphere. Integration of this function over the
range of longitudes and latitudes corresponding to some domain of orientations on the sphere gives the spherical
image of the patch. For example, integration over the northern hemisphere gives the area of the hemisphere 2.
Now consider the subset of lines in three dimensional space that all share the same orientation, as shown in Figure
3a. This positional aspect to defining a line in three dimensional space may be visualized by constructing a plane
that is perpendicular to the orientation of the line set. A Cartesian coordinate system in this plane facilitates
defining the location of a particular line. A particular line is then represented by a point (x,y) on this plane. A set
of lines that lie in some region on this plane (e.g., the set of lines that intersect a particular particle of in the
specimen) is represented by the set of points (x,y) contained in that domain. The measure of this set of oriented
lines is thus the measure of this set of points, which is the area of the domain on the plane.
Figure 3. Arrays of parallel lines (a) and planes (b) in 3D space as discussed in the text.
Thus, a line in three dimensional space requires four variables for its complete specification: two orientation
variables (, ) and, for a given (, ) two position variables (x,y). The measure of a set of orientations is an area
on the unit sphere and, for a given orientation, the measure of a set of positions is an area on the plane which is
perpendicular to the orientation direction. In selecting a set of lines to be included in a sample from this four-
variable population it is necessary that the two sets of points represented in the construction be sampled uniformly.
A sampling strategy, called the method of vertical sections, provides a simple and automatic procedure for
guaranteeing such a uniform sample of positions and orientations of line probes from the population of lines in
three dimensional space.
Planes in Three Dimensional Space
Like a line, a plane in three dimensional space has an orientation and a position, as shown in Figure 3b. The
orientation of a plane is unambiguously described by the direction of a line that is perpendicular to the plane, called
the normal to the plane. Since the plane normal is a line, the measure of a set of plane orientations is identical with
that described for lines in three dimensional space, i.e., the area of the spherical image of the plane normals in the
set.
Specification of the orientation of a plane in space by the angles (, ) does not complete the description of the
plane. There is an infinite set of parallel planes that all share a given orientation. A specific plane in such a set of
parallel planes may be specified by constructing a convenient Cartesian coordinate system and constructing the line
OA through its origin that is parallel to the direction given by (, ). The position of a plane is given by its
distance p from the origin along that line. The measure of the set of planes in some interval on OA is the measure
of the set of points that lie in that interval. We have seen earlier that the measure of the set of points in an interval
on a line is the length of the interval.
Thus, a particular plane in three dimensional space is described by three numbers in some convenient Cartesian
coordinate system: (p, , ). The measure of the set of orientations is an area on the unit sphere of orientations
and the measure of the set of positions is a length on a line in the direction of the plane normal. In designing a
sample of a set of planes in a stereological experiment it is required that their orientations and positions be
uniformly chosen from these measures.
Producing a uniform sample of orientations and positions of plane probes in three dimensional is perhaps the most
challenging of the sampling problems in stereology. Chapter 7 describes procedures that have been used.
Disectors in Three Dimensional Space
The disector sample consists of a rectangular prism with areas A of the top and bottom faces and a known
thickness, h, as shown in Figure 4. It is used to sample a volume V = A
0
h. An individual disector may be
specified by the coordinates (x, y, z) of a convenient point in the box, e.g., one of its corners. There are no
orientation variables involved in this specification. Since for every disector there is one such point and for every
point in space there is a unique disector, the population of the set of disectors in space is identical with the
population of the set of points in three dimensional space.
Figure 4. The populations of disectors in a three dimensional space is identical to the population of points,
since each disector may be located by the position of one of its corners. The volume analyzed by each is A
0
h.
A uniform sample may be selected from the population by selecting points that locate the disector from the uniform
population of points in the specimen.
Sampling Strategies in Stereology
In order for the mean of a sample of stereological measurements to provide an unbiased estimate of the expected
value of that measurement for the probe population that corresponds to the measurement it is necessary that the set
of probes chosen for examination be drawn uniformly from the population of those probes in three dimensional
space. The population of probes may be described by position variables, and by orientation variables. For point
probes and disector probes the set of sample probes must be drawn uniformly from the population of points that
occupy the specimen. For line and plane probes the sample must also be drawn uniformly from the set of points
on the unit sphere of orientation. Once an appropriate sample of probes has been obtained, the corresponding
counts are made and the fundamental relations of stereology are then applied to estimate the geometric property
that is of interest in the experiment.
Producing such a uniform sample may at first appear to be a daunting task. One might imagine that it would
require a large number of fields to produce such a sample, or to prepare a number of sections from several places
in the specimen. However, the sampling strategies discussed in this section do not require measurements on
significantly more fields than might be needed to produce an estimate for a structure which is itself uniform. It is
only necessary to plan ahead in sectioning the specimen and collecting a random, uniform set of the pieces. In
some cases this sample set of pieces can all be mounted together in one mount for subsequent preparation and
measurement.
An additional principle which is important in fostering inclusion of a representative uniform sample of probes
from the their population is "Do more less well" (Gundersen & Osterby, 1981). It is better to choose
magnification and grid scales so that a few counts are made on a large number of fields rather than a larger number
of counts on a few fields. This strategy not only includes more regions of the specimen in the sample but, by
encouraging collection of a few counts on the typical field, helps to minimize counting errors.
A primary strategy used to sample uniformly with respect to position has been called the fractionator (Howard &
Reed, 1998b). The specimen is cut into a number N of pieces; depending upon the application, these pieces may
be sliced, then cut into sticks or rods and or into smaller pieces. Some preliminary experiments determine roughly
what fraction, f, of these pieces will be included for mounting and examination. These pieces are chosen randomly
and uniformly from the population of pieces using a random systematic sampling strategy similar to that discussed
in Chapter 4.
Spread out the pieces in a row on a table. Suppose N = 29, and on the basis of preliminary study it is decided that
a total of about six of these pieces will form a convenient mounting for analysis. Imagine the pieces on the table
are numbered 1 through 29. An exhaustive collection of samples from the set of pieces may be obtained by
selecting every fifth piece starting with, respectively,
A 1 6 11 16 21 26
B 2 7 12 17 22 27
C 3 8 13 18 23 28
D 4 9 14 19 24 29
E 5 10 15 20 25 -
Sample set E has only 5 members because 29 is not divisible by 5, but that still satisfies our choice of "about six"
pieces for analysis. Choose a random number from 1 to 5 to select sample A, B, C, D or E for analysis. Suppose
the number comes up 4, so that sample D is chosen. Then the pieces
D 4 9 14 19 24 29
will be mounted for preparation and subsequent analysis.
If the 29 pieces were laid out on the table without paying attention to where they came from in the specimen,
sample D (or for that matter, any of the samples) provides a random sample of the population of pieces. Every
piece in the set of 29 pieces has an equal probability of being included in the sample. If on the other hand the
pieces are laid out in some order with respect to position in the original specimen (e.g., slices from "top" to
"bottom") then the selection of one sample, D, constitutes a random systematic sample of the population of, in this
case, slices. In this case every sample (A, B, C, D, E) has an equal probability of being included in the
measurement.
This difference has been found to be important in the design scheme for stereological experiments. In the simple
random sample there is some chance that the pieces in a given sample chosen for measurement may all come from
near the top of the specimen, or be otherwise localized. In the random systematic sample each sample set has
pieces that are uniformly distributed from top to bottom. Both samples produce unbiased estimates of population
expected values as required by the stereological relationships.
However experience has shown that the variance associated with the set of sample means is very much smaller (by
a factor of 2 or 4 or perhaps more {Gundersen & Jensen, 1987}) for the random systematic sample than for the
simple random sample. This result has important practical implications for the level of effort required to obtain a
given precision in the statistical experiments of stereology. The precision of an estimate of a mean value is given
by the confidence interval assigned to the estimate of the mean. The confidence interval is a range about the
estimated mean defined by
C. I. = x 2
x
(6.1
where
x
is the standard error of the mean computed from the sample. This "95% confidence interval" means
that the probability that the true mean of the population lies within this interval is 0.95. The precision with which
the mean of the population is estimated is thus determined by
x
. This property is computed from the standard
deviation sample measurements,
x
, given by
x
=

x
n
(6.2
where n is the number of readings (fields) in the sample. The smaller the confidence interval, the more precise is
the estimate. Precision can be increased in two ways:
1. Increasing the number of fields n measured in the sample: (Taking four times as many measurements cuts
the confidence interval in half, making the estimate "twice as precise");
2. Decreasing the standard deviation of the sample, which is an estimate of the square root of the variance of
the population.
Applying the strategy of the random systematic sample decreases the variance (in comparison with a simple
random sample), decreasing
x
and thus
x
and the confidence interval for a sample of a given size, n. Halving
the variance cuts the confidence interval in half.
Applying the strategy of the random systematic sample permits attainment of a given precision with fewer counts,
or, for the same number of counts, increases the precision. This sampling strategy is now widely used in
stereology because it significantly increases the efficiency of these techniques. Implementation of this strategy
requires only conscious planning of the choices made in designing a sample for analysis, i.e., where the specimen
should be cut and how the pieces should be chosen for preparation and examination.
This fractionator approach can be implemented for each of the stereological probes that have been described.
1. In using the Cavalieri method to estimate the volume of an object the pieces may be slices obtained by
microtoming the sample.
2. The point count may be also applied to this fractionated sample of slices through the structure. On a given slice
fields chosen for measurement may be chosen from the set of possible fields by another fractionating process.
3. The method of vertical sections may be applied to a fractionated sample of pieces obtained from the specimen
by cutting it as you would to make french fries from a potato. This is illustrated in Figure 5. The vertical direction
in the sample is the long dimension of these stick shaped pieces. Rotating the sticks randomly or using a
systematic random rotation of the pieces about the vertical axis when placing them in the mounting powder with
long (vertical) axes parallel provides a uniform sample of longitudinal directions as well as a clear indication of the
vertical direction in every field. Using a cycloid test line uniformly samples latitude directions. A single section
reveals structure that uniformly samples position in the x, y, and z directions and orientation in and . Random
systematic sample of all of the possible fields that may be constructed on this one section provides a uniform
isotropic random sample for analysis.
4. It mat be possible to produce an isotropic, uniform random sample by dicing the specimen into small pieces that
are equiaxed, (e.g., cubes), fractionating them and mounting them with uniform random rotations as they are
placed in the mounting medium. Then any plane section through the structure will produce an IUR sample of
points, lines and planes. The practical difficulty associated with such a method is contained in the phrase
"mounting them with random uniform rotations".
Figure 5. A procedure for obtaining IUR vertical sections: a) the specimen is cut into vertical samples; b) a
structured random procedure for selecting an appropriate subsample of these is used; c) the selected prisms
are rotated either randomly or by structured random angles; d) the rotated prisms are embedded and
sectioned. On the resulting section image the use of cycloid grids will provide isotropic, uniform and
random sampling of orientations in the microstructure.
These examples are not practical in all cases and are presented here to illustrate the principle that, once the ground
rules are clearly formulated for obtaining a "representative" sample from a population of probes, a little ingenuity
applied in the original design of the experiment may solve the sampling problem in stereology without a great deal
of additional effort expended in making the measurements. Chapter 7 discusses some of the practical sampling
procedures that may be used.
Summary
Each of the stereological relationships assumes that the population of probes used in the experiment is uniform
over the domain of the specimen. Knowledgeable design of stereological experiments requires that the nature of
these probe populations be clearly understood. Each class of probes has a population that is described by the
variables necessary to define an individual probe.
The population of point probes in two dimensions is the set of all possible position pairs (x,y) in the
specimen. The measure of the set of point probes in a given region is the area of that region.
The population of point probes in three dimensions is the set of all possible position triplets (x,y,z) in the
specimen. The measure of the set of point probes in a given region is the volume of that region.
The population of line probes in three dimensions is the set of all possible points (, ) on the sphere of
orientation and, for a given such orientation, the set of points (x, y) on a plane perpendicular to that
orientation. The measure of the set of orientations in a region of orientations is an area on the sphere of
orientation. For a set of lines, the range of orientations is an area on the hemisphere of orientation. The
measure of the set of positions of lines with a given orientation is an area on the plane perpendicular to that
orientation.
The population of plane probes in three dimensions is the set of points (, ) on the unit sphere or
hemisphere designated by the normal to any given plane, and the set of positions p of points along a line in
that direction. The measure of the set of orientations is an area on the sphere and the measure of the set of
positions of planes is a length along their normal directions.
The population of disectors in three dimensional space is the same as the population of points, since each
disector may be uniquely located by one of its corner points.
The fractionator strategy envisions cutting the specimen into a large number of pieces then, using a simple random
or a random systematic method, selects a uniformly representative small sample from the population of pieces for
analysis.
With careful planning in the design of the sample it is possible to satisfy the isotropic uniform random sampling
requirement of stereology without significant additional effort. Chapter 7 discusses some representative
implementations of such strategies.
Chapter 7 - Procedures for IUR Sampling
Chapter 6 emphasized the requirement that sampling of a three-dimensional structure with points, lines or planes
be isotropic, uniform and random (IUR). Accomplishing this is not easy, and much of the thrust of recent
stereological development has been to explore sampling strategies that provide such results. It is useful to
summarize here just what IUR means (without recapitulating the detailed discussion in Chapter 6). Point probes
do not have to be isotropic, because the points themselves have no direction. It is sufficient to disperse a set of
points through the structure with an equal probability of sampling all regions (e.g., not points that are concentrated
near one end or near the surface), and which are randomly placed with respect to the structure.
The idea of randomness is not always an easy one to test. If the structure itself has no regular spacings or
repetitive structure, a regular grid of points can be used (and often is, for convenience in counting and for
efficiency of sampling). But if the structure does have some regularity, then the use of a regular grid of points will
produce a biased result. Figure 1 shows a cross section of a man-made fiber composite. While not perfectly
regular in their arrangement, the fibers are clearly not randomly distributed in the microstructure. Attempting to
measure the volume fraction of fibers with a grid of points would produce a biased result because that grid and the
arrangement of the fibers would interfere to produce a result in which points systematically hit or missed the
fibers. For an image such as this one a routine that placed points at random on the image would be required.
Typically this is done by generating two uniform random numbers using a computer pseudo-random number
generator (a mathematical routine seeded by the system clock; we will not consider the intricacies of such routines
here). The two numbers are typically provided as decimal fractions between 0 and 1, and can conveniently be
scaled to the dimensions of the image to locate points in X, Y coordinates.
Figure 1. Cross section image of a man-made fiber composite, with an approximately regular array of fibers.
Chapter 7 - Procedures for IUR sampling Page # 111
Such random sampling can be used for any structure of course, even one that has no regularities. But there is a
more efficient procedure that produces equivalent results. It converges on the same answer as more and more
points are sampled and converges more rapidly than the random sampling approach (Gundersen & Jensen, 1987).
This is equivalent to saying that it has a better precision for a given amount of effort. The method (described in
earlier chapters) is called structured random sampling, which at first seems to be an oxymoron. It can be applied to
many types of sampling with points, lines and planes, but will be explained first as it applies to the point sampling
of an image.
Volume fraction
To determine the volume fraction of a phase in a structure, assuming that the sampling plane is representative (we
will see below how to deal with that requirement), the fundamental stereological relationship is V
V
= P
P
, or the
volume fraction equals the point fraction. The precision of a counting measurement is a standard deviation equal to
the square root of the number of events counted. For instance, if asked to estimate the number of cars per hour
traveling along a highway we could count the cars and measure the time required for 100 cars to pass. The number
of cars divided by the time T (expressed as a fraction of one hour) would give the desired answer. The standard
deviation of the number counted is 10, so the relative standard precision would be 10/100 = 10%. By counting
1000 cars we could reduce the standard deviation to 1000 = 31.6, or a relative standard precision of 3.16%.
Counting 10,000 cars would give a relative standard precision of 1%.
Usually in a given experiment we know what precision is required or desired. Consider for the moment a desired
precision of 10%. This requires counting 100 events as described above. For point counting, this means that there
are 100 points in the grid or random distribution that hit the phase of interest. If the phase represents (e.g.) 25% of
the structure, this would require placing 400 points onto the image to achieve 100 hits. However, there is one
additional assumption hidden in this procedure: each of the points is required to be an independent sample of the
structure. This means that the points should be separated from each other by enough distance that they do not
repeat the same measurement, which is usually taken to mean that they do not lie closer together than a
characteristic distance in the structure such as the mean intercept length.
However, random points generated by the procedure outlined above will not all conform to that assumption. As
shown in Figure 2, random distributions of points on an image inevitably produce some points that are quite close
together. These are not all independent samples, and so the actual precision of the measurement is not as good
would be predicted based on the number of points. The clustering of points produced by a random (Poisson)
generation process is used in another context to study the degree to which features in images have tendencies
toward clustering or self-avoidance. The standard deviation of the P
P
value obtained using random points will be
greater (worse) than with a regular grid by a factor of e (2.718). Notice that this does not mean that the result
obtained with such a random point array is biased or in error, just that it takes more work (more points have to be
sampled) to obtain an equivalent measurement precision.
The use of regular grids of points to count P
P
is widely used, of course. Grids of points are available as reticles to
fit within microscope eyepieces, or applied to photographic prints as transparent overlays, or superimposed on
images using computer displays. This is not so much a consequence of the greater measurement efficiency as a
recognition of the ease of counting such points manually by having the eye track along a regular grid.
Figure 2. An example of 200 random points. Note the presence of clusters of points that
oversample some regions while large gaps exist elsewhere.
The proper way to use such a grid while still obeying the requirements of random sampling according to the
structured random sampling method is as follows:
1. Determine, based on the desired level of precision and the estimated volume fraction of the target phase, the
number of points to be sampled in the image. Note that in many real cases not all of the points will be sampled
in a single image - there will be many fields and many section planes in order to meet the requirements of
uniform sampling of the entire structure. There is also the restriction against using too many points on one
image based on the requirement that the points must be separated far enough to be independent samples,
meaning as noted above that multiple points do not fall into the same region of the structure.
2. The resulting number of points form a regular grid. Typically these grids are square so that the number of
points is a perfect square (25, 49, 100, etc.) with fairly small numbers used for purely manual counting
because larger numbers foster human counting errors. In the example shown in Figure 3, the grid is 5x5=25
points.
3. Generate a pair of random numbers that define the X,Y coordinates for the placement of the first point of the
grid within a square on the image. This will automatically place all of the other grid points at the same relative
offset.
4. Perform the counting of P
P
.
5. For other fields of view, generate new random numbers for the offset and repeat steps 3 and 4, until enough
fields of view, section planes, etc. have been examined to produce uniform sampling of the structure and
enough points have been counted to produce the desired precision.
Figure 3. A 5x5 grid placed on an image area. The upper left corner is displaced randomly
within the red square to accomplish structured random sampling of the area.
This procedure is a straightforward application of structured random sampling. Several examples will be detailed
below that extend the same method to other situations and types of probes. In all cases note that it is important to
estimate beforehand the number of images (sections planes and fields of view) that must be examined in order to
obtain the desired precision. This requires an estimate of the quantity being measured, in this example the volume
fraction. Typically this parameter is known at least approximately from previous sampling of the structure, prior
results on other similar specimens, or independent knowledge about the objects of interest.
Sampling planes
The same procedure as detailed above can be applied to the selection of the sampling planes themselves. For
example, consider the problem of determining the volume fraction of a phase within an irregular 3D object as
shown in Figure 4. The measurement in this case must measure the volume of the object as a whole as well as that
of the phase in order to obtain the volume fraction. If the phase is expected to be about 20% of the volume, and a
measurement precision of 5% is desired, this means that about 400 points should hit the phase and hence 2000
points should hit the object. If the object is 5 inches long and about 3 inches in diameter, a rough estimate of its
volume can be made by assuming a more-or-less ellipsoidal shape. The volume of an ellipsoid is about half (/6 =
0.524) of the volume of the bounding box. This means that grid should have about 4000 points. A cubical grid
spacing of 0.2 inches will produce a total of 15x15x25 = 5625 points, quite suitable for this measurement.
Figure 4. A representative irregular object (potato) to be sampled as discussed in the text,
and one section to be used for counting with a portion of the grid superimposed.
This sampling grid can be achieved by sectioning the specimen every 0.2 inches and placing a grid with that
spacing onto each section for counting. The placement of the grid uses the method described above. A similar
method is used to position the planes: A single random number can be used to determine the position of the first
plane within the first 0.2 inches of the specimen, and then all of the other planes are regularly spaced with respect
to this first one. In this way structured random sampling is extended to all three dimensions.
This represents a fairly large number of points and a high sampling density; most stereological experiments use
much lower sampling density and achieve lower precision on each specimen, but average over many specimens.
In the example of Figure 4, a lower sampling density has been used for clarity. the planes are spaced 1/2 inch
apart and the grids have a 1/2 inch spacing, producing a total of 360 points, of which about 180 can be expected to
fall within the object and hence about 36 within the phase of interest. This corresponds to a standard deviation due
to counting statistics of 6, or a relative precision of 6/36 = 17%.
In many cases the sections of the specimen are obtained with a microtome, in which case the procedure is to take
every Nth section from the series to obtain the desired spacing, starting with a randomized initial section.
Likewise, if the examination procedure is to use M fields of view on each section, the procedure is to create a grid
of field positions which is offset by a random location on each section. The procedure can also be extended to
sampling specimens within a population in the same way.
Isotropic planes
Unlike point probes, lines and planes have a direction and so it is also necessary to make their orientation isotropic
within the structure. This is much more difficult to accomplish (and may be very important, as few natural
structures are themselves isotropic). Line probes are used to sample surfaces and volumes, either by counting the
intersections of the line probes with surfaces or measuring the intercept length with the volumes. Because
counting is a much easier and hence preferred technique, and the precision of counting experiments can be
predicted as discussed above, most modern stereological experiments emphasize counting of intercepts and so we
discuss here the use of line probes for that purpose.
Lines cannot in general be passed through structures without first cutting planes on which to draw the lines. If all
of the planes were cut as in the preceding experiment as a series of parallel sections, it would not be possible to
draw lines in any direction except those included in the planes. Hence the most commonly used and efficient
method of cutting parallel plane sections through a specimen cannot be used when line probes are to be employed
for surface area measurement.
It is instructive to consider first the procedures for placing an IUR set of planes through an object. This is required
when the measurement probe is a plane, for instance when measuring the size and shape of plane intersections
with features or counting the intersections of the plane with features in the volume. Plane probes are primarily
used for counting the number of intersections per unit area with linear structures such as fibers, which are rarely
isotropic in space, in order to measure the total length of the linear structures. IUR planes can also be used for
drawing line probes through structures, although we will see later that there is an easier way to accomplish this
when the purpose is simply to count intersections of the lines with surfaces.
In order for sampling to be uniform, it will still be necessary to obtain a series of sections that cover the extent of
the feature. There is a tendency in visualizing the orientation of section planes to deal with those that pass through
the geometric center of the object being sectioned, which of course does not provide uniform sampling of the
extremities. Since many real structures are not only anisotropic but also have gradients, the need for uniform
sampling is very important.
Using a series of section planes (distributed as discussed above using the principles of structured random
sampling) naturally limits the sampled planes to a single orientation, because once cut they cannot be reassembled
to permit sampling again in another orientation. This usually means that multiple specimens must be used to obtain
many different sampling orientations, which in turn requires that specimens within a population must have some
definable or discernible axis of orientation of their own that can be used as a basis for selecting orientations. In
biological systems, which is the primary field of application of these techniques, this is usually straightforward.
For materials or geological specimens there are also natural axes (typically perpendicular to deposited planes or
parallel to a cooling gradient or deformation direction) but these may be more difficult to ascertain before
sectioning.
In any case, assuming that there is some natural axis, one approach is to define a different axis orientation for
sampling each member of the population using a scheme that distributes the orientations randomly and uniformly
in three dimensions to achieve isotropic sampling. This is not so easy to do for an arbitrary number of
orientations. We have already seen the efficiency of the structured random sampling approach, so a method based
on random tumbling of the specimen is not desired. Visualization of the problem may be assisted by realizing that
each direction can be mapped as a point onto a half-sphere of orientations. Is there an arrangement of such points
so that they are regularly and equally spaced over the surface of the sphere?
Figure 5. The five platonic solids (regular polyhedra).
Figure 6. Orientation vectors for the dodecahedron, plotted on the sphere of orientation.
The arrangements which are exactly equal and regular correspond to the surface normals of the regular polyhedra,
of which there are only five (Figure 5): the tetrahedron with four triangular faces, cube with size square faces,
octahedron with eight triangular faces, dodecahedron with twelve pentagonal faces, and icosahedron with twenty
triangular faces. Taking just the faces whose normal directions point into the upper half of the sphere of
orientations, the number of orientations is half of the number of faces (except for the tetrahedron). For instance,
the dodecahedron has 12 faces but they provide only six sampling orientations. If one of these solids corresponds
to the desired number of orientations to be used for sampling, a useful strategy would be to embed each entire
object to be sectioned into such a structure, the number of such objects corresponding to the number of faces
(e.g., six organs from a population placed into six dodecahedra). This would place the natural axis at some
random orientation, which would however fall within the area of one of the faces. Identify this face, and select a
unique and different face for each of the polyhedra. Figure 6 shows the normal directions for the dodecahedron.
Use the surface normal of the selected face as a sectioning axis for that object, using the usual procedure as
presented above to determine the number of sections to be used and the location of fields of view within each
section.
This procedure is fairly easy to implement but is appropriate only when there are multiple objects representing a
population which is to be measured. When a single object must be sampled isotropically with planes, an
equivalent procedure is to first subdivide the object into portions, for example cutting it into six (for the
dodecahedron) or ten (for the icosahedron) parts. Then each part can be embedded into the regular polyhedron and
sectioned as described above. The danger in this approach is that orientations of structure within the object may
vary from place to place, and since the sampling orientation also varies this can result in measurement bias.
Correcting such bias requires performing the procedure on several specimens with different orientations selected
for each portion. In this case, it is just as efficient to use the first approach with sectioning of each specimen in one
orientation.
Figure 7. Vertical sections cut to include the vertical axis of a specimen. Each section includes
the vertical direction (arrow) and is rotated randomly to a different azimuthal angle.
If it is not required that the specimen be uniformly sampled at each orientation, then another sampling method can
be employed to obtain section planes that are isotropic. First cut a so-called vertical section that includes the
specimens natural axis (or some identifiable direction) and is uniformly random with respect to orientation, as
shown in Figure 7. Then cut a surface perpendicular to this surface but at an angle that is not uniformly random
but is instead sine-weighted. Sine weighting is accomplished by generating a uniform random number in the range
-1..1 and then calculating the angle whose sine has that value = Arc Sin (Random). This angle varies from -90 to
+90 degrees and represents the angle from vertical. It is also easy to implement this procedure by creating a grid of
radial lines that are not uniformly spaced but instead have the appropriate sine weighting, and then to select one
using a uniform random number from 0 to 99 (Figure 8). This procedure is called the Orientator (Mattfeldt, quoted
in Gundersen et al., 1988). Figure 9 shows the process applied to the object from Figure 4. It produces section
planes that are isotropically uniform. Of course, this produces only a single section that passes through the object,
and so does not provide spatially uniform sampling.
Figure 8. The Orientator. Place a horizontal cut surface (perpendicular to the assigned vertical direction
in a specimen) along the 0-0 line with a vertical cut (parallel to the vertical direction and uniformly random
with respect to rotation). Generating a random number from 0-99 to select a cutting direction as shown.
Repeating this procedure produces planes that are isotropic (uniformly sample orientations).
a
b
Figure 9. Implementation of the orientator: a) cutting a vertical section;
b) cutting the examination plane using the sine-weighted grid.
Isotropic line probes
Fortunately, there are few stereological procedures that require IUR planes as the sampling probes. IUR lines are
much easier to generate. If the generated planes in the specimen were themselves IUR then drawing lines on them
that were also IUR would produce line probes having an IUR distribution in 3D space. Such a grid of lines would
be produced for example by drawing a set of parallel lines with an appropriate spacing, shifting them by a random
fraction of that spacing, and rotating them to an angle given by a uniform random number between 0 and 180
degrees. The structured random sampling approach lends itself directly to the positioning of the lines in this way.
Note that a grid of radial lines with uniform angular spacing drawn from the center of the field of view or of the
specimen does not satisfy the IUR requirement because it is not uniform across the area - more of the lines sample
positions near the center than at the periphery.
Given the difficulty of drawing the IUR planes in the first place, another easier approach is generally used. It
relies on the idea of a vertical section (Baddeley et al., 1986), the same as mentioned above. This is a plane that
includes some natural axis of the specimen or some readily identifiable direction within it. It does not matter
whether this orientation is actually vertical in a geocentric sense, and the name comes from the fact that the
placement of images of the vertical section plane for viewing and measurement often positions this direction in the
vertical orientation.
Figure 10. A series of planes cut parallel to one vertical section.
A vertical section plane can be cut through a specimen parallel to the vertical direction without necessarily passing
through the center of the specimen. In fact, while most diagrams and illustrations of vertical sectioning tend to
draw the plane as passing through the center, for IUR sampling it is of course important that this restriction not be
present. A series of parallel vertical section planes (Figure 10) with one uniformly random rotation angle about the
vertical axis can be cut using the same principles of uniform random sampling discussed above by calculating an
appropriate plane spacing and a random offset of a fraction of that spacing. The uniform random sampling must
then also be performed by cutting a similar set of section planes on additional specimens (or portions of the same
specimen) at angles offset from the first chosen angle.
On all of the vertical section planes cut at different rotational angles, directions near the vertical direction are
sampled. Plotted onto a sphere of directions (as shown in Figures 11 and 12), it can be seen that these lines cluster
near the north pole of the figure while directions at low latitudes near the equator are sparsely sampled. The
compensation for this is to use the same sine-weighting as discussed above. By generating lines with an angle
from the vertical calculated as Arc Sine (R) where R is a uniform random number between -1 and +1, the
directions are spread uniformly over the latitudes on the sphere (Figure 12).
Figure 11. Radial lines drawn on a vertical section; top) uniformly distributed every 15 degrees
(these lines are not isotropic in 3D space); bottom) the same number of lines drawn
with sine-weighting, which does produce lines isotropic in 3D space.
Figure 12. Direction vectors in space mapped as points on a half-sphere of orientations: a) uniformly
distributed angles at every 15 degrees along each vertical section corresponding to Figure 11a; the vectors
are close together near the north pole. b) sine-weighted angles along the same vertical section
corresponding to Figure 11b; the same number of directions are now uniformly distributed.
This approach fits well with structured random sampling because the number of angles in the horizontal (uniform)
direction, the vertical (sine-weighted) direction, the number of lines and their spacing can all be calculated based
on the required precision. However, it requires more horizontal orientations and hence more vertical section cuts
than a modified procedure that uses cycloids rather than straight lines. This obviously does not apply to
experiments in which intercept lengths are to be measured, but is quite appropriate for the counting of intersections
between the line probes (the cycloids) and surfaces within the structure.
Cycloids are the correct mathematical curve for this application because they have exactly the same sine weighting
as used to generate the straight lines in the method above. A cycloid is the path followed by a point on the rim of a
rolling circle, and can be generated using the mathematical procedure shown in Figure 13. The curve has only a
small fraction of line with vertical orientation and considerably greater extent that is near horizontal, and exactly
compensates for the vertical bias in the vertical section planes. Distributing a series of cycloidal arcs (in all four
possible orientations obtained by reflection) across the vertical section and shifting the grid of these lines
according to the usual structured random sampling guidelines produces isotropic uniform random sampling in
three dimensions. Note that the grid of cycloids (Figure 14) is not square, but wider than it is high; the shifting
using random offsets must displace the grid by a random fraction of the grid size in each direction. As usual, the
size of the grid must be such that in few cases will more than one line intersect the same element of structure. In
many examples of this technique the statement is made that as few as three sets of planes at angles of 60 degrees
are sufficient for many purposes. Obviously, this premise can be tested in any particular experiment by sampling
at higher density and seeing whether the results are affected.
Figure 13. A cycloidal arc and the procedure for drawing it. The are has a width of ,
a height of 2 and a length of 4 (twice its height).
For projected images through transparent volumes, it is possible to generate IUR surfaces using cycloids. As
described in detail in Chapter 14 on finite section thickness, a set of cycloidal lines drawn on the projected image
represent surfaces through the volume. Choosing a vertical direction (which can be arbitrary) for the volume and
rotating it about that axis while collecting projected images causes the same bias in favor of directions near the
vertical axis as that produced by vertical sectioning. Elements of area of these surfaces have orientations that
compensate for the bias so that isotropic sampling of the structure is obtained. As shown in the examples in that
chapter, the result is an ability to obtain isotropic uniform random sampling of intersections of linear features to
measure N
A
, from which L
V
can be calculated.
Figure 14. A grid of cycloidal arcs. Placed on vertical sections (the vertical direction in the
specimen is vertical on the figure), the lines isotropically sample directions in 3D space.
Volume probes - the Disector
One of the first developments in the so-called new stereology that emphasizes design of probes and sampling
strategies rather than classical methods such as unfolding of size distributions based on shape is the Disector
(Sterio, 1984). The disector requires comparing two parallel section planes to detect features that are present in
one but not the other. But while it is implemented using section planes, it is actually a volume probe (the volume
between the planes). Since volumes have no orientation isotropy is not an issue, although requirements for
uniform random sampling remain (and can be satisfied using the same methods described above).
The initial and still primary use of the disector is to determine the number of objects per unit volume. Point, line
and plane probes cannot accomplish this without bias because they are more likely to intersect large features than
small ones. As noted in the first chapter, the number of objects present in a region is a topological property, and
cannot be determined by probes of lower dimension than the volume. The disector provides a surprisingly simple
and direct way to count objects that is unbiased by feature size and shape. It relies on the fact that features can be
counted by finding some unique characteristic that occurs only (and always) once per feature. In this case that
characteristic is taken to be the topmost point (in some arbitrary direction considered to be up) of each feature.
For illustration, consider counting people in a room. For most of them the topmost point would be the top of the
head, but in some cases it might be the tip of an raised hand, or the nose (someone lying on his back on the floor).
Regardless of what the point is, there is only one. Counting those points gives the number of people. Of course,
in this example the procedure must be able to look throughout the volume of the room. When three-dimensional
imaging is used, as discussed in Chapter 15, this is the method actually used. The disector provides an efficient
method using just sets of two parallel planes.
The disector can be implemented either with physical sectioning (e.g., microtoming) to produce two thin sections,
or by sequential viewing of two polished surfaces of an opaque material, or by optical sectioning of a transparent
volume using confocal microscopy. In all cases the spacing between the two sections must be known, which can
be difficult particularly in the case of microtomed sections. For polished opaque materials one method for
accomplishing this is to place hardness indentations (which have a known shape, typically either spherical, conical
or pyramidal) in the surface and measure the change in size with polishing, from which the depth of polishing can
be determined. These hardness indentations also help to align the two images. Similar fiducial marks can be used
for the microtomed sections. Optical sectioning is generally the easiest method (when applicable) because spacing
can be measured directly and image alignment is not required.
The key to the disector is to have the two parallel images be close enough together (small spacing between them)
that the structure between them is relatively simple. No entire feature of interest can be small enough to hide in that
volume, and as we will see no branching of networks can have branch lengths smaller than that distance. It must
be possible to infer what happens in the volume by comparing the two images. Only a small number of basic
topological events can be allowed to occur, which can be detected by comparing the images:
1. A feature may continue from one plane to the other with no topological events (the size of the intersection can
change, but this is not a topological change).
2. A feature may end or begin between the planes, appearing in one but not the other.
3. A feature may branch so that it appears as a single intersection in one plane and as two (or more) in the other.
4. Voids and indentations within a feature may also continue, begin or end, or branch.
Figure 15. Diagram of the disector (cases illustrated are discussed in the text).
Figure 15 shows a diagram illustrating several of these possibilities. The critical assumption is made that
familiarity with the structures will enable identification by a human to detect features in the two sections that are
matched. For automatic analysis the planes must be spaced closely enough together that feature overlaps can be
used to identify feature matches. Features of type 6 (in the figure) are considered to be continuations of the same
object with no topological events occurring. Events of type 3 and 4 represent the start or end of a feature
(depending on which of the two planes is taken to be the floor and which the ceiling). Events of type 1 and 2
represent simple branching. The type 5 event reveals the bottom of an internal void.
If the spacing between the planes is small so that no complex or uninterpretable events can occur in the volume
between them, then in real images most objects will continue through the two planes (type 6) and these non-events
are ignored. For counting features, events of type 3 and 4 are of interest. The number of these events divided by
two since we are now counting both beginnings and endings of features, and divided by the volume between the
planes (the area of the images times their spacing) gives the number of features per unit volume directly. As noted
above, this value is unbiased since feature size or shape does not affect the count (Mayhew & Gundersen, 1996).
Figure 16. Guard frame and exclusion lines for the disector. Only the red features are considered.
The method is only simple for convex objects. When features may branch, or are long and slender so that they
may cross and re-cross through the sampled volume, it becomes necessary to keep track of all of the parts of the
feature so that it is only counted once. Since the images are finite in area, attention must be given to the problems
that the edges of the image introduce. As shown in Figure 16, this is accomplished by defining exclusion edges
and a guard region around the active counting area so that features are ignored if they extend across the exclusion
edge (Gundersen et al., 1988). Of course, as noted above it is necessary to follow features that branch or extend
laterally to detect any crossing of the exclusion edges (this is why the exclusion edges are extended as shown by
the arrows in the figure). The requirement for a small spacing between planes to eliminate confusion about
connectivity means that only a few topological events are detected, so that a large area or many fields of view are
required to obtain enough counts for useful statistical precision.
Networks
As noted above, the disector can be used to count the number per unit volume (N
V
) when the objects are convex,
or at least relatively compact and well separated. Objects that are long, twisted, branched and intertwined create
difficulties in identifying the multiple sections that occur as part of the same object. On the other hand, the disector
is also very useful for dealing with an extended network such as the pore structure in a ceramic, blood vessels or
neurons in the body, etc. The topological property of interest for these structures is the connectivity or genus of
the network. This is a measure of how branched the structure is, or more specifically of the number of
connections (per unit volume) or paths that are present between locations.
Topological properties such as number of objects and connectivity of pore networks require volume probes. The
use of full three-dimensional imaging (Chapter 15) offers one approach to this, but the disector offers another that
is elegant and efficient. It will be useful first to digress slightly and revisit some aspects of topology and genus,
and define some terms and quantities, which were introduced more comprehensively in earlier chapters.
Points on the surfaces of objects in three-dimensional space have normal vectors (perpendicular to the local
tangent plane) that identify their orientation. Each such vector can be represented by a point on the surface of a
sphere, which is called the spherical image of the point. A patch or region on the curved surface of the object
corresponds to a patch of orientations on the sphere. For the surface of a convex, closed object every direction on
the sphere corresponds to one (and only one) point somewhere on the surface of the object. Since the spherical
image of the convex surface exactly covers (or is mapped onto) the unit sphere, its spherical image value is 4 (the
area of the unit sphere), independent of any details about the shape of the body. The spherical image is an
important topological property since it has a value that is independent of size and shape.
Figure 17. Convex, concave and saddle curvatures
of surface elements.
For bodies that are not totally convex we must recall the idea of negative spherical image introduced in Chapter 5.
This is the projection of points on saddle surfaces (Figure 17), which have two principle radii of curvature with
different signs. With this convention, the total spherical image of any simply-connected closed surface is 4 (and
the total spherical image of N objects would be 4N). If the body has a hole through it (for instance a donut or
torus, Figure 18), the surface around the hole is saddle surface and covers the sphere exactly once, and the surface
on the outside is convex and also covers the sphere once, so the new spherical image of a donut is 0.
Figure 18. A torus; the green shaded area is saddle surface with a negative spherical image,
the red surface is convex with a positive spherical image.
The hole in the donut changes the topological quantity called the genus of the object. It becomes possible to make
one cut through the object without disconnecting it into separate parts. Every hole introduced into the object adds a
spherical image of -4, so the general result is that the spherical image of an object is related to the genus or
connectivity by the relationship Spherical Image = 4(1C). The total spherical image of a set of N objects with a
total connectivity of C would be 4(NC). We are usually primarily interested in the two extreme cases when
C=0 (a set of separate, simply connected objects which need not be convex but have no holes) or N=1 (a single
extended network whose genus we wish to determine).
Imagine a plane sweeping through the volume of the structure and note the occurrences when the plane is
momentarily tangent to the surface. There can be three different kinds of tangent events as illustrated in Chapter 5
(DeHoff, 1987; Zhao & MacDonald, 1993; Roberts et al., 1997):
1. The plane may be tangent to a locally convex surface (both radii of curvature point inside the object). This is
called a T
++
event and the total number of them per unit volume is denoted T
V
++
.
2. The plane may be tangent to a locally concave surface (both radii of curvature point outside the object). This is
called a T
V
.
3. The plane may be tangent to a local patch of saddle surface (the two radii of curvature lie on opposite sides of
the plane). This is called a T
+
V
+
.
The disector can be used to count these tangent events. The appearance of features in the two planes allows us to
infer that a tangent event of one type or another occurred in the volume between the planes. Referring back to the
diagram in Figure 15, types 1 and 2 correspond to T
+
events, types 3 and 4 to T
++
events, and type 5 to a T

event. The total number of counts per unit volume (the product of the area of the image and the spacing between
the planes) can be obtained by counting.
The sum of these tangent counts (T
V
++
+ T
V
T
V
+
) is called the net tangent count T
V
, and the total spherical
image of the structure is just 2T
V
. Consequently the difference between the number of features present N and
the connectivity or number of holes C is just N C = T
V
/2. For a network structure with N=1, this gives the
connectivity of the structure directly. Because topological properties and the volume probe used cannot be
anisotropic, orientation considerations do not arise in using the disector (but of course averaging of samples to
obtain uniform representation of the structure is still necessary).
Chapter 8 - Statistical Interpretation of Data
Introduction - sources of variability in measurement
Figure 1. Effects of accuracy and precision in shooting at a target.
It is commonplace to observe that repeated measurements of what seems to be the same object or phenomenon do
not produce identical results. Measurement variation arises from a number of sources, but one root cause is often
the finite precision of the measuring tool. If a simple yardstick is used to measure carpet, we expect to obtain a
result no better than perhaps 1/4 inch, the smallest division on the measurement scale, but this is entirely adequate
Chapter 8 - Statistical Interpretation of Data Page # 130
for our purpose. If we try to measure the carpet to a higher resolution, say 1/64 inch, we are likely to find that the
roughness of the edge of the carpet causes uncertainty about just where the boundary really is, and measurements
at different locations produce different answers. To push this particular example just a bit farther, if the
measurement is performed without taking care that the ruler is perpendicular to the edges of the carpet, the
measurement will be biased and the result too large.
The limitations of the measurement tool and the difficulty in defining just what is to be measured are two factors
that limit the precision and accuracy of measurements. Precision is defined in terms of repeatability, the ability of
the measurement process to duplicate the same measurement and produce the same result. Accuracy is defined as
the agreement between the measurement and some objective standard taken as the "truth." In many cases, the latter
requires standards which are themselves defined and measured by some national standards body such as NIST or
NPL (for example, measures of dimension). Arguing with a traffic officer that your speedometer is very precise
because it always reads 55 mph when your car is traveling at a particular velocity will not overcome his certainty
that the accuracy of his radar detector indicated you were actually going 60 mph (arguing about the accuracy of the
calibration of the radar gun may in fact be worthwhile- the calibration and standardization of such devices can be
rather poor!). Figure 1 suggests the difference between precision and accuracy - a highly precise but inaccurate
measurement is generally quite useless.
In a few cases, such as counting of events, it is possible to imagine an absolute truth, but even in such an
apparently simple case the ability of our measurement process to yield the correct answer is far from certain. In
one classic example of counting errors, George Moore from the National Bureau of Standards (before it became
NIST) asked participants to count the occurrences of the letter "e" in a paragraph of text. Aside from minor
ambiguities such as whether the intent was to count the capital "E" as well as lower case, and whether a missing
"e" in a misspelled word should be included, the results (Figure 2) showed that many people systematically
undercounted the occurrences (perhaps because we read by recognizing whole words, and do not naturally dwell
on the individual letters) while a significant number counted more occurrences than were actually present.
A computer spell checker program can do a flawless job of counting the letter "e" in text of course, provided that
there are no ambiguities about what is to be counted. This highlights one of the advantages that computer-assisted
measurements have over manual ones. But the problem remains one of identifying the things that are to be
counted. In most cases involving images of microstructure (particularly of biological structures which tend to be
more complex than man-made ones) this is not so easy. The human visual system, aided by knowledge about the
objects of interest, the system in which they reside, and the history of the sample, can often outperform a
computer program. It is, however, also subject to distractions and results will vary from one person to another or
from one time until the next. Tests in which the same series of images are presented to an experienced observer in
a different orientation or sequence and yield quite different results are often reported.
In addition to the basic idea that measurement values can vary due to finite precision of measurement, natural
variations also occur due to finite sampling of results. There are practically no instances in which a measurement
procedure can be applied to all members of a population. Instead, we sample from the entire collection of objects
and measure a smaller number. Assuming (a very large assumption) that the samples are representative of the
population, the results of our measurements allow us to infer something about the entire population. Achieving a
representative sample is difficult, and many of the other chapters deal to greater or lesser degrees with how
sampling should be done in order to eliminate bias.
Nearly all laboratories where there is occasion to use extensive manual
measurement of micrographs, or counting of cells or particles therein, have
reason to note that certain workers consistently obtain values which are
either higher or lower than the average. While it is fairly easy to count all
the objects in a defined field, either visually or by machine, it is difficult
to count only objects of a single class when mixed with objects of other
classes. Before impeaching the eyesight or arithmetic of your colleagues, see
if you can correctly count objects which you have been trained to recognize
for years, specifically the letter 'E.' After you have finished reading these
instructions, go back to the beginning of this paragraph and count all the
appearances of the letter 'e' in the body of this paragraph. You may use a
pencil to guide your eyes along the lines, but do not strike out or mark E's
and do not tally individual lines. Go through the text once only; do not
repeat or retrace. You should not require more than 2 minutes. Please stop
counting here!
Please write down your count on a separate piece of paper before you forget it
or are tempted to "improve" it. You will later be given an opportunity to
compare your count with the results obtained by others. Thank you for your
cooperation!
0
5
10
15
20
25
30
35
F
r
e
q
u
e
n
c
y
7
0
7
5
8
0
8
5
9
0
9
5
1
0
0
1
0
5
1
1
0
1
1
5
1
2
0
1
2
5
Count Reported
Figure 2. The "e" counting experiment, and some typical results. The true answer is 115,
and the mean (average) from many trials is about 103.
The human population affords many illustrations of improper sampling. It would not be reasonable to use the
student body of a university as a sample to determine the average age of the population, because as a group
students tend to be age selected. It would not be reasonable to use the population of a city to represent an entire
nation to assess health, because both positive and negative factors of environment, nutrition and health care
distinguish them from rural dwellers. In fact, it is very difficult to figure out a workable scheme by which to
obtain a representative and unbiased sample of the diverse human population on earth. This has been presented as
one argument against the existence of flying saucers: If space aliens are so smart that they can build ships to travel
here, they must also understand statistics; if they understand the importance of proper random sampling, we
would not expect the majority of sightings and contact reports to come from people in the rural south who drive
pickup trucks and have missing teeth; Q.E.D.
For the purposes of this chapter, we will ignore the various sources of bias (systematic variation or offset in
results) that can arise from human errors, sampling bias, etc. It will be enough to deal with the statistical
characterization of our data and the practical tests to which we can subject them.
Distributions of values
Since the measurement results from repeated attempts to measure the same thing naturally vary, either when the
operation is performed on the same specimen (due to measurement precision) or when multiple samples of the
population are taken (involving also the natural random variation of the samples), it is natural to treat them as a
frequency distribution. Plotting the number of times that each measured value is obtained as a function of the
value, as was done in Figure 2 for the "e" experiment, shows the distribution. In cases where the measurement
value is continuous rather than discrete, it is common to "bin" the data into ranges. Such a distribution plot is often
called a histogram.
0
10
20
30
40
50
60
70
80
90
C
o
u
n
t
17 18 19 20 21 22 23
Measured Value
0
5
10
15
20
25
30
35
40
C
o
u
n
t
17 18 19 20 21 22 23
Measured Value
0
2
4
6
8
10
12
C
o
u
n
t
17 18 19 20 21 22 23
Figure 3. Histogram plots of 200 data points in 4,
12 and 60 bins
Usually for convenience the bin ranges are either scaled to fit the actual range of data values, so as to produce a
reasonable number of bins (typically 10 to 20) for plotting, or they are set up with boundary limits that are
rounded off to an appropriate degree of precision. However this is done, the important goal is to be sure that the
bins are narrow enough and there are enough of them to reveal the actual shape of the distribution, but there are
not so many bins that there are only a few counts in each, which as we will see means that the heights of the bins
are not well defined. Figure 3 shows cases of plotting the same data with different numbers of bins. A useful
guide is to use no more bins than about N/20 where N is the number of data points, as this will put an average of
20 counts into each bin. Of course, it is the variation in counts from one bin to another that makes distributions
interesting and so there must be some bins with very few counts, so this offers only a starting point.
Plotting histogram distributions of measured data is a very common technique that communicates in a more
succinct way than tables of raw data how the measurements vary. In some cases, the distribution itself is the only
meaningful way to show this. A complicated distribution with several peaks of different sizes and shapes is not
easily reduced to fewer numbers in a way that retains the complexity and permits comparison with other
populations.
Usually the reason that we perform the measurements in the first place is to have some means of comparing a set
of data from one population with a set of data from another group. This may be to compare two groups that are
both being measured, or to compare one with data taken at another time and place, or to compare real data with
some theoretical model or design specification. In any case, plotting the two histograms for visual comparison is
not a very good solution. We would prefer to have only a few numbers extracted from the histogram that
summarize the most important aspects of the data, and tools that we can employ to compare them.
The comparisons will most often be of the form that can be reported as a probability - usually the probability that
the observed difference(s) between the two or more groups are considered to be real rather than chance. This is a
surprisingly difficult concept for some people to grasp. We can almost never make a statement that group A is
definitely different from group B based on a limited set of data that have variations due to the measurement
process and to the sampling of the populations. Instead we say that based on the sample size we have taken there
is a probability that the difference we actually observe between our two samples is small enough to have arisen
purely by chance, due to the random nature of our sampling.
If this probability is very high, say 30 % or 50 %, then it is risky to conclude that the two samples really are
different. If it is very low, say 1% or 5 %, then we may be encouraged to believe that the difference is a real
reflection of an underlying difference between the two populations. A 5 % value in this case means that only one
time in twenty would we expect to find such a large difference due solely to the chance variation in the
measurements, if we repeated the experiment. Much of the rest of this chapter will present tools for estimating
such probabilities for our data, which will depend on just how great the differences are, how many measurements
we have made, how variable the results within each group are, and what the nature of the actual distribution of
results is.
Let us consider two extreme examples by way of illustration. Imagine measuring the lengths of two groups of
straws that are all chosen randomly from the same bucket. Just because there is an inherent variation in the
measurement results (due both to the measurement process and the variation in the straws), the average value of
the length of straws in group A might be larger than group B. But we would expect that as the number of straws
we tested in each group increased, the difference would be reduced. We would expect the statistical tests to
conclude that there was a very large probability that the two sets of data were not distinguishable and might
actually have come from the same master population.
Next, imagine measuring the heights of male and female students in a class. We expect that for a large test sample
(lots of students) the average height of the men will be greater than that for the women, because that is what is
usually observed in the adult human population. But if our sample is small, say twenty students with 11 men and
9 women, we may not get such a clear-cut result. The variation within the groups (men and women) may be as
large or larger than that between them. But even for a small group the statistical tests should conclude that there is
a low probability that the samples could have been taken from the same population.
When political polls quote an uncertainty of several percent in their results, they are expressing this same idea in a
slightly different way. Most of us would recognize that preferences of 49 and 51% for two candidates are not
really very different if the stated uncertainty of the poll was 4 percentage points, whereas preferences of 35 and
65% probably are different. Of course, this makes the implicit assumption that the poll has been unbiased in how
it selected people from the pool of voters, in how it asked the questions, and so forth.
Expressing statistical probabilities in a meaningful and universally understandable way is not simple, and this is
why the abuse of statistical data is so widespread. When the weatherman says that there is a 50% chance of
showers tomorrow, does this mean that a) 50% of the locations within the forecast area will see some moment of
rain during the day; b) every place will experience rain for 50% of the day; c) every location has a 50% probability
of being rained upon 50% of the time; or d) something else entirely?
The mean, median and mode
The simplest way to reduce all of the data from a series of measurements to a single value is simply to average
them. The mean value is the sum of all the measurements divided by the number of measurements, and is familiar
to most people as the average. It is often used because of a belief that it must be a better reflection of the "true"
value than any single measurement, but this is not always the case; it depends on the shape of the distribution.
Notice that for the data on the "e" experiment in Figure 2, the mean value is not a good estimate of the true value.
There are actually three parameters that are commonly used to describe a histogram distribution in terms of one
single value. The mean or average is one; the median and mode are the others. For discrete measurements such as
the "e" experiment the mode is the value that corresponds to the peak of the distribution (108 in that example). For
a continuous measurement such as length, it is usually taken as the central value within the bin that has the highest
frequency. The meaning of the mode is simple - it is the value that is most frequently observed.
0
5
10
15
20
25
F
r
e
q
u
e
n
c
y

(
%
)
5
0
5
5
6
0
6
5
7
0
7
5
8
0
8
5
9
0
9
5
Value
Mean = 72.7
Median = 75
Mode = 80
Figure 4. Distribution showing the mean, median and mode.
The median is the value that lies in the middle of the distribution in the sense that just as many measured values are
larger than it as are smaller than it. This brings in the idea of ranking in order rather than looking at the values
themselves, which will be used in several of the tests in this chapter.
If a distribution is symmetric and has a single peak, then the mean or average does correspond to the peak of the
distribution (the mode) and there are just as many values to the right and left so this is also the median. So, for a
simple symmetrical distribution the mean, median and mode are identical. For a distribution that consists of a
single peak but is not symmetric, the mode lies at the top of the peak, and the median is always closer to the mode
than the mean is. This is summarized in Figure 4.
When distributions do not have very many data points, determining the mode can be quite difficult. Even the
distribution in Figure 2 is "noisy" so that repeated experiments with new groups of people would probably show
the mode varying by quite a lot compared to the mean. The mean is the most stable of the three values, and it is
also the most widely used. This is partly because it is the easiest to determine (it is just the average), and partly
because many distributions of real data are symmetrical and in fact have a very specific shape that makes the mean
a particularly robust way to describe the population.
The central limit theorem and the Gaussian distribution
One of the most common distributions that is observed in natural data has a particular shape, sometimes called the
"normal" or "bell" curve; the proper name is Gaussian. Whether we are measuring the weights of bricks, human
intelligence (the so-called IQ test), or even some stereological parameters, the distribution of results often takes on
this form. This is not an accident. The Central Limit Theorem in statistics states that whenever there are a very
large number of independent causes of variation, each producing fluctuations in the measured result, then
regardless of what the shape of each individual distribution may be, the effect of adding them all together is to
produce a Gaussian shape for the overall distribution.
The Gaussian curve that describes the probability of observing a result of any particular value x, and which fits the
shape of the histogram distribution, is given by equation (8.1).
G(x, , ) =
1
2
e
( x )
2
2
2
(8.1
The parameters and are the mean and standard deviation of the distribution, which are discussed below. The
Gaussian function is a continuous probability of the value x, and when it is compared to a discrete histogram
produced by summing occurrences in bins or ranges of values, the match is only approximate. As discussed
below, the calculation of the and values from the data should properly be performed with the individual values
rather than the histogram as well, although in many real cases the differences are not large.
0
2
4
6
8
10
12
14
16
C
o
u
n
t
40 45 50 55 60 65
533( mm)
0
2
4
6
8
10
12
14
C
o
u
n
t
40 45 50 55 60 65
503( mm)
0
5
10
15
20
25
C
o
u
n
t
40 45 50 55 60 65
501( mm)
Figure 5. Distributions of normally distributed data with differing means and standard deviations.
Figure 5 shows several histogram distributions of normally distributed (i.e. Gaussian) data, with the Gaussian
curve described by the mean and standard deviation of the data superimposed. There are three data sets
represented, each containing 100 values generated by simulation with a Gaussian probability using a random
number generator. Notice first of all that several of the individual histogram bins vary substantially from the
smooth Gaussian curve. We will return to this variation to analyze its significance in terms of the number of
observed data points in each bin later.
There are certainly many situations in which our measured data will not conform to a Gaussian distribution, and
later in this chapter we will deal with the statistical tests that are properly used in those cases. But when the data do
fit a Gaussian shape, the statistical tools for describing and comparing sets of data are particularly simple, well
developed and analyzed in textbooks, and easily calculated and understood.
Variance and standard deviation
In Figure 5, notice that the three distributions have different mean values (locations of the peak in the distribution)
and different widths. Two of the distributions (b and c) have the same mean value of 50, but one is much broader
than the other. Two of the distributions (a and b) have a similar breadth, but the mean values differ. For the
examples in the figure, the means and standard deviations of the populations from which the data were sampled
are:
Figure
5.a 53.0 3.0
5.b 50.0 3.0
5.c 50.0 1.0
The mean value, as noted above, is simply the average of all of the measurements. For a Gaussian distribution,
since it is symmetrical, the mode and median are equal to the mean. The only remaining parameter needed to
describe the Gaussian distribution is the width of the distribution, for which either the standard deviation ( in
equation 8.1) or the variance (the square of the standard deviation) is used. These descriptive parameters for a
distribution of values can be calculated whether it is actually displayed as a histogram or not.
The standard deviation is defined as the square root of the mean (average) value of the square of the difference
between each value and the mean (hence the name "root-mean-square" or rms difference). However, calculating it
according to this definition is not efficient. A procedure for doing so with a single pass through the data is shown
in Figure 6 (this also includes several parameters discussed in the next section).
// assume data are held in array value[1..N]
for i = 1 to 5 do
sum[i] = 0.0;
for j = 1 to N
{ temp = 1.0
for i = 1 to 5 do
{ sum[i] = sum[i] + temp;
temp = temp * value[j];
}
}
mean = sum[2]/sum[1];
variance = sum[3]/sum[1]-mean*mean;
std_dev = sqrt(variance);
skew = ((sum[4]-3*mean*sum[3])/sum[1]+2*mean**3)/variance**1.5;
kurtosis = ((sum[5]-4*sum[4]*mean+6*sum[3]*mean**2)/sum[1]-
3*mean**4)/variance**2;
Figure 6. Example computer procedure to calculate statistical parameters
Because it has the same units as the mean, the standard deviation is often used to denote the spread of the data in
the form (e.g.) 50.0 3.0 cm, the units in which the value was measured. For data that are normally distributed,
this means that 68% of the measured values are expected to fall within the range from 47.0 (50 3.0) to 53.0 (50
+ 3.0) cm, 95% within the range 44.0 (50 2 3.0) to 56.0 (50 + 2 3.0) cm, and 99% within the range 41.0
(50 3 3.0) to 59.0 (50 + 3 3.0) cm.
The values 68, 95 and 99% come directly from integrating the mathematical shape of the Gaussian curve within
limits of 1, 2 and 3 times the standard deviation. As written in equation (8.1), the integral of the Gaussian curve
is 1.0 (meaning that the probability function is normalized and the total probability of all possible measurement
values being observed is unity, which makes the statisticians happy). The definite integral between the limits of
1, 2 and 3 gives results of 0.6827, 0.9545, and 0.9973, respectively. Tables of the value and integral of the
Gaussian function are widely available in statistics books, but will not be needed for the analytical tests discussed
in this chapter.
The variance is just the square of the standard deviation and another measure of the distribution width. It is used in
some statistical tests discussed later on. Because in most cases the analyzed data set is but a sample of the
population of all possible data, the estimated variance of the population is slightly greater than that calculated in the
procedure of Figure 6 for the data set. The variance of the population is N/(N1) times the sample variance, where
N is the number of values in the analyzed data set.
The mean value calculated from the data values sampled from a larger population is also only an estimate of the
true value of the entire population. For example, the data shown in Figure 5b come from a simulated population
with a mean of exactly 50, but the sample of 100 data points have a calculated mean value of 50.009. We also
need to be able to evaluate how well our calculated mean value estimates that of the population, and again the
result depends on how many data points we have taken. The standard error of the mean is just / N where is
the calculated standard deviation of the sample and N is the number of values used. It is used in the same way as
the standard deviation, namely we expect to find the true population mean within 1, 2 or 3 standard errors of the
calculated sample mean 68, 95 and 99 % of the time, respectively. It is important to understand the difference
between the standard deviation of our data sample () and the standard error of the calculated mean value .
The mean and standard deviation are also used when measurement data are combined. Adding together two sets of
data is fairly straightforward. If one set of measurements on one population sample determine a mean and standard
deviation for a particular event as
1
and
1
, and a second set of measurements on another sample determine the
mean and standard deviation for second event as
2
and
2
, then the mean probability of either event is the sum of
the two individual means (
1
+
2
) and the standard deviation is the square root of the sum of the two variances
(
1
2
+
2
2
)
1/2
. To give a concrete example, if the fraction of vehicles passing on the highway is determined in one
time period as 50 6.5 buses per hour, and in a second time period we count 120 8.4 trucks per hour, then the
combined rate of "large vehicles" (busses plus trucks) would be estimated as 170 10.6. The 170 is simply 50 +
120 and the 10.6 is the square root of 112.8 = (6.5)
2
+ (8.4)
2
.
The situation is a bit more complicated and certainly much more unfavorable if we try to combine two events to
determine the difference. Imagine that we counted 120 8.4 trucks per hour in one time period and then 75 7.2
eighteen-wheel transports (a subclass of trucks) in a second period. Subtracting the second class from the first
would give us the rate of small trucks, and the mean value is estimated simply as 45 per hour (= 120 75). But
the standard deviation of the net value is determined just as it was for the case of addition; multiple sources of
uncertainty always add together as the square root of the sum of squares. The standard deviation is thus 11.1 (the
square root of 122.4 = (8.4)
2
+ (7.2)
2
) for an estimated rate of 45 11.1, which is a very large uncertainty. This
highlights the importance of always trying to measure the things we are really interested in, and not determining
them by difference between two larger values.
Testing distributions for normality - skew and kurtosis
In order to properly use the mean and standard deviation as descriptors of the distribution, it is required that the
data actually have a normal or Gaussian distribution. As can be seen from the graphs in Figure 5, visual judgment
is not a reliable guide (especially when the total number of observations is not great). With little additional
computational effort we can calculate two additional parameters, the skew and kurtosis, that can reveal deviations
from normality.
The skew and kurtosis are generally described as the third and fourth moments or powers of the distribution. Just
as the variance uses the sum of (x )
2
, the skew uses the average value of (x )
3
and the kurtosis uses the
average value of (x )
4
divided by
4
. Viewed in this way, the mean is the first moment (it describes how far
the data lie on the average from zero on the measurement scale) and the variance is the second moment (it uses the
squares of the deviations from the mean as a measure of the spread of the data). The skew uses the third powers
and the kurtosis the fourth powers of the values, according to the definitions:
Skew = m
3
/m
2
3/2
(3rd moment)
Kurtosis = m
4
/m
2
2
(4th moment)
where m
k
= (x
i
)
k
/N. An efficient calculation method is included in the procedure shown in Figure 6.
The skew is a measure of how symmetrical the distribution is. A perfectly symmetrical distribution has a skew of
zero, while positive and negative values indicate distributions that have tails extending to the right (larger values)
and left (smaller values) respectively. Kurtosis is a measure of the shape of the distribution. A perfectly Gaussian
distribution has a Kurtosis value of 3.0; smaller values indicate that the distribution is flatter-topped than the
Gaussian, while larger values result from a distribution that has a high central peak. A word of caution is needed:
some statistical analysis packages subtract 3.0 from the calculated kurtosis value so that zero corresponds to a
normal distribution, positive values to ones with a central peak, and negative values to ones that are flat-topped.
It can be quite important to use these additional parameters to test a data set to see whether it is Gaussian, before
using descriptive parameters and tests that depend on that assumption. Figure 7 shows an example of four sets of
values that have the same mean and standard deviation but are very different in the way the data are actually
distributed. One set (#3 in the figure) does contain values sampled from a normal population. The others do not -
one is uniformly spaced, one is bimodal (with two peaks), and one has a tight cluster of values with a few
outliers. The skew and kurtosis reveal this. The outliers in data set #1 produce a positive skew and the clustering
produces a large kurtosis. The kurtosis values of the bimodal and uniformly distributed data sets are smaller than
3. For the sample of data taken from a normal distribution the values are close to the ideal zero (skewness) and 3
(kurtosis).
140 130 120 110 100 90 80 70
Value
Mean
1 Standard Deviation
Data Set #1
Data Set #2
Data Set #3
Data Set #4
Data Set # Mean= Variance=
2
Skew Kurtosis
1 100 140 1.68 5.06
2 100 140 0 1.12
3 100 140 0.352 2.929
4 100 140 0 1.62
Figure 7. Four data sets with the same mean and standard deviation values,
but different shapes as revealed by higher moments (skew and kurtosis).
But how close do they actually need to be, and how can we practically use these values to test for normality? As
usual, the results must be expressed as a probability. For a given value of skewness or kurtosis calculated for an
actual data set, what is the probability that it could have resulted due to the finite sampling of a population that is
actually normal? The graphs in Figures 8 and 9 show the answers, as a function of the number of data values
actually used. If we apply these tests to the 100 data points shown in Figure 5b we calculate a skew value of 0.09
and a kurtosis of 2.742, well within the values of 0.4 and range of 2.4 - 3.6 that the graphs predict can happen
one time in twenty (5%). Consequently there is no reason to expect that the data did not come from a normal
population and we can use that assumption in further analysis. As the data set grows larger, the constraints on
skew and kurtosis narrow.
0.00
0.25
0.50
0.75
1.00
1.25
0
1
0
0
2
0
0
3
0
0
4
0
0
5
0
0
N
Skewness
1%
5%
Figure 8. The probability that the absolute value of skewness will exceed various values for
random samples from a Gaussian distribution, as a function of the number of observations.
1.0
2.0
3.0
4.0
5.0
0
5
0
0
1
0
0
0
1
5
0
0
2
0
0
0
N
Kurtosis
1%
5%
5%
1%
Figure 9. The probability that the kurtosis will exceed various values for random
samples from a Gaussian distribution, as a function of the number of observations.
Some other common distributions
The Gaussian or normal curve is certainly not the only distribution that can arise from simple physical processes.
As one important example, many of the procedures we use to obtain stereological data are based on counting, and
a proper understanding of the statistics of counting is therefore important. Consider a simple event counting
process, such as determining the number of cars per hour that pass on a particular freeway. We could count for an
entire hour, but it seems simpler to count for a short period of time and scale the results up. For example, if we
counted the number of cars in a 5 minute period (e.g., 75 cars) and multiplied by 12, it should give the number of
cars per hour (12*75 = 900). But of course, the particular five minute period we chose cannot be perfectly
representative. Counting for a different 5 minute period (even assuming that the average rate of traffic does not
change) would produce a slightly different result. To what extent can we predict the variation, or to put it another
way how confident can we be that the result we obtain is representative of the population?
When we count events, it is not possible to get a negative result. This is immediately a clue that the results cannot
have a Gaussian or normal distribution, because the tails of the Gaussian curve extend in both directions. Instead,
counting statistics are Poisson. The Poisson function is
P(x, ) =
x
x!
e
(8.2
where is the mean value and x! indicates factorial. Notice that there is no term in this expression, as there was
for the Gaussian distribution. The width of the Poisson distribution is uniquely determined by the number of
events counted; it is the square root of the mean value.
This simple result means that if we counted 75 cars in our 5 minute period, the standard deviation that allows us to
predict the variation of results (68% within 1 standard deviation, 95% within 2, etc.) is simply 75 = 8.66. The
five minute result of 758.66 scales up to an hourly rate of 900103.9, which is a much less precise result than
we would have obtained by counting longer. Counting 900 cars in an hour would have given a standard deviation
of 900 = 30, considerably better than 104. On the other hand, counting for 1 minute would have tallied
approximately 900/60 = 15 cars, 15 = 3.88, and 153.88 scales to an estimated hourly rate of 900232.8 which
is much worse. This indicates that controlling the number of events (e.g., marks on grids) that we count is vitally
important to control the precision of the estimate that we obtain for the desired quantity being measured.
Figure 10 shows a Poisson distribution for the case of =2 (a mean value of two counts). The continuous line for
the P(x,) curve is only of mathematical interest because counting deals only with integers. But the curve does
show that the mean value (2.0) is greater than the mode (the highest point on the curve), which is always true for
the Poisson distribution. So the Poisson function has a positive skew (and also, incidentally, a kurtosis greater
than three).
Fortunately, we usually deal with a much larger number of counts than 2. When the mean of the Poisson function
is large the distribution is indistinguishable from the Gaussian (except of course that the standard deviation is
still given by the square root of the mean ). This means that the function and distribution becomes symmetrical
and shaped like a Gaussian, and consequently the statistical tools for describing and comparing data sets can be
used.
0
0.10
0.20
0.30
0.05
0.15
0.25
0 1 2 3 4 5 6 7 8 9 10
Figure 10. Poisson function and distribution for a mean of 2.
However, there is one important arena in which the assumption of large numbers is not always met. When a
distribution of any measured function is plotted, even if the number of bins is small enough that most of them
contain a large number of counts, there are usually some bins at the extremities of the distribution that have only a
few counts in them. If these bins are compared from one distribution to another, it must be based on their
underlying Poisson nature and not on an assumption of Gaussian behavior. Comparing the overall distributions
by their means and standard deviations is fine as a way to determine if the populations can be distinguished.
Trying to detect small differences at the extremes of the populations, such as the presence of a small fraction of
larger or smaller members of the population, is much more difficult.
0
10
20
30
40
50
60
70
80
C
o
u
n
t
0 10 20 30 40 50 60
Linear Scale
0
20
40
60
80
100
120
C
o
u
n
t
1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25
Logarit hmic Scale
Figure 11. Linear and logarithmic scale histogram plots of the same set of log-normally distributed values.
Another distribution that shows up often enough in stereological measurement to take note of is the log-normal
distribution. This is a histogram in which the horizontal axis is not the measured value but rather the logarithm of
the measured value, but the resulting shape of the distribution is normal or Gaussian. Note that the bins in the
distribution are still of uniform width, which means they each cover the same ratio of size values (or whatever the
measurement records) rather than the same increment of sizes. This type of distribution is often observed for
particle sizes, since physical processes such as the grinding and fracturing of brittle materials produce this
behavior. Figure 11 shows a histogram distribution for log-normally distributed data; on a linear scale it is
positively skewed.
For a thorough discussion of various distributions that occur and the tools available to analyze them, see P. R.
Bevington (1969) Data Reduction and Error Analysis for the Physical Sciences McGraw Hill, New York
Comparing sets of measurements - the t-test
As mentioned at the beginning of this chapter, one common purpose of statistical analysis is to determine the
likelihood that two (or more) sets of measurements (for example, on different specimens) are the same or
different. The answer to this question is usually expressed as a probability that the two samples of data could have
come from the same original population. If this probability is very low, it is often taken as an indication that the
specimens are really different. A probability of 5% (often written as =0.05) that two data sets could have come
from the same population and have slightly different descriptive parameters (mean, etc.) purely due to sampling
variability is often expressed as a 95% confidence level that they are different. It is in fact much more difficult to
prove the opposite position, that the two specimens really are the same.
When the data are normally distributed, a very efficient comparison can be made with student's t-test. This
compares the mean and standard deviation values for two populations, and calculates a probability that the two
data sets are really drawn from the same master population and that the observed differences have arisen by chance
due to sampling. Remember that for a Gaussian distribution, the mean and standard deviation contain all of the
descriptive information that is needed (the skew and kurtosis are fixed).
Given two normal (Gaussian) sets of data, each with n
i
observations and characterized by a mean value
i
and a
standard deviation
i
, what is the probability that they are significantly different? This is calculated from the
difference in the mean values, in terms of the magnitude of the standard deviations and number of observations in
each data set, as shown in equation (8.3); is the number of degrees of freedom, which is needed to assess the
probabilities in Figure 12.
T =
1
2
n
1
2
2
n
2
=
1
2
n
1
+
2
2
n
2

2
1
2
n
1

2
n
1
1
+
2
2
n
2

2
n
2
1
(8.3
The larger the difference of means (relative to the standard deviations) and the larger the data sets, the more likely
it is that the two sets of observations could not have come from a single population by random selection. The
procedure is to compare the value of the parameter T to the table of student's t values shown by the graph in
Figure 12 for the number of degrees of freedom and probability . If the magnitude of T is less than the table
value, it indicates that the two data sets could have come from the same population, with the differences arising
simply from random selection. If it exceeds the table value, it indicates the two sets are probably different at the
corresponding level of confidence 100 (1 ); for example, =0.01 corresponds to a 99% confidence level that
the two groups are not the same.
20 15 10 5 0
0
1
2
3
4
5
6
7
a=0.10
a=0.05
a=0.01
Degrees of freedom
C
r
i
t
i
c
a
l

v
a
l
u
e

o
f

t
Figure 12. Critical values for t-test comparison as a function of the number of degrees of freedom.
The value of is given for a double-sided test (the two means are "different"). In a single sided test (deciding
whether one mean is "greater" or "less" than the second), the value of is halved. Notice that the curves of critical
values level off quickly as the number of degrees of freedom increases. For typical data sets with at least tens of
observations in each, only the asymptotic limit values are needed. Hence a value of T greater than 1.282 indicates
that the groups are not the same with a confidence level of 90% (=0.10), and values greater than 1.645 and
1.960 give corresponding confidence values of 95% (=0.05) and 99% (=0.01) respectively. Common and
convenient use is made of the easily remembered approximate test value T2.0 for 99% confidence.
Comparing the two sets of data in Figure 5a and 5b using the t-test gives the following results:
Data Set n
a 52.976 3.110 100
b 50.009 3.152 100
The calculated value of T is 57.83, the number of degrees of freedom is large enough to consider just the
asymptotic values, and since T exceeds 1.96 the probability that the two data sets are significantly different is
greater than 99%.
The Analysis of Variance or ANOVA test is a generalization of the t-test to more than 2 groups, still making the
assumption that each of the groups has a normal data distribution. The ANOVA test compares the differences
between the means of several classes, in terms of their number of observations and variances. For the case of two
data sets, it is identical to the t-test.
To perform the ANOVA test, we calculate the following sums-of-squares terms from the observations y
ij
(i=class,
j=observation number). y
i
* is the mean of observations in class i, and y
mean
is the global average. There are n
i
observations in each class, k total classes, and t total observations.
SST (total sum of squares of differences) = (y
ij
- y
mean)
2
SSA (sum of squares of difference within the classes) = (y
i
* y
mean
)
2
= n
i
(y
i
* y
mean
)
2
(8.4
SSE (difference between the total variation and that within classes) = SST SSA
From these, an F value is calculated as F = (SSA/n
1
) / (SSE/n
2
)
where the degrees of freedom are n
1
= k 1 and n
2
= t k
n
2
n
1
=3 n
1
=5 n
1
=10 n
1
=40 n
1
=
3 9.28 9.01 8.79 8.59 8.53
5 5.41 5.05 4.74 4.46 4.36
10 3.71 3.33 2.98 2.66 2.54
40 2.84 2.45 2.08 1.69 1.51
2.60 2.21 1.83 1.39 1.00
Figure 13. Critical values of F for =0.05 (ANOVA test).
This value of F is then used to determine the probability that the observations in the k classes could have been
selected randomly from a single parent population. If the value is less than the critical values shown in tables, then
the difference between the groups is not significant at the corresponding level of probability. Figure 13 shows
critical values for F for the case of =0.05 (95% confidence that not all of the data sets come from the same parent
population).
Nonparametric comparisons
The t-test and ANOVA test are flawed if the data are not actually Gaussian or normal since the mean and
standard deviation do not then fully describe the data. Nonparametric tests do not make the assumption of
normality and do not use parameters such as mean and standard deviation to characterize the data. One set of
methods is based on using the rank order of the measured data rather than their actual numerical values. As applied
to two data sets, the Wilcoxon test sorts the two sets of values together into order based on the measurement
values as shown schematically in Figure 14. Then the positions of the observations from the two data sets in the
sequence are examined. If all of the members of one set are sorted to one end of the stack, it indicates that the two
groups are not the same, while if they are intimately mixed together the two groups are considered
indistinguishable. This test is also called the Mann-Whitney test, which generalized the original method to deal
with groups of different sizes.
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
Sorted
Object
Parameter
Value
Sorted
Object
Parameter
Value
B
A
Indistinguishable Distinguishable
Figure 14. Principle of the Wilcoxon or Mann-Whitney test based on rank order.
This idea is much the same as examining the sequence of red and black cards in a deck of playing cards to decide
whether it has been well shuffled. The binomial theorem allows calculation of the probability of any particular
sequence of red and black cards occurring. Shuffles that separate the red and black cards are much less likely to
occur than ones with them mixed together, and will therefore happen only rarely by chance. To illustrate the
procedure, consider the two extreme cases of data shown in Figure 15. Case A has the two groups well mixed and
Case B is completely segregated. The sum of rank positions of the groups are tallied and whichever is smaller is
then used to calculate the test statistic U.
U = W
1
n
1
n
1
+1 ( )
2
(8.5
Rank Case A Case B
1 1 1
2 2 1
3 1 1
4 2 1
5 1 1
6 2 2
7 1 2
8 2 2
9 1 2
10 2 2
Case W
1
=Rank Sum n
1
U
A 25 5 10 0.6015
B 15 5 0 0.0090
Figure 15. Example of two extreme cases for Wilcoxon comparison.
where W
i
is the sum of rank values and n
i
is the number of observations in the groups. If the value of U is less
than a critical value that depends on the number of observations in the two data sets and on a the probability of
chance occurrence by random sampling of values from a single population, then the two groups are considered to
be different with the corresponding degree of confidence. In the example, the two groups in Case A are not
distinguishable (there is a 60% probability that they could have come from the same population) and those in Case
B are (there is a 0.9% probability that they came from the same population). Figure 16 shows the critical test
values of U for =0.01 and =0.05 (99 and 95% confidence respectively) for several values of n
i
.
Figure 16. Critical values for the Wilcoxon (Mann-Whitney) test.
The Kruskal-Wallis test is a generalization of the Wilcoxon or Mann-Whitney test to more than two groups, in
much the same way that the ANOVA test generalizes the t-test. The data sets are sorted into one list and their rank
positions used to calculate the parameter H based on the number of groups k, the number of objects n (and
number in each group n
i
), and the rank order of each observation R (summed for those in each group). This test
value H is compared to the critical values (which come from the chi-squared distribution) for the number of
degrees of freedom (k1) and the probability that this magnitude of H could have occurred purely by chance for
observations all sampled randomly from one parent group.
H =
n (n + 1)
12

i=1
k
(
n
i
R
i
2
) 3 (n + 1)
(8.6
Figure 17 shows a tiny data set to illustrate the principle of the Kruskal-Wallis test. Three sets of measurement
values are listed with the groups identified. Sorting them into order and summing the rank order for each group
gives the values shown. Based on the number of degrees of freedom (df=k1), the probability that the calculated
value of H could have occurred by chance in sampling from a single parent population is 43.5%, so we would not
conclude that the three data sets are distinguishable. Figure 18 shows a table of critical values for H for several
values of df and different values of . H must exceed the table value for the difference to be considered significant
at the corresponding level of confidence.
Length Group
24.0 1
16.7 1
22.8 1
19.8 1
18.9 1
23.2 2
19.8 2
18.1 2
17.6 2
20.2 2
17.8 2
18.4 3
19.1 3
17.3 3
19.7 3
18.9 3
18.8 3
19.3 3
17.3 3
Class n
i
Rank sum
1 5 61
2 6 63.5
3 8 65.5
k 3
df 2
n 19
H 1.663
0.4354
Figure 17. Kruskal-Wallis example (3 groups of data).
df 0.05 0.025 0.01
1 3.841 5.024 6.635
2 5.991 7.378 9.210
3 7.815 9.348 11.345
4 9.488 11.143 13.277
5 11.070 12.832 15.086
6 12.592 14.449 16.812
7 14.067 16.013 18.475
8 15.507 17.535 20.090
9 16.919 19.023 21.666
10 18.307 20.483 23.209
Figure 18. Critical value table for the Kruskal-Wallis test.
The Wilcoxon (Mann-Whitney) and Kruskal-Wallis tests rely on sorting the measured values into order based on
their numerical magnitude, but then using the rank order of the values in the sorted list for analysis. Sorting of
values into order is a very slow task, even for a computer, when the number of observations becomes large. For
such cases there is a more efficient nonparametric test, the Kolmogorov-Smirnov test, that uses cumulative plots
of variables and compares these plots for two data sets to find the largest difference.
0
50
100
150
200
C
o
u
n
t
16 17 18 19 20 21 22 23 24
Lengt h
0
10
20
30
40
50
60
70
80
90
100
P
e
r
c
e
n
t
16 17 18 19 20 21 22 23 24
Lengt h
Figure 19. Cumulative plot compared with usual differential histogram display.
As shown in Figure 19, the cumulative distribution plot shows the fraction or percentage of observations that have
a value (length in the example) at least as great as the value along the axis. Cumulative plots can be constructed
with binned data or directly from the actual measurement values. Because they are drawn with the vertical axis in
percent rather than the actual number of observations, it becomes possible to compare distributions that have
different numbers of measurements.
Data Set Length Values
A 1.023, 1.117, 1.232, 1.291, 1.305, 1.413, 1.445, 1.518,
1.602, 1.781, 1.822, 1.889, 1.904, 1.967
B 1.019, 1.224, 1.358, 1.456, 1.514, 1.640, 1.759, 1.803,
1.872
2.0 1.8 1.6 1.4 1.2 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Length
Greatest Difference
Data Set A
Data Set B
n
1
14
n
2
9
Max.Diff. 0.214
A (=0.05)
1.22
Test Value 0.521
Figure 20. Example of applying the Kolmogorov-Smirnov test.
Figure 20 shows the step-by-step process of performing the Kolmogorov-Smirnov test. The two data sets are
plotted as cumulative distributions showing the fraction of values as a function of length. Once the plots have been
obtained, the greatest vertical difference between them is located. Since the vertical axis is a fraction, there are no
units associated with the difference value. It does not matter where along the horizontal axis the maximum
difference occurs, so the actual magnitude of the measurement values is unimportant.
The maximum difference is compared to a test value calculated from the number of observations in the groups n
i
S = A
n
1
+ n
2
n
1
n
2

1
2
(8.7
where the parameter A is taken from the table in Figure 21 to correspond to the desired degree of confidence. For
the specific case of =0.05 (95% probability that the two data sets did not come from sampling the same
population), A is 1.22 and the test value is 0.521. Since the maximum difference of 0.214 is less than the test
value, the conclusion is that the two data sets cannot be said to be distinguishable.
0.10 0.05 0.025 0.010

A 1.07 1.22 1.36 1.52
Figure 21. Critical Values for the Kolmogorov-Smirnov test.
For large data sets, the Kolmogorov-Smirnov test is a more efficient nonparametric test than the Wilcoxon because
much less work is required to construct the cumulative distributions than to sort the values into order. Figure 22
shows an example of a comparison of two large populations (n
1
=889, n
2
=523). The greatest difference in the
cumulative histograms is 0.118 (11.8%). For a confidence level of 99%, =0.01 and A=1.52, so the test value S
is 0.84. Since the measured difference is greater than the test value, we conclude that the two populations are
distinguishable and not likely to have come from the same population.
For a complete discussion of these and other nonparametric analysis tools, see J. D. Gibbons (1985)
Nonparametric Methods for Quantitative Analysis, 2nd Edition American Sciences Press, Columbus, OH.
Nonparametric tests are not as efficient at providing answers of a given confidence level as parametric tests, but of
course they can be applied to normally distributed data as well as to any other data set. They will provide the same
answers as the more common and better-known parametric tests (e.g., the t-test) when the data are actually
normally distributed, but they generally require from 50% more to twice as many data values to achieve the same
confidence level. However, unless the measured data are known to be Gaussian in distribution (such as count data
when the numbers are reasonably large), or have been tested to verify that they are normal, it is generally safer to
use a nonparametric test since the improper use of a parametric test can lead to quite erroneous results if the data
do not meet the assumed criterion of normality.
0
20
40
60
80
100
Dat aSet N
0
20
40
60
80
100
Dat a Set C
0
20
40
60
80
2 4 6 8 10 12 14
Over l ay
Maximum Diamet er
Figure 22. Overlay comparison of cumulative plots of two populations for the Kolmogorov-Smirnov test.
Linear regression
When two or more measurement parameters are recorded for each object, it is usually of interest to look for some
relationship between them. If we recorded the height, weight and grade average of each student we might search
for correlations. It would be interesting to determine the probability that there is a real correlation between the first
two variables but not between them and the third one. Again, we will want to express this as a confidence limit or
a probability that any observed correlation did not arise from chance as the data were selected from a truly random
population.
Figure 23. Presentation modes for data correlating
two variables: a) point plot, b) flower plot, c) two-
way histogram.
The first tool used is generally to plot the variables in two (or more) dimensions to look for visual trends and
patterns. Figure 23 shows some of the common ways that this is done. The point plot works well if the individual
points representing data measured from one object are not so dense that they overlap. Flower plots are produced
by binning the data into cells and counting the number within each cell, while the two-way histogram shows quite
graphically where the clusters of points lie but in doing so hides some parts of the plot. With interactive computer
graphics, the scatter plot can be extended to handle more than two dimensions by allowing the free rotation of the
data space, and color coding of points allows comparison of two or more different populations in the same space.
Since the human visual system, abetted by some prior knowledge about the problem being studied, is very good at
detecting patterns in quite noisy data, this approach is often a very useful starting point in data analysis. However,
humans are often a bit too good at finding patterns in data (for instance, constellations in the starry sky), and so
we would like to have more objective ways to evaluate whether a statistically significant correlation exists.
Linear regression makes the underlying assumption that one variable (traditionally plotted on the vertical y axis) is
a dependent function of the second (plotted on the horizontal x axis), and seeks to represent the relationship by an
equation of the form y = m x + b, which is the equation for a straight line. The process then determines the
values of m and b which give the best fit of the data to the line in the specific sense of minimizing the sum of
squares of the vertical deviations of the line from the points. For N data points x
i
, y
i
the optimal values of m and b
are
m =
N x
i
y
i
x
i
y
i
N x
i
2
x
i
( )
2
b =
y
i
m x
i

N
(8.8
This is conventional linear regression. When it is used with data that are not all of the same value (weighted
regression), or where the data have been previously transformed in some way (for example by taking the
logarithm, equivalent to plotting the data on log paper and fitting a straight line), the procedure is a bit more
complex and beyond the intended scope of this chapter. Figure 24 shows a computer procedure that will determine
m and b and also calculate the standard deviation of both values.
// assumes data are held in arrays x[1..N], y[1..N]
sumx = 0;
sumy = 0;
sumx2 = 0;
sumy2 = 0;
sumxy = 0;
for i = 1 TO N
{ sum = sum+1;
sumx = sumx+x[i];
sumy = sumy+y[i];
sumx2 = sumx2+x[i]*x[i];
sumy2 = sumy2+y[i]*y[i];
sumxy = sumxy+x[i]*y[i];
}
dx = sum*sumx2-sumx*sumx;
dy = sum*sumy2-sumy*sumy;
d2 = sum*sumxy-sumx*sumy;
m = d2/dx; //slope
b = (sumy-a*sumx)/sum; //intercept
ss = 1.0/(sum-1)*(sumy2+sum*b*b+a*a*sumx2-2*(b*sumy-
a*b*sumx+a*sumxy));
sigma_b = sqrt(ss*sumx2/dx); //intercept standard deviation
sigma_m = sqrt(sum*ss/dx); //slope standard deviation
r2 = d2/sqrt(abs(dx*dy)); //correlation coefficient
Figure 24. Example computer procedure to calculate linear regression
But how good is the line as a description of the data (or in other words how well does it fit the points)? Figure 25
shows two examples with the same number of points. In both cases the procedure outlined above determines a
best fit line, but the fit is clearly better for one data set than the other. The parameter that is generally used to
describe the goodness of fit is the correlation coefficient R or R
2
. This is a dimensionless value that can vary
between zero (no correlation whatever - any line would be as good) and one (a perfect fit with the line passing
exactly through all of the points). The sign of the R value can be either positive (y increases with x) or negative (y
decreases as x increases) but only the magnitude is considered in assessing the goodness of fit. R is calculated
from the product of two slopes, one treating y as a dependent function of x (minimizing the squares of the vertical
deviations of the line from the points) and one treating x as a dependent function of y (minimizing the sum of
squares of the horizontal deviations).
R =
N x
i
y
i
x
i
y
i

N x
i
2
x
i
( )
2
N y
i
2
y
i
( )
2
(8.9
For the examples in Figure 25, the "good fit" data have an R value of 0.939 and the "poor fit" data a value of
0.271. How are we to assess these values? It depends upon the number of points used in calculating the fit as well
a the magnitude of R, and again we use as the probability that a fit with the same R might occur by chance if we
were fitting the line to truly random points like salt grains sprinkled on the table. For the case of 12 data points,
the value of 0.939 would be expected to occur only once in 10000 times (=0.01%), while a value of 0.271
would be expected to occur nearly 2 times in 5 (=39.4%). We would consider the first to be statistically
significant but not the second.
Figure 25. Data sets with high and low correlation coefficients.
Figure 26 shows the relationship for evaluating the fit. For any given number of points, an R value above the
corresponding line means that the fit is significant with the corresponding level of confidence (i.e. did not occur
by chance with a probability ). As the number of points increases, the value of R required for any selected
degree of confidence is reduced. Alpha is the probability that the apparent fit arose by chance from uncorrelated
values. The R
2
value is a measure of the percentage of the variation in the y (dependent variable) values that is
explained is a statistical sense by the fit to the independent variable x.
Linear regression can be extended relatively straightforwardly to deal with more than two variables. Multiple
regression tries to express one dependent parameter z as a linear combination of many others (z=a
0
+ a
1
x
1
+
a
2
x
2
+ ...). The procedure for efficiently determining the a
i
values is very similar to the matrix arithmetic usually
employed to calculate analysis of variance for multiple parameters. If the x
i
parameters are actually values of a
single parameter raised to successive powers, then the relationship is z=a
0
+ a
1
x
1
+ a
2
x
2
+ ... and we have
polynomial regression. An excellent reference for regression methods is N. Draper, H. Smith (1981) Applied
Regression Analysis, 2nd Edition J. Wiley & Sons, New York.
Figure 26. Probability of significance of values of the linear correlation coefficient R for N observations.
Stepwise multiple regression starts with a list of independent variables x and adds and removes them one at a time,
keeping only those which have a values that are statistically significant. We will not cover the details of the
calculation here but many computer statistics packages provide the capability. A closely related technique provided
by quite a few programs plots all of the data points in a high dimensionality space (corresponding to the number of
measured parameters) and then finds the orientation of axes that best projects the data onto planes that give high
correlation coefficients. The "principal components analysis" method is another way, like stepwise multiple
regression, that identifies which of the available independent variables actually correlate with the dependent
variable.
The main difference is that stepwise regression treats the independent variables separately while principal
components analysis groups them together algebraically. Neither method tells the user whether the correlation
reveals any physical significance, whether the assumed dependent variable is actually causally related to the
independent one, or vice versa, or whether both may be dependent on some other (perhaps unmeasured)
parameter. Attempting to infer causality from correlation is a common error that provides some of the more
egregious examples of the misuse of statistical analysis.
Nonlinear regression
The previous section dealt with the mathematics of fitting straight lines to data, and referenced methods that fit
polynomials and other fixed mathematical relationships. These all assume, of course, that the user has some idea
of what the appropriate relationship between the independent and dependent variables is (as well as correctly
identifying which is which). The assumption of linearity is convenient from a calculation standpoint, but there are
few reasons to expect genuine physical circumstances to correspond to it.
When there is no reason to expect a particular functional form to the data, we would still like to have a tool to
assess the degree to which the data are correlated (one increases monotonically with variation in the other).
Spearman or rank correlation accomplishes this in a typical nonparametric way by using the rank order of the
values rather than their numerical values. The data are sorted into rank order and each point's numerical values for
the x and y parameters is replaced by the integer rank position of the value for the corresponding parameter. These
rank positions are then plotted against each other (as illustrated in Figure 27) and the R value calculated and
interpreted in the usual way. This method also has advantages in analyzing data that are strongly clustered at one
end of a plot or otherwise distributed nonuniformly along a conventional linear regression line.
-1.5
- 1
- . 5
0
.5
1
1.5
2
2.5
D
e
p
.

V
a
r
.
0 20 40 60 80 100
Indep. Var.
R=0.9099
a
0
20
40
60
80
100
R
a
n
k
e
d

D
e
p
.

V
a
r
.
0 20 40 60 80 100
Ranked Indep. Var.
R=0.9975
b
Figure 27. Comparison of regression using actual values (a) and rank order of values (b).
Chapter 9 - Computer-Assisted Methods
There are relatively few applications in which completely automatic computer-based methods are easily employed
to perform the various counting and measuring operations that yield stereological data. In practically all cases
some human effort is required at least to oversee the image processing operations that make it ready for automatic
measurement, to verify that the features that are of interest are actually extracted from the more complex image that
is acquired. In quite a few cases, the computer is used only to tally human counting operations (e.g., mouse
clicks) while the human powers of recognition are used (or sometimes misused) to identify the features of interest.
Even in this mode, the use of the computer is often worthwhile for image acquisition and processing to enhance
visibility of the structure, and to keep the records accurately for later analysis. This chapter discusses the use of a
computer to aid manual analysis, while the next one deals with computer-based measurements.
Getting the image to the computer
A typical computer-assisted system consists of the components shown in Figure 1 (Inoue, 1986). In the majority
of cases, the camera is a conventional video camera, usually a solid state camera. It may be either color or
monochrome ("black and white") depending on whether the application deals with color images. In most cases,
biological tissue uses colored stains to delineate structures whereas in materials science color is less useful. The
camera is connected to a "frame grabber" (analog to digital converter, or digitizer board) installed in the computer.
This reads the analog voltage signal from the camera and stores a digital representation in the form of pixels within
the computer. This is then displayed on the computer screen, stored as desired, printed out, and used for
computation.
Optics
Camera Digitizer Memory Display
Storage
Printout
Processing
User
Figure 1. Block diagram of a typical computer-based imaging system.
There are many choices involved in most of these operations. Video cameras come in two principal flavors, tube
type and solid state. The vidicon is a classic example of the former, the CCD of the latter. Tube cameras have a
continuous light sensitive coating that is capable of higher resolution than the array of discrete transistors
fabricated on the chip in a solid state camera, and have a spectral response that matches the human eye and
conventional photography well. But most of the other advantages belong to the solid state camera. It has very little
distortion (especially if electrical fields from a nearby transformer are present to distort the electron beam scanning
in the tube camera), is inherently a linear device in terms of output signal versus light intensity, does not suffer
Chapter 9- Computer Assisted Measurements Page # 159
from "bloom" in which bright areas spread to appear larger than they actually are, can be mounted in any
orientation required by the microscope, and is generally less expensive for equivalent performance because of the
economics of manufacturing high-end consumer cameras in quantity.
Color sensitivity can be achieved in three ways: by splitting the incoming light to fall on three separate detectors
(tube or CCD) each filtered to see just the red, green or blue; by using filters to sequentially capture the red, green
and blue images; or by depositing a filter on the solid state chip that allows columns of transistors to detect the
different colors (there are other more efficient geometries possible but they are more expensive to fabricate). The
latter method gives only about one-half the lateral resolution of the former (it is acceptable for broadcast video
because the color information has lower resolution than the brightness variations). The single chip color camera is
not so satisfactory for most technical applications, but much less expensive. Even with a monochrome camera, the
use of a filter to eliminate infrared light, to which the solid state cameras are sensitive, can be very important
because the imaging optics will not focus the light beyond the visible range to the same plane as the useful image.
Figure 2. The effect of changing the size of a pixel in a grey scale and a color image (clockwise
from upper left pixels are doubled in size) is to obscure small features and boundaries.
The signal from the video camera is the same as used for standard broadcast television, and effectively limits the
horizontal resolution of the image to less than 330 points and the grey scale resolution to less than 60 grey levels.
Figure 2 illustrates the effect of changing pixel size on image representation, and Figure 3 shows the effect of
varying the number of grey levels used. Digitizers often store the image in an array 640 pixels wide by 480 high
because this corresponds to the dimensions of many computer screens, and use 256 grey levels because it
corresponds to the size of one byte of memory, but this is partially empty magnification in both cases. If color
information is being used, the composite signal produced by low end cameras is greatly inferior in color
bandwidth to component (also called S-video) signals with separate wires for luminance and chrominance. The
consequence is blurring of color boundaries, which directly affects image measurements. Some high end cameras
provide separate red, green and blue (R G B) signals.
Figure 3. The effect of changing the number of bits used for grey scale and color plane representation is to
posterize the image. Depending on the quality of the image reproduction on screen or book page, the segments
of the grey scale image with a large number of discrete steps may not be fully resolved. The color image shows
that colors are significantly altered with only 2, 4 or 8 levels for red, green and blue planes.
Video digitizers are called frame grabbers because they record a single video frame lasting 1/30 (in Europe 1/25)
of a second. Standard video is broadcast using interlace, so the frame actually consists of two fields, one with the
odd number scan lines (1, 3, 5, 7, ...) and then 1/60th of a second later the even scan lines (2, 4, 6, 8, ...). For
rapidly changing images, this can cause additional distortion of features. But in most applications the image is
stationary and the rapid speed of frame grabbing is not needed; indeed it causes a definite loss of quality because
of electronic noise. Averaging several video frames, or (with more specialized cameras not used for standard
video) collecting light for a longer period of time, produces much better quality images with more distinguishable
grey levels.
Special cameras with many more transistors that can record images with more than 1000x1000 pixels are
available, but cost more than standard video cameras. Cooled cameras (to reduce electrical noise) with a large pixel
array can be used for computer imaging, but in addition to their cost suffer from relatively slow image transfer.
Most of these perform the digitzation within the camera and transmit digital values to the computer, and the time
required to send millions of values limits either the portion of the image or the number of updates that can be
viewed per second, so that focusing and field selection can be difficult. The great advantage of digital cameras of
this type is that with cooling of the chip and electronics, thermal noise is reduced and it becomes practical to
integrate the photons striking the transistors for many seconds. This allows capturing very dim images;
astronomical image and fluorescence microscopy are the two major applications.
There is another group of digital cameras that are aimed more at the consumer and general photography market
than at the microscopy market. At the lower end of the price range, these have built in lenses that are not easily
removed or adapted to microscope connection, and arrays that produce full color digital images of about 320x240
resolution (sometimes the images can be made larger, but it is empty magnification). Recall that this is about the
same as the resolution of a typical consumer level video camera connected to a frame grabber (indeed, the camera
CCD is identical to that used in the video camera), but the cost is very low and no frame grabber is required. The
interface to the computer is generally a relative slow (several seconds for image transfer) serial link, and many of
the cameras come with cables and software for both Macintosh and Windows platforms. These offer some very
interesting possibilities for capturing images that are to be used for interactive marking of points to count. For
occasional use, simply attaching the camera so that it looks through the microscope eyepiece may be satisfactory.
The principal limitation of the low-end units is distortion of the image due to the limited optics.
Higher end units consist of a camera back that can be attached to standard lens mounts, which does allow their
connection to microscopes. These may either capture full color images in a single pass, or use color filters to
capture three images (red, green, and blue) sequentially which are then recombined in the computer. These can
either be three separate filters inserted with a filter wheel, or a more costly LCD filter that has no moving parts and
changes it transmission characteristics to produce the desired band pass. The image quality with such a setup can
be quite high, approaching the resolution of 35 mm film.
Cameras in the bottom end of the range are available at this writing from many of the companies in the
traditional photographic camera and consumer electronics businesses, such as Casio, Olympus, Kodak, etc.
Higher performance units are sold by Sony, Nikon, Kodak, Polaroid and others. The evolution of consumer
handy-cam cameras to ones with digital capability is also underway. The latest generation of these units use
digital tape to record the images (and sound) instead of recording the analog signals. Many of these units are
already capable of being computer controlled and it seems only a matter of time until the images can also be read,
either from the tape or from the camera directly into computer memory. This would approximately double the
resolution of images from these cameras, since the principal current limitations are in the electronics and not the
CCD or optics.
There is another type of digital camera that does not require the large CCD array (which is the source of much of
the cost of most units). The MicroLumina camera from Leaf/Scitex uses three parallel linear CCD arrays (one for
red, one for green, one for blue) which are scanned across the film plane in the camera back (it mounts wherever
Nikon backs can be attached) to record the image. With 12 bits each (a range of 2
12
or 4096:1) and a full
resolution of 3400x2700 pixels, these images rival film performance at a cost considerably below the cameras that
use a large CCD array. They are not much more costly than a good quality color video camera and frame grabber
board. The limitation, of course, is that the image capture time is over a minute (somewhat dependent on computer
and hard disk speed) so the image must be a stationary one with adequate brightness, since each point is read for
only a momentary time interval.
Display and storage
One of the reasons that transferring the image to the computer in digital form is desirable is the flexibility with
which it can be displayed on the computer screen (Foley & Van Dam, 1984). Some frame grabber boards also
function as the display boards for the computer and directly show the image on the screen, but in most cases the
image is held in memory and transferred to the display memory under program control. This allows for various
modifications and enhancements that can increase the visibility of details, to assist the human operator in
recognizing, locating and marking the features of interest. The processing possibilities are summarized below.
Even without such steps, simple modification of the display brightness to maximize contrast range, adjust it
nonlinearly (gamma adjustments) to increase contrast in either the bright or dark areas, adjust colors for changes
in lighting, substitute colors (pseudo color) for grey scale values to make small variations visible, are more
easily and reproducibly accomplished in the digital realm than by using analog circuits. They can also be
accomplished without altering the original data.
Storing images for archival or reference purposes is one area where great care is needed in assembling a system.
Images are large. A full scanned color image from the MicroLumina camera mentioned above occupies more than
26 megabytes of storage, either in ram memory or on disk. Obviously, this means that a computer with adequate
memory to hold the entire image for user access, a large display to show many pixels at a time, and a fast
processor to be able to access all of the data, are desirable. But computer systems have advanced rapidly in all of
these areas and are expected to continue to do so. A system with a fast Pentium or Power PC processor, 128 Meg
of ram or more, a display screen with at least 800 and perhaps more than 1000 pixel width and capable of true
color (8 bits or 256 levels each for R, G and B), and a reasonably large (several gigabyte) hard disk can be had in
a laptop at this writing.
The major questions have to do with long term storage of the images. Any hard disk will be filled quickly with
images if many are to be kept. Less expensive, slower access, more permanent storage is therefore desirable. Tape
systems have been widely used for computer disk backup, but because access is sequential and the tapes
themselves are subject to stretching and wear if they are used often, this does not seem attractive for an image
storage facility. Writable and rewritable CD-ROM disks are widely regarded as the most suitable medium at the
moment, with the latest generation of CDs providing expanded storage on a single disk from 600 Meg by about an
order of magnitude, enough to store a useful number of images on a single platter. Access speed is good, but the
only practical way to use CD-R (as opposed to CD-RW) is to write all of the disk at one time, which means that
conventional hard disk storage of the same size is required to assemble the files to be written.
The greatest problem with archiving images is having some way to find them again when they are needed. If
pictures can be identified based on some independent scheme such as patient identification in medical imaging,
date and sample number in forensics, etc., then the problem is relatively simple. In a research setting things get
more complicated. It is very difficult to set up a data base that uses words to describe all of the important
characteristics of each particular image. The dictionary of words must be very consistent and fairly small so that a
word search of the data based will locate all of the relevant images. The discipline to create and maintain such a
data base is hard to instill, and consistency between operators is poor.
It would be more desirable to actually have the ability to see the image, since recollection and recognition
processes in human memory are extremely powerful. But archival storage implies that the images themselves are
off-line, and only the data base with some means of identification is available to the user. A partial solution to this
dilemma is offered by image compression. A very much smaller (in terms of storage requirements) version of an
image can be produced using encoding algorithms that take advantage of redundancies in the pixel values within
the image, or discard some frequency components. Compression methods that achieve useful compression ratios
of 50:1 or higher are lossy, meaning that some of the data in the original image are permanently lost in the
compressed version. These can be very important for measurement purposes - small features are missing, color
and brightness values are distorted, and boundaries are blurred and shifted.
We will want to ensure that our archival storage system does not use a lossy compression method for the original
versions of the images. But a compressed version may be quite suitable for inclusion in the image data base to
help the user locate a particular image that cannot be completely or adequately described in words. The
compression methods have been developed for digital video, putting movies onto CDs, transmission of images
over the Internet, and other similar purposes. The emphasis has been on keeping those bits of the image that seem
to be most important to human vision and discarding things that the human does not particularly notice. A casual
glance at an original image and a compressed version that requires only 1 or 2 percent of the storage space does
not reveal substantial differences, which only become apparent when side-by-side higher magnification displays
are used.
The compression methods most often used for still color or monochrome images are JPEG (Joint Photographers
Expert Group) and fractal compression. Fractal compression breaks the image into small blocks and seeks
matches between blocks of different sizes. As these are found, the larger blocks can be discarded and replaced by
duplicating the smaller ones. Even for compression ratios of 100:1 or more, the method offers fair visual fidelity.
Because of patent issues, and because it is a very slow process that requires serious computation to compress the
original images (decompression to view them after compression is very fast, however), fractal methods are not so
widely used.
Figure 4. Comparison of a 2x enlarged fragment of a light microscope image before and after
it has been JPEG compressed with a ratio of 40:1. Color shifts, blurring or omission of lines,
and block-shaped artefacts are all present.
The JPEG method also breaks the image into blocks. Each 8 pixel wide square is transformed into frequency
space using a discrete cosine transform, and the magnitude of the various frequency terms examined. Ones that are
small, below a threshold set to accomplish the desired level of compression, are discarded. Repetitions or
redundancies in the remaining terms are eliminated and the values stored in a sequence that allows the image to be
reconstructed as the data are received rather than needing the entire image to be reconstructed at once (the method
was developed to deal with image transmission in digital high resolution video). Both compression and
decompression use similar computer routines, and are also supported by specialized hardware that can perform the
process in real time. Many standard programs for image handling on standard computers perform JPEG
compression and decompression entirely in software. The speed is quite satisfactory for the intended use for
visual recognition in an image data base, as long as the original uncompressed images are still available in case
measurements are to be made. However, as shown in Figure 4, highly compressed images are not suitable for
measurement purposes.
Transferring images to a computer also opens up many possibilities for printing them out, for example to include
in reports. The quality of printers has steadily improved, and in particular dye-sublimation (also called dye-
diffusion) printers offer resolution of at least 300 pixels per inch with full color at each point. This should not be
confused with laser printers, for which the "dots per inch" (dpi) specification may be higher. Because the "dots"
from a laser printer are either on or off, it takes an array of them to create the illusion of grey scale or (for color
versions) colors. The effective resolution is consequently much poorer than the dpi specification, and even the
magazine or book images printed by high end image setters with dot resolution of well over 1200 dpi do not stand
up to close scrutiny as photographic representations. Furthermore, color aliasing is particularly difficult to control.
Figure 5. Additive (RGB) colors which begin with a black background and combine to produce
other colors and white, with subtractive (CMY) colors which begin with a white background
and combine to produce other colors and black.
Color printers work by depositing cyan, magenta and yellow inks or powders (the complementary colors to red,
green and blue) onto white paper. Figure 5 compares the behavior of additive (RGB) and subtractive (CMY)
colors. By combining the inks in various proportions, a color "gamut" is achieved that is never as wide as additive
color displays that can appear on video screens, particularly with respect to fully saturated primary colors. Adding
black ink (CMYK printing) does produce somewhat improved dark tones. In the dye-sublimation process, the
inks from three (or four) separate sources are combined by diffusion into a special coating on the print stock that
allows them to mix and produce a true color instead of relying on the juxtaposition of colored dots to fool the eye.
Some ink-jet printers also allow for blending of the inks on paper, rather than simply depositing them side by
side. While not as high in resolution as a photographic print, such hardcopy results of images are relatively fast
and not too expensive to produce. The main drawback is that every copy must be printed out separately. On the
other hand, computer programs to process the images or at the very least to conveniently label them are widely
available.
It is possible, of course, to record the computer display onto film. This can either be done directly using an image
setter (which performs any necessary color separations and directly exposes film to produce the masters for
printing), or a film recorder. The latter records the full color image onto a single piece of color film using a high
resolution monochrome monitor. The image is separated into red, green and blue components and three exposures
are superimposed through corresponding color filters. This is much more satisfactory than trying to photograph
the computer screen, because it avoids the discrete red, green and blue phosphor dots used on the color video
display, which trick the human eye into seeing color when they are small but can become noticeable when the
image is enlarged. It also overcomes geometric distortions from the curved screens, as well as glare from reflected
light and other practical problems.
Image processing
Image processing is an operation that starts with an image and ends with another corrected or enhanced image, and
should not be confused with image analysis. The latter is a process that starts with an image and produces a
concise data output, reducing the very large amount of data that is required to store the original image as an array
of pixels to a much smaller set of (hopefully relevant) data. Image processing includes a family of operations that
modify pixel values based on the original pixels in the image. It usually produces as its output another image that
is as large as the original, in which the pixel values (brightness or color) have changed.
The overview of processing methods presented here is necessarily brief. A much more complete discussion of
how and why these procedures are used can be found in John C. Russ (1998) "The Image Processing Handbook,
3rd Edition" CRC Press, Boca Raton FL (ISBN 0-8493-2532-3. All of the examples shown in this chapter and
the next were created using Adobe Photoshop, an inexpensive image processing program that supports most kinds
of image capture devices and runs on both Macintosh and Windows platforms, using the Image Processing Tool
Kit, a set of plug-ins for Adobe Photoshop distributed on CD-ROM that implements the various processing and
measurement algorithms from The Image Processing Handbook. Information on the CD is available over the
world-wide-web (http://members.aol.com/ImagProcTK). There are numerous books available that describe the
mathematics behind the steps involved in image processing, for instance Castleman, 1979; Gonzalez & Wintz,
1987; Jain, 1989; Pratt, 1991; Rosenfeld & Kak, 1982.
The steps used in image processing may usefully be categorized in several ways. One classification is by
operations that correct for various defects that arise in image capture (a common example is nonuniform
illumination), operations that improve visibility of structures (often by relying on the particular characteristics of
human vision) and operations that enhance the visibility or measurability of one type of feature by suppressing
information from others (an example is enhancing boundaries or texture in an image while reducing the brightness
difference between regions). We will examine operations in each of these categories in this chapter and the next.
Another basis for classification is how the operations function. This classification cuts across the other, with the
same functional procedures capable of being used for each of the different purposes.
Point processes replace each pixel value (color or brightness) with another value that depends only on the original
local value. Examples are increases in contrast and pseudo-coloring of grey scale images.
Neighborhood operations modify the pixel value based on the values of nearby pixels, in a region that is typically
a few pixels across (and is ideally round, although often implemented using a small square region for ease of
computation). There are two subsets of these operations: ones that use arithmetic combinations of the pixel values,
and rank operations that rank the values in the neighborhood into order and then keep a specific value from the
ranked results.
Global operations typically operate in frequency space, by applying a Fourier transform to the image and keeping
information based on the orientation and frequency of selected wave patterns of brightness in the image. These are
particularly useful for removing period noise that can arise in image capture, or detecting periodic structures such
as high resolution atom images in the presence of noise.
The most common imaging defects are:
Inadequate contrast due to poor adjustment of light intensity and camera settings;
Nonuniform brightness across the image resulting from lighting conditions, variations in sample thickness (in
transmission viewing) or surface orientation (particularly in the SEM), or from optical or camera problems such as
vignetting;
Noisy images, due to electronic faults or more commonly to statistical fluctuations in low signal levels (especially
in SEM X-ray imaging, fluorescence microscopy, autoradiography, etc.)
Geometrical distortions resulting from a non-normal viewing orientation or non-planar surfaces (particularly in the
SEM and TEM because of their large depth of field), or from nonlinear scan generators (particularly in the atomic
force microscope and its cousins)
Requirement for several different images taken with different lighting conditions (different polarizations, different
color filters, etc.) or other signals (e.g. multiple X-ray maps in SEM) to be combined to reveal the important
details.
It might be argued that some of these are not really imaging defects but rather problems with specimens or
instruments that might be corrected before the images are acquired, and when possible this is the preferred course.
But in reality such corrections are not always possible or practical and images are routinely acquired that have
these problems, which need to be solved before analysis.
Contrast manipulation
The brightness range of many images is greater than the discrimination capability of the human eye, which can
distinguish only about 20 grey levels at a time in a photograph. The contrast of a stored image can be manipulated
by constructing a transfer function that relates the stored brightness value of each pixel to the displayed value. A
straight line identity relationship can be varied in several ways to enhance the visibility of detail. These
manipulations are best understood by reference to the image histogram, which is a plot of the number of pixels
having each of the possible brightness levels. For a typical stored grey scale image this will be 256 values from
white to black, and for a typical color image it will be three such plots for the red, green and blue planes, or the
equivalent conversion to HSI space.
Figure 6. A light microscope color image separated into its red, green, blue (RGB) at the left,
and hue, saturation, intensity (HSI) planes at the right. Algebraic calculation allows conversion
from one representation to the other.
HSI (Hue, Saturation and Intensity) space offers many advantages for image processing over RGB. Image
capture and display devices generally use red, green and blue sensitive detectors and phosphors to mimic the three
receptors in the human eye, which have sensitivity peaks in the long (red), medium and short (blue) portions of
the visible spectrum. But our brains do not work directly with those signals, and artists have long known that a
more useful space for thinking about color corresponds to hue (what most people mean by the "color", an angle
on the continuous color wheel that goes from red to yellow, green, blue, magenta and back to red), saturation (the
amount of color, the difference for example between pink and red) and intensity (the same brightness information
recorded by panchromatic "black and white" film). For microscopists, hue corresponds to the color of a stain,
saturation to the amount of stain, and intensity to the local density of the section, for instance. Figure 6 shows an
image broken down into the RGB and HSI planes.
Figure 7a shows an example of a histogram of a grey scale image. Note that the actual values of pixels do not
cover the entire available range, indicating low contrast. Linearly stretching the brightness range with a transfer
function that clips off the unused regions at either end (Figure 7b) improves the visible contrast significantly. This
particular histogram also has several peaks (which typically correspond to structures present in the specimen) and
valleys. These allow spreading the peaks to show variations within the structures, using histogram equalization
(Figure 7c). The equalization transfer function is calculated from the histogram and therefore is unique to each
image.
Figure 7. An SEM image with low contrast is shown with its histogram (a). Linearly expanding
the display brightness range (b) increases the visual contrast, and equalization (c) broadens
the peaks to show variations within the uniform regions.
Gamma adjustments to the image contrast compress the display range at either the bright or dark end of the range
to allow more visibility of small changes at the other end. This is particularly useful when linear CCD cameras are
used, as they record images that are visibly different than film or human vision (which have a logarithmic
response). Figure 8 shows the effect of gamma adjustment. The same nonlinear visual response means that
features are sometimes more easily distinguished in a negative image than in a positive, and this reversal of
contrast is easily applied in the computer. Solarization, a technique borrowed from the photographic darkroom,
combines contrast reversal at either the dark or bright end of the contrast range with expanded positive contrast for
the remainder, to selectively enhance visibility in shadow or bright areas (Figure 8c).
Figure 8. Gamma adjustments nonlinearly change the display of grey values, in this light microscope
example (a) increasing the brightness to show detail in shadow areas (b). Solarization (c) reverses the
contrast at the end of the brightness scale to create negative representations, in this example in dark areas.
Each of these techniques can typically be applied to color images as well. It is not usually wise to alter the RGB
planes separately as this will produce radically shifted colors in the image. Transforming the color image data to
HSI space allows many kinds of image processing to be performed on the separate hue, saturation and intensity
planes. Many systems apply them to the intensity plane alone (which corresponds to the monochrome image that
would be recorded by panchromatic film).
It is usually possible to modify a selected portion of an image, rather than the entire area, but this requires manual
intervention to select the regions. When the image does not have uniform illumination, for any of the reasons
mentioned above, it makes it difficult to recognize the same type of feature in different locations.
Correcting nonuniform brightness
Figure 9. Portion of a nonuniformly illuminated metallographic microscope image (a) with the background
(b) produced by fitting a polynomial to the brightest points in a 9x9 grid and the result of leveling (c).
Leveling the image brightness is typically done by dividing by or subtracting a reference image that represents the
variation. Subtraction is the more common approach, but technically should only be used when the image has
been acquired by a logarithmic recording device (film, a densitometer, some tube type video cameras, or a CCD
camera with appropriate circuitry).
There are several approaches to getting the reference image to use. Sometimes it can be acquired directly by
removing the specimen and recording variations in illumination and camera response. More often the specimen
itself causes the variation and the reference image must be derived from the original image. There are three basic
methods used:
1. If there is some structure present throughout the image (either features or background) that can be assumed to
be the same, the brightness of those points can be used to generate a polynomial curve of background as a
function of location. This is easiest to automate when the structure comprises either the brightest or darkest
pixels in each local area. Figure 9 shows an example. This approach works best when the nonuniform
brightness results from lighting conditions and varies as a smooth function of position.
2. If the features can be assumed to be small in at least one direction, a rank operator can be used. This is a
neighborhood operator that replaces each pixel in the image with the (brightest or darkest) value within some
specified distance, usually a few pixels. If the features can be replaced by the background in this way, the
resulting image is suitable for leveling. Figure 10 shows an example. This technique can handle abrupt
changes in lighting that often result from specimen variations but requires that the features be small or narrow.
3. Filtering the image to remove high frequency changes in brightness can be performed either in the spatial
domain of the pixels or by using a Fourier transform to convert the image to frequency space. The spatial
domain approach requires calculating a weighted average of each pixel with its neighbors out to a considerable
distance in all directions, to smooth out local variations and generate a new background image that has only the
gradual variations. This method is slow and has the limitations of both methods above and none of their
advantages, but is sometimes used if neither of the other methods is provided in the software.
Figure 10. Use of a rank operator to produce a background image from Figure 9a by replacing each pixel
with the brightest neighbor within a radius of 4 pixels (a), and the leveled result (b).
Reducing image noise
Noisy images are usually the result of very low signals, which in turn usually arise when the number of photons,
electrons or other signals is small. The best solution is to integrate the signal so that the noise fluctuations average
out. This is best done in the camera or detector itself, avoiding the additional noise and time delay associated with
reading the signal out and digitizing it, but this depends upon the design of the camera. Video cameras for instance
are designed to continuously read out the entire image every 1/30 or 1/25 of a second, so that averaging can only
take place in the computer.
0 0 0 0 1 0 0 0 0
0 0 2 4 6 4 2 0 0
0 2 8 21 28 21 8 2 0
0 4 21 53 72 53 21 4 0
1 6 28 72 99 72 28 6 1
0 4 21 53 72 53 21 4 0
0 2 8 21 28 21 8 2 0
0 0 2 4 6 4 2 0 0
0 0 0 0 1 0 0 0 0
Figure 11. A noisy fluorescence microscope image (a) and comparison of the result of smoothing it (b)
using a Gaussian kernel of weights (c) with a standard deviation of 1.6 pixels vs. the application of a
median filter (d) that replaces each pixel value with the median of the 9-pixel-wide circular neighborhood.
If the signal strength cannot be increased and temporal averaging of the signal is not possible, the remaining
possibility is to perform some kind of spatial averaging. This approach makes the assumption that the individual
pixels in the image are small compared to any important features that are to be detected. Since many images are
digitized with empty magnification, we can certainly expect to do some averaging without losing useful
information. The most common method is to use the same weighted averaging mentioned above as a way to
generate a background image for leveling, but with a much smaller neighborhood. A typical procedure is to
multiply each pixel and its neighbors by small integer weights and add them together, divide the result by the sum
of the weights, and apply this method to all pixels (using only the original pixel values) to produce a new image.
Figure 11 shows this process applied to an image with evident random noise, using the "kernel" of weights
shown. These weights fit a Gaussian profile and the technique is called Gaussian filtering. It is equivalent to a low
pass filter in frequency space (and indeed it is sometimes implemented that way).
Although it is easily understood and programmed, this kind of smoothing filter is not usually a good choice for
noise reduction. In addition to reducing the image noise, it blurs edges and decreases their contrast, and may also
shift them. Since the human eye relies on edges - abrupt changes in color or brightness - to understand images and
recognize objects, as well as for measurement, these effects are undesirable. A better way to eliminate noise from
images is to use a median filter (Davies, 1988). The pixel values in the neighborhood (which is typically a few
pixels wide, larger than the dimension of the noise being removed) are ranked in brightness order and the median
one in the list (the one with an equal number of brighter and darker values) is used as the new value for the central
pixel. This process is repeated for all pixels in the image (again using only the original values) and a new image
produced (Figure 11). As discussed in Chapter 8 on statistics, for any distribution of values in the neighborhood
the median value is closer to the mode (the most probable value) than is the mean (which the averaging method
determines). The median filter therefore does a better job of noise removal, and accomplishes this without shifting
edges or reducing their contrast and sharpness.
Both the averaging and median filtering methods are neighborhood operators, but ranking takes more computation
than addition and so the cost of increasing the size of the neighborhood is greater. For color images the operations
may either be restricted to the intensity plane of an HSI representation, or in some cases may be applied to the hue
and saturation planes as well. The definition of a median in color space is the point whose distances from all
neighbors gives the lowest sum of squares.
Rectifying image distortion
Distorted images of a planar surface due to short focal length optics and nonperpendicular viewpoints can be
rectified if the details of the viewing geometry are known. This can be accomplished either from first principles
and calculation, or by capturing an image of a calibration grid. In the latter case, marking points on the image and
the locations to which they correspond on a corrected image offers a straightforward way to control the process.
Figure 12 shows an example. Each pixel address in the corrected image is mapped to a location in the original
image. Typically this lies between pixel points in that image, and the choice is either to take the value of the nearest
pixel, or to interpolate between the neighboring pixels. The latter method produces smoother boundaries without
steps or aliasing, but takes longer and can reduce edge contrast.
Figure 12. Foreshortening due to an angled view with a short focal length lens (a). The vignetting is first
corrected by leveling (b) and then the central portion of the image rectified using 20 control points (c).
Performing this type of correction can be vital for SEM images, due to the fact that specimen surfaces are normally
inclined and the lens working distance is short. Tilted specimens are also common in the TEM, and low power
light microscopes can produce images with considerable radial distortion. Even with ideal viewing conditions and
optics the image may still be foreshortened in the horizontal or vertical direction due to errors in the digitization
process. The electronics used for timing along each scan line have tolerances that allow several percent distortion
even when properly adjusted, and some older frame grabbers made no attempt to grab pixels on an ideal square
grid. Fortunately most systems are quite stable, and the distortion can be measured once using a calibration grid
and a standard correction made to images before they are measured.
When the viewed surface is not planar, or worse still is not a simple smooth shape, the process is much more
complicated. If the user has independent knowledge of what the surface shape is, then each small portion of it can
be corrected by the same process of marking points and the locations to which they map. Effectively, these points
subdivide the image into a tesselation of triangles (a Voronoi tesselation). Each triangle in the captured image must
be linearly distorted to correspond to the dimensions of the corrected planar image, and the same mapping and
interpolation is carried out. Sometimes the shape can be determined by obtaining two views of the surface and
identifying the same points in each. The parallax displacements of the points allow calculation of their position in
space. This is stereoscopy, rather than stereology, and lies outside the scope of this text.
Real images may also suffer from blur due to out-of-focus optics or motion of the specimen during exposure.
These can be corrected to some degree by characterizing the blur and removing it in frequency space but the details
are beyond the intended scope of this text, and also beyond the capability of most desktop computer based
imaging systems. The Hubble telescope pictures suffered from blurring due to an incorrect mirror curvature. As
this defect was exactly known (but only after launch), the calculated correction was able to be applied to produce
quite sharp images (the remaining problem of collecting only a small fraction of the desired light intensity was
corrected by installing compensating optics). Blurring of images from confocal microscopes due to the light
passing through the overlying parts of the specimen can be corrected in much the same way, by using the images
from the higher focal planes to calculate the blur and iteratively removing it.
Enhancement
The same basic methods for manipulating pixel values based on the values of the pixels themselves and other
pixels in the neighborhood can also be used for enhancement of images to reveal details not easily viewed in the
original. Most of these methods become important when considering automatic computer-based measurement and
are therefore discussed in the next chapter. The human visual system is quite a good image processing system in
its own right, and can often extract quite subtle detail from images without enhancement, whereas computer
measurement algorithms require more definite distinctions between features and background (Ballard & Brown,
1982).
Figure 13. Light micrograph (a) showing the effect of a Laplacian filter (b) to increase the contrast of
edges and sharpen the image appearance.
There are some enhancement methods that take advantage of the particular characteristics of the human visual
system to improve visibility (Frisby, 1980). The most common of these is sharpening, which increases the
visibility of boundaries. Local inhibition within the retina of the eye makes us respond more to local variations in
brightness than to absolute brightness levels. Increasing the magnitude of local changes and suppressing overall
variations in brightness level makes images look crisper and helps us to recognize and locate edges.
Figure 13 shows a very simple example. The neighborhood operator that was used is based on the Laplacian,
which is a second derivative operator. The filter multiplies the central pixel by 5 and subtracts the four neighbor
pixels above, below and to the sides; this is repeated for every pixel in the image, using the original pixel values.
For pixels in smooth areas where the brightness is constant or gradually changing, this produces no change at all.
Wherever a sudden discontinuity occurs, such as an edge running in any direction, this increases the local
contrast; pixels on the dark side of the edge are made darker and those on the bright side are made brighter.
The more general approach than the Laplacian that mimics a photographic darkroom technique is the unsharp
mask. Subtracting a smoothed version of the image from the original both increases contrast, reduces some local
background fluctuations, and increases edge sharpness. Figure 14 shows an example in which this method is
applied to the intensity plane of a color image.
Figure 14. Color light micrograph (a) showing the effect of an unsharp mask (b) to increase contrast and
sharpen edges. A Gaussian smooth of the intensity plane from the original image with a diameter of six
pixels is subtracted from the original; hue and saturation are not altered.
Other edge enhancement operators that use combinations of first derivatives are illustrated in the next chapter. So
are methods that respond to texture and orientation in images, and methods that convert the grey scale or color
image to black and white and perform various operations on that image for measurement purposes.
Figure 15. Example of grids overlayed on images on the computer screen: a) a black cycloid grid on a
color light microscope image; b) a colored grid on a monochrome metallographic microscope image.
Overlaying grids onto images
As discussed in Chapter 1 on manual counting, grids provide ways to sample an image. The lines or points in the
grid are probes that interact with the structure revealed by the microscope section image. Counting (and in some
cases measuring) the hits the grids make with particular structures generates the raw data for analysis. There are
several advantages to using the computer to generate grids to overlay onto the image. Compared to placing
transparent grids onto photographic prints, the computer method avoids the need for photography and printing.
Compared to the use of reticles in the microscope, it is easier to view the image on a large screen and to have the
computer keep track of points as they are counted and provide feedback on where counting has taken place. The
computer is also able to generate grids of many different types and sizes as needed for particular images and
purposes (Russ, 1995a), and to generate some kinds of grids such as random points and lines which are not easy
to obtain otherwise. It can also adjust the grid appearance or color for easy visibility on different images. Perhaps
the greatest hidden advantage is that the computer places the grid onto the image without first looking at what is
there; humans find it hard to resist the temptation to adjust the grid position to simplify the recognition and
counting process, and the result is practically always to bias the resulting measurement data.
Overlaying a grid onto an image so that the user can conveniently use it to mark points requires that the grid show
up well and clearly on the image yet not obscure it. One approach that works well in most cases is to display either
black or white grids on color images, and color grids on grey scale pictures. The selection of black or white is
usually based on whether there are many areas within the color image that are very dark or very light; if so, choose
a white or black grid, respectively. Color grids on black and white images are usually easiest to see if they are
colored in red or orange. Blue colors often appear indistinguishable from black, because the eye is not very
sensitive at that end of the spectrum. On the other hand, sensitivity to green is so high that the colors may appear
too bright and make it difficult to see the image (the green lines may also appear wider than they really are). But
allowing the user to choose a color for the grid is easily supported, and solves problems of individual preference
or color blindness. Figure 15 shows color and grey scale images with grids superimposed.
In generating the grid, there are several different kinds of grids that are particularly suitable for specific purposes.
For example, a grid used to count points can consist of a network of lines, with the points understood to lie at the
line intersections (or in some cases at the ends of the line segments), or a series of small circles, with the points of
interest understood to lie at the centers of the circles. The circles have the advantage that they do not obscure the
point being marked, which can be important for deciding which points to mark or count. The lines have the
advantage that they impose a global order on the points and make it easier to follow from one point to the next, so
that no points are accidentally overlooked. Sometimes both methods are used together. Figure 16 shows several
specific kinds of grids to generate grids of lines and arrays of points. The routine for generating these grids is
shown in Appendix 1. The listings shown there are in a specific Pascal-like macro language used by the public
domain NIH-Image program, but serves to document the algorithms required. They are easily translated to other
systems and languages. The identical algorithms are included in the set of Photoshop plug-ins mentioned earlier
(in that case they were written in C so that they could be compiled to run on both Macintosh and Windows
versions of Photoshop).
Figure 16. Some other types of grids that can be used: a) square array of points; b) staggered array of
points, c) randomly placed points; d) circular lines; e) random lines.
The example of a point grid in Figure 16a is a simple array of points marked by crosses and hollow circles
arranged in a square pattern with a spacing of 50 pixels. The similar example in Figure 16b places the points in a
staggered array with a higher point density. It is most efficient to select a point density for sampling so that
neighboring points only rarely fall within the same structural unit. When the structure being sampled by the grid is
itself random, then a regular array of grid provides random sampling, and is easiest for a human user to traverse
without missing points. But for applications to structures that have their own regularity, such as man-made
composites, it is necessary for the grid to provide the randomization. The point grid in Figure 16c distributes
random points across the image to be used in such cases. This is done by generating random pairs of X, Y
coordinates (and rejecting overlapping ones) until 50 points are marked. Counting with a random grid is more
difficult than a regular one because it is easier to miss some points altogether. Changing this number to provide
reasonable and efficient sampling of the structure is straightforward. A common strategic error in counting of
images using grids is to use too many points. It is almost always better to count only a few events or occurrences
of structure in each image, and to repeat this for enough different images to generate the desired statistical
precision. This helps to assure that reasonable sampling of the entire specimen occurs.
The two examples of line grids shown in Figures 16d and 16e supplement those in Figure 15. A square grid can
be used either to count intersections of structure with the lines (P
L
) or to count the cross points in the grid as a
point grid (P
P
). If the image is isotropic (in the two dimensional plane of sampling), then any direction is
appropriate for measurement and a square grid of lines in easiest for the user to traverse. However, when the
structure itself may have anisotropy it is necessary to sample uniformly in all directions, which the circle grid
provides. If the structure also has any regularity, then the grid must be randomized. The random line grid
generates a series of random lines on the sample. If the section is itself a random one in the specimen, then the
random line grid samples three-dimensional structures with uniform, isotropic and random probes.
The cycloid shape is appropriate for lines drawn on so-called vertical sections (Baddeley et al., 1986). The spatial
distribution of the lines is uniform in three-dimensional space when the section planes are taken with random
rotation around a fixed vertical axis in the specimen. This method is particularly useful in measuring structures
in specimens that are known not to be isotropic. Examples include metals that have been rolled or have surface
treatments, specimens from plant and animal tissue, etc. The cycloid is easily drawn as a parametric curve as
shown in the appendix. The individual arcs are drawn 110 pixels wide and 70 pixels high (110/70 = 1.571, a very
close approximation to /2 which is the exact width to height ratio of the cycloid). The length of the arc is twice its
height, or 140 pixels. Alternating cycloids are drawn inverted and flipped to more uniformly sample the image.
For all of these grids, the number of points marked on the image, or the total length of grid lines drawn, is shown
along with the image area. These values may be used later to perform the stereological calculations.
There are two principal methods used by computer programs to keep track of the users marks on the image
employing a grid. One is to count the events as they occur. Typically these will be made using some kind of
interactive pointing device, which could be a touch screen, light pen, graphics pad, etc., but in most cases will be
a mouse as these are now widely used as the user interface to a graphical operating system (or the track pad, track
ball or pointing stick that are employed as mouse substitutes). Counting the clicks as they occur gives a real-time
updated count of events, but makes editing to correct mistakes a bit more difficult and requires some other form of
user action to select between multiple categories of features that can be counted at once. It also requires some way
to mark the points as they are identified by the user, and for this purpose colored labels are often placed on the
image on the screen.
Figure 17. Example of counting with a grid. Three different types of interface between the white and grey,
black and grey and black and white phases have been marked along the circular grid lines in different
colors. The report of the total length of the grid lines, the area of the image and the number of each
class of mark allows calculation of the surface area per unit volume of each type of interface and
the contiguity between phases.
The second common method for counting user marks is to just allow placing any kind of convenient mark onto the
screen, (Figure 17) selecting different colors to distinguish different classes of countable events, and then have the
computer tally them up at the end. Given that most drawing programs have some kind of undo capability to edit
mistakes, the ability to change the size and color of marks, and tools to erase marks or cover them up with one of
a different color, this allows the user quite a lot of freedom to mark up the image using fairly intuitive and
straightforward tools. Of course, since the totaling up is performed at the end there is no continuously updated
result.
Counting the features present in an image requires a computer algorithm to connect together all of the touching
pixels that may be present. A simplified approach that can be used for the counting of marks is available if it can
be assumed that the cluster of pixels constituting the mark is always convex and contains no holes. It is this
simplified logic that is shown in the Appendix. Each pixel is examined in each line of the image. Pixels that match
the selected classification color value are compared to the preceding pixel and to the three touching pixels in the
line above. If the pixel does not touch any other identical pixels, then it represents the start of a mark and is
counted. Additional pixels that are part of marks already counted are passed over. A more general approach is to
use the same logic as employed for feature measurement and labeling in the next chapter, which is capable of
dealing with arbitrarily complex shapes.
The number of marks in each class is reported to the user, who must combine it with knowledge of the grid
(length of lines, area of image, number of grid points, etc.) to obtain the desired structural information. Other
parameters such as the placement of the marks (to assess nonuniformities and gradients, for instance) are not
usually extracted from the user marks, which means that they do not actually have to be placed on the exact grid
points but can be put nearby leaving the actual points available for viewing to confirm accuracy.
Basic Stereological Calculations.
The two most common stereological measures that illustrate the use of counting user marks made employing a grid
are the determination of volume fraction of each phase present, and the measurement of the surface area per unit
volume. These and other stereological measures are discussed in much more detail in earlier chapters. There may
be several different type of boundaries evident as lines in the image, each of which corresponds to a one kind of
surface separating the structure in three dimensions. The user can select different colors to measure the various
kinds of boundaries that are of interest.
The volume fraction of a phase is estimated by the number of grid points counted as lying within the phase,
divided by the total number of points in the grid. The word "phase" as used in the context of stereology, refers to
any recognizable structure that may be present, even voids. If the volume fraction of one structure within another
is desired, that is the ratio of the number of points in the inner structure divided by the sum of those points and
those which lie within the outer structure. For example, the volume fraction of a cell occupied by the nucleus
would be measured by marking points within the nucleus in one color, and points within the cell but outside the
nucleus in a second color. Then the volume fraction of the cell occupied by the nucleus is N
1
/(N
1
+N
2
). The same
method would be used if there were many cells and nuclei within the image.
The surface area per unit volume is estimated from the number of places where the grid lines cross the boundary
line corresponding to that surface. If the number of points is N, and the total length of the grid lines is L (in
whatever units the image is calibrated in), then the surface area per unit volume is 2N/L. This measurement has a
dimension, and so the image calibration is important. The dimension of area per unit volume is (1/units).
All stereological techniques require that the grid be randomly placed with respect to the features in the
microstructure. When the structure itself and the acquisition of the image are random, this criterion is fulfilled by a
regular grid of points or lines. There is one important and common case where orientation of the section plane is
not randomized. In the so-called vertical sectioning case, the surface of the sample that is examined is
perpendicular to an exterior surface, but multiple sections can be taken in different orientations that include that
surface normal (actually, the method works whenever some defined direction within the structure can be
consistently defined as the vertical direction, which lies in all section planes imaged). In that case, in order to
count intersections with lines that are uniformly oriented in three-dimensional space, the cycloid grid should be
used. This is discussed in more detail elsewhere.
One of the common microstructural measurements used to characterize materials is the so-called "grain size." This
is actually a misnomer, since it does not actually have anything directly to do with the size of the grains. There are
two different definitions of grain size included in the ASTM standard. One is derived from the surface area per
unit volume of the grain boundaries in the material. If this are measured by counting N intersections of a grid of
total length L (in millimeters) with the grain boundaries, then the ASTM standard grain size can be calculated as
G = 6.65 Log (L/N) 3.3
where the logarithm is base ten. Note that depending on the units of the image calibration, it may be necessary to
convert L to millimeters.
The second grain size method counts the number of grains visible in the image. The manual marking procedure
can also be employed to mark each grain, without regard to the grid chosen. For grains that intersect an edge of
the field of view, you should count those that intersect two edges, say the top and left, and ignore those that
intersect the other two, say the bottom and right. By this method, if the number of grains is N and the image area
is A (converted to square millimeters), then the grain size is
G = 3.32 Log (N/A) 2.95
Note that these two methods do not really measure the same characteristic of the microstructure, and will in
general only approximately agree for real structures. Usually the grain size number is reported only to the nearest
integer.
More information about the interpretation of the counts, their conversion to microstructural information, and the
statistical confidence limits that are a function of the number of counts can be found in other chapters.
Stereological calculations from counting experiments are extremely efficient, and provide an unbiased
characterization of microstructural characteristics. It is important to remember the statistical advantage of counting
the features that are actually of interest rather than trying to determine the number by difference. However, for
counting a grid with a fixed total number of points it is best to mark only the minor phase points and obtain the
number of counts for the major phase by subtracting the number of marked points from the (known) total number
of points in the grid.
Appendix - Computer Assisted Measurements
{Manual stereology macros for NIH Image.
Overlay grids on an image with arrays of lines or points (reports the number of points or the length of the
lines in image units). Grids provided include three different point arrays and four line arrays, one of
which is cycloids and one sine-weighted radial lines for vertical section method. Then use paintbrush set to
any of the fixed colors (up to 6) to mark locations to be counted (e.g., where line grids cross feature
boundaries). Finally, use macro to count marks in each class, and use results for stereological
calculations. For more details, see the paper "Computer-Assisted Manual Stereology" in Journal of Computer
Assisted Microscopy, vol. 7 #1, p. 1, Mar. 1995
1995-1998 John C. Russ - may be freely distributed provided that the documentation is included.}
Macro 'Point Grid';
Var
k,x,y,xoff,pwd,pht,nrow,ncol: integer;
area,ppx: real;
un: string;
Begin
GetPicSize(pwd,pht);
nrow:=pht Div 50;
ncol:=pwd Div 50;
xoff:=(pwd - 50*ncol) Div 2;
If (xoff<25) Then xoff:=25;
y:=(pht - 50*nrow) Div 2;
If (y<25) Then y:=25;
SetLineWidth(1);
k:=0;
Repeat {Until >pht}
x:= xoff;
Repeat {Until >pwd}
MoveTo (x-5, y);
LineTo (x-1, y);
MoveTo (x+1, y);
LineTo (x+5, y);
MoveTo (x, y-5);
LineTo (x, y-1);
MoveTo (x, y+1);
LineTo (x, y+5);
k:=k+1; {counter}
x:=x+50;
Until ((x+10)>pwd);
y:=y+50;
Until ((y+20)>pht);
GetScale(ppx,un);
MoveTo (2,pht-6);
SetFont('Geneva');
SetFontSize(10);
Write('Total Points=',k:3);
area:=pwd*pht/(ppx*ppx);
MoveTo (2,pht-18);
Write('Total Area=',area:10:3,'sq.',un);
End;
Macro 'Staggered Grid';
Var
i,k,x,y,xoff,yoff,pwd,pht,nrow,ncol: integer;
area,ppx: real;
un: string;
Begin
nrow:=pht Div 34;
ncol:=pwd Div 50;
If (xoff<25) Then xoff:=25;
yoff:=(pht - 34*nrow) Div 2;
If (yoff<25) Then yoff:=25;
SetLineWidth(1);
k:=0;
i:=0;
y:=yoff;
Repeat {Until >height}
x:= xoff;
If (2*(i Div 2)=i)
Then x:= x + 25;
Repeat {Until >width}
MoveTo (x-5, y);
LineTo (x-2, y);
MoveTo (x+2, y);
LineTo (x+5, y);
MoveTo (x, y-5);
LineTo (x, y-2);
MoveTo (x, y+2);
LineTo (x, y+5);
MakeOvalRoi(x-2,y-2,5,5);
DrawBoundary;
KillRoi;
k:=k+1; {counter}
x:=x+50;
Until ((x+25)>pwd);
y:=y+34;
i:=i+1;
Until ((y+25)>pht);
GetScale(ppx,un);
MoveTo (2,pht-6);
SetFont('Geneva');
SetFontSize(10);
Write('Total Points=',k:3);
MoveTo (2,pht-18);
End;
Macro 'Cycloids';
Var
h,i,j,k,x,y,xoff,yoff,pwd,pht,nrow,ncol,xstep,ystep: integer;
len,area,ppx,pi,theta: real;
un: string;
Begin
pi:=3.14159265;
nrow:=pht Div 90;
ncol:=pwd Div 130;
{cycloids are 110 pixels wide x 70 high, length 140}
SetLineWidth(1);
h:=0;
For j:=1 To nrow Do
Begin
y:=yoff + j*90-10;
For i:=1 To ncol Do
Begin
x:=xoff+(i-1)*130+10;
If (h Mod 4)=0 Then
Begin
MoveTo (x,y);
For k := 1 To 40 Do
Begin
theta:=(pi/40) *k;
xstep:=Round(35*(theta-Sin(theta)));
ystep:=Round(35*(1.0-Cos(theta)));
LineTo (x+xstep,y-ystep);
End;
End;
If (h Mod 4)=1 Then
Begin
MoveTo (x,y-70);
For k := 1 To 40 Do
Begin
theta:=(pi/40) *k;
LineTo (x+xstep,y-70+ystep);
End;
End;
If (h Mod 4)=2 Then
Begin
MoveTo (x+110,y);
For k := 1 To 40 Do
Begin
theta:=(pi/40) *k;
LineTo (x+110-xstep,y-ystep);
End;
End;
If (h Mod 4)=3 Then
Begin
MoveTo (x+110,y-70);
For k := 1 To 40 Do
Begin
theta:=(pi/40) *k;
LineTo (x+110-xstep,y-70+ystep);
End;
End;
h:=h+1;
End; {for i}
End; {for j}
GetScale(ppx,un);
len:=h*140/ppx;
MoveTo (2,pht-6);
SetFont('Geneva');
SetFontSize(10);
Write('Total Length=',len:10:4,' ',un);
MoveTo (2,pht-18);
Write('Total Area=',area:10:3,' sq.',un);
End;
Macro 'Square Lines';
Var
i,j,x,y,xoff,yoff,pwd,pht,nrow,ncol: integer;
len,area,ppx: eal;
un: string;
Begin
nrow:=pht Div 100;
ncol:=pwd Div 100;
If (xoff=0) Then
Begin
xoffset:=50;
ncol:=ncol-1;
End;
If (yoff=0) Then
Begin
yoff:=50;
nrow:=nrow-1;
End;
SetLineWidth(1);
For j:=0 To nrow Do
Begin
y:= yoff + j*100;
MoveTo (xoff, y);
LineTo (pwd-xoff-1, y);
End;
For i:=0 To ncol Do
Begin
x:= xoff + i*100;
MoveTo (x,yoff);
LineTo (x,pht-yoff-1);
End;
GetScale(ppx,un);
len:=(nrow*(ncol+1)+ncol*(nrow+1))*100/ppx;
MoveTo (2,pht-6);
SetFont('Geneva');
SetFontSize(10);
MoveTo (2,pht-18);
End;
Macro 'Circle Grid';
var
i,j,x,y,xoff,yoff,pwd,pht,nrow,ncol: integer;
len,area,ppx,pi: real;
un: string;
Begin
SetLineWidth(1);
pi:=3.14159265;
nrow:=pht Div 120;
ncol:=pwd Div 120;
For j:=1 To nrow Do
Begin
y:= yoff + 15 + (j-1)*130;
For i:=1 To ncol Do
Begin
x:= xoff + 15 + (i-1)*130;
MakeOvalRoi(x,y,101,101);
DrawBoundary;
KillRoi;
End;
End;
GetScale(ppx,un);
Len:=nrow*ncol*pi*100/ppx;
MoveTo (2,pht-6);
SetFont('Geneva');
SetFontSize(10);
Write('Total Length=',Len:10:4,' ',un);
MoveTo (2,pht-18);
End;
Macro 'Radial Lines';
var
step,i,j,pwd,pht:integer;
x,y,theta,temp:real;
Begin
step:=GetNumber('Number of Lines',16,0);
SetPalette('GrayScale',6);
AddConstant(7);
SetForegroundColor(1);
setlinewidth(1);
For i:=0 To step Do
Begin
theta:=i*3.14159265/step;
x:=(pwd/2)*Cos(theta);
y:=(pht/2)*Sin(theta);
MoveTo(pwd/2-x,pht/2+y);
LineTo(pwd/2+x,pht/2-y);
End;
End;
Function ArcSin(X:real) : real;
var
temp:real;
Begin
if (X=0) then temp:=0
else if (X=1) then temp:=3.14159265/2
else if (X=-1) then temp:=-3.14159265/2
else temp:=ArcTan(x/sqrt(1-x*x));
ArcSin:=temp;
End;
Macro 'Sine-wt. Lines';
var
step,i,j,pwd,pht:integer;
x,y,theta,temp:real;
Begin
step:=GetNumber('Number of Lines',16,0);
AddConstant(7);
setlinewidth(1);
For i:=0 To step Do
Begin
temp:=(-1+2*i/step);
theta:=ArcSin(temp);
x:=(pwd/2)*Cos(theta);
y:=(pht/2)*Sin(theta);
MoveTo(pwd/2-x,pht/2+y);
LineTo(pwd/2+x,pht/2-y);
End;
End;
Macro 'Horiz. Lines';
var
space,i,pwd,pht:integer;
Begin
space:=GetNumber('Line Spacing',5,0);
AddConstant(7);
setlinewidth(1);
i:= (pht mod space)/2;
if (i<2) then i:=(space/2);
while (i<pht) do
Begin
MoveTo(0,i);
LineTo(pwd-1,i);
i:=i+space;
End;
End;
Macro 'Vert. Lines';
var
space,i,pwd,pht:integer;
Begin
space:=GetNumber('Line Spacing',5,0);
AddConstant(7);
setlinewidth(1);
i:= (pwd mod space)/2;
if (i<2) then i:=(space/2);
while (i<pwd) do
Begin
MoveTo(i,0);
LineTo(i,pht-1);
i:=i+space;
End;
End;
Macro 'Random Points';
Var
x,y,k,i,pwd,pht,limt: integer;
ppx,area: real;
un: string;
collide: boolean;
Begin
limt:=50;{number of points}
k:=1;
Repeat
x:=Random*(pwd-20); {10 pixel margin around borders}
y:=Random*(pht-20);
collide:=false;
If (k>1) Then {avoid existing marks}
For i:=1 To (k-1) do
If (Abs(x-rUser1[i])<5) and (Abs(y-rUser2[i])<5)
Then collide:=true;
If Not collide Then
Begin
rUser1[k]:=x;
rUser2[k]:=y;
MakeOvalRoi(x+6,y+6,7,7);
DrawBoundary;
KillRoi;
k:=k+1;
End;
Until (k>limt);
GetScale(ppx,un);
SetFont('Geneva');
SetFontSize(10);
MoveTo (2,pht-18);
MoveTo (2,pht-6);
Write('Total Points=',k-1:4);
End;
Macro 'Random Lines';
Var
i,j,k,x1,x2,y1,y2,pwd,pht,len,limt: integer;
x,y,theta,m,area,ppx,dummy: real;
un: string;
Begin
len:=0;
k:=0;
limt:=3*(pwd+pht); {minimum total length in pixels}
Repeat {Until length>limt}
x:=Random*pwd;
y:=Random*pht;
theta:=Random*3.14159265;
m:=Sin(theta)/Cos(theta);
x1:=0;
y1:=y+m*(x1-x);
If (y1<0) Then
Begin
y1:=0;
x1:=x+(y1-y)/m;
End;
If (y1>pht) Then
Begin
y1:=pht;
x1:=x+(y1-y)/m;
End;
x2:=pwd;
y2:=y+m*(x2-x);
If (y2<0) Then
Begin
y2:=0;
x2:=x+(y2-y)/m;
End;
If (y2>pht) Then
Begin
y2:=pht;
x2:=x+(y2-y)/m;
End;
MoveTo(x1,y1);
LineTo(x2,y2);
len:=len+Sqrt((x2-x1)*(x2-x1)+(y1-y2)*(y1-y2))
k:=k+1;
Until (len>limt);
GetScale(ppx,un);
SetFont('Geneva');
SetFontSize(10);
MoveTo (2,pht-18);
len:=len/ppx;
MoveTo (2,pht-6);
End;
Macro 'Count Marks';
{note - this routine is VERY slow as a macro because it must access each pixel. The Photoshop drop-in is
much faster for counting features, and when used by NIH Image will perform exactly as this does and count
the number of marks in each of the six reserved colors.}
VAR
i,j,k,pwd,pht,valu,nbr,newfeat: integer;
Begin
For i:= 1 To 6 Do
rUser1[i]:=0;
MoveTo(0,0);
For i:=1 To pht Do
Begin
GetRow(0,i,pwd);
newfeat:=0; {start of a new image line - nothing pending}
For j:=1 To (pwd-1) Do {skip edge pixels}
Begin
valu:=LineBuffer[j]; {test pixel}
If ((valu=0) or (valu>6)) Then
Begin {pixel is not a fixed color}
If (newfeat>0) Then {End of a line}
rUser1[newfeat]:=rUser1[newfeat]+1;
newfeat:=0;
End;
If ((valu>=1) and (valu<=6)) Then {a fixed color point}
Begin
nbr:=LineBuffer[j-1]; {left side}
If (nbr<>valu) Then {test continuation of line}
Begin
If (newfeat>0)Then {prev touching color}
rUser1[newfeat]:=rUser1[newfeat]+1;
newfeat:=valu;{start of a chord}
End;
For k:=(j-1) To (j+1) Do {check prev line}
Begin
nbr := GetPixel(k,i-1);
If (nbr = valu) Then
Begin
newfeat:=0;{touches}
End;
End;
End;
End; {for j}
LineTo(0,i); {progress indicator because getpixel is very slow}
End; {for i}
ShowMessage('Class#1=',rUser1[1]:3,'\Class#2=',rUser1[2]:3,
'\Class#3=',rUser1[3]:3,'\Class#4=',rUser1[4]:3,
'\Class#5=',rUser1[5]:3,'\Class#6=',rUser1[6]:3);
{can substitute other output procedures as needed}
End;
Chapter 10 - Computer Measurement of Images
The preceding chapter discussed the display of microscope images on a computer screen along with the
superposition of grids and overlays. Interactive marking by a human able to recognize the various features present
provides the input for the computer to tally the different counts, from which calculation of stereologically useful
data can be carried out. The calculations are straightforward, usually requiring no more than a simple spreadsheet
and often performed manually.
Some computer-based image processing operations may be useful in preparing the image for display, to aid the
operator in recognizing the structures of interest, but these are primarily limited to correcting image acquisition
defects such as poor contrast, nonuniform illumination, non-normal viewing angles, etc. Some of the methods
used to perform those processes, such as neighborhood operations that combine or rank pixel values in each small
region of the image to produce new values that form a new image, can also be used to carry out the more
aggressive image processing operations discussed in this chapter.
The intent of this chapter is to consider ways that the computer can directly perform measurements on the images
to obtain data that can be used to characterize the structure. This is not really automatic computer-based
measurement since there is still a considerable amount of user interaction needed to specify the operations based
on visual recognition of structure. Selection of the appropriate processing and measurement steps requires more,
not less human involvement and knowledge than the simple grid counting methods. However, it becomes possible
to measure things that are not otherwise available. For example, humans can count events such as the intersections
of grid lines with features, but are not good at measuring lengths or angles.
Measurement using grids
The grids of points and lines used in the preceding chapter served only as visual guides to mark points of interest
on the image. Sometimes the counting process can be automated, or direct measurements obtained by combining
the grid with the image. These methods usually rely on first converting the grey scale or color image to a binary or
black and white image. The convention used here is that black pixels are part of the features of interest and white
represents the background (about half of the systems on the market use this convention, and the other half the
opposite convention that black is background and white represents features; the latter is a hangover from screen
displays that used bright characters on a dark background).
Reducing a grey scale image to black and white is most directly accomplished by thresholding, selecting a range of
grey (or color) values by setting markers on the histogram and treating any pixel whose value lies within that
range as foreground, and vice versa. In fact, there are not too many real images in which such a simple procedure
actually works, and several methods will be described later in this chapter for processing images so that
thresholding can be used. But to illustrate the use of grids for direct counting we will use the illustrative image in
Figure 1, which can be thresholded to delineate the pink-colored "phase." This is a generic name used in
stereology for any structure of interest, and in many cases does not correspond to a phase in the chemical or
physical sense (a region homogeneous in chemical composition and atomic or crystallographic structure)
Chapter 10 - Computer Measurement Figures - Page # 193
Figure 1. Example of an image (a) in which a structure can be thresholded by brightness or color to
delineate it as a phase for measurement, represented as a binary image (b).
Figure 2. Combining the binary phase from Figure 1b with a grid of points (a) using a Boolean AND
operation (b). This allows automatic counting of the point fraction to estimate the volume fraction.
To determine the area fraction of the selected phase by manual grid counting, the grid would be overlaid on the
image, the user would mark those points that fall on the foreground regions, these would be counted by the
computer (either by counting mouse clicks, for example, or by counting the marks made on the image in some
unique color). Then the number of points marked divided by the number of points in the grid is P
P
, whose
expected value is equal to the volume fraction of the phase. If the image is a binary image, and the grid is
considered to be another binary image, then the two can be combined using a Boolean AND operation. This
examines the individual pixels in the two images and creates a new image in which a pixel is made black only if
both of the two original pixels at that location are black.
Figure 3. Processing the binary image from Figure 1b to get just the outline of the phase boundaries (a).
Combining these with a grid of lines with known total length (b) using a Boolean AND (c) allows
counting of the points to estimate the surface area per unit volume of the phase.
The grid points that fall onto the black phase are kept, the others are erased. Counting the number of points that
remain is a simple operation, which can be performed directly from the image histogram. Even if the points in the
grid are marked by more complex shapes (to aid in visual recognition), counting the marks is the same process as
discussed in the previous chapter. As shown in Figure 2, ANDing a point grid (which can be regular or random as
appropriate to the sample) with the binary image gives an immediate estimated result for P
P
.
If a line grid is used to determine the number of points P
L
at which the lines cross the phase boundary, this
requires a single additional step. The binary image of the phase of interest is converted to its outline form. This is
another example of a neighborhood operator, but one of a class of operators often used with binary images called
morphological operators. They examine the pattern of neighboring pixels and turn the central pixel on or off
depending on that pattern. Typical criteria are the number of touching neighbors (used in erosion and dilation) and
whether or not the neighbors that are foreground points themselves are all touching each other (used in
skeletonization).
In this particular case, we want to keep the outline of the phase regions, so we keep every pixel that is black
(foreground) that touches at least one white (background) pixel, testing the four closest neighbors to the left, right,
top and bottom. The result, shown in Figure 3, erases all internal pixels within the regions and keeps just the
outlines. ANDing this image with that of a grid of lines keeps just those points where the lines cross the edges.
Counting these points and dividing by the length of lines in the grid (a known constant) gives P
L
. Note that the
points are not necessarily single pixels, since the lines may be tangent to the boundaries so that the points of
intersection cover more than one pixel; counting all of the marks (groups of touching pixels) gives the correct
answer.
There is another seemingly more efficient way to perform this same measurement without first getting the outlines,
by just ANDing the grid lines with the original features and counting the number of line segments. Since each line
segment has two ends, the number of points must be twice the number of segments. But there is a problem with
this method because some line segments may end at the edge of the image inside the foreground region, which
would produce an erroneous extra count. Dealing with the edges of images is always a problem, and although it
can be solved (in this example by testing the ends of the lines to see whether they lie on black or white pixels and
adjusting the count accordingly) it requires additional logic.
Measurement of structure dimensions, rather than just counting, can also be performed using a grid of lines.
Figure 4 shows one simple example. The image shows a cross section of a coating on a metal. Thresholding the
image to delineate just the coating is possible, so no additional image processing is required. This fortunate
circumstance is often possible in metallography by the judicious use of chemical etchants to darken one phase and
not the other. The same approach is used with chemical staining of biological tissues, but as these tend to be more
complex in structure the selectivity of the stains is not as great.
Once the coating is isolated as the foreground, ANDing a grid of lines oriented in the direction normal to the metal
surface produces a set of line segments that can be measured. The number of pixels in each line, converted to a
distance based on the image magnification, is a measure of the thickness of the coating at that point. The
distribution of line lengths can be analyzed statistically to determine the mean coating thickness and its variation.
Of course, creating other templates of lines to measure in various orientations, including radial dimensions, is
straightforward.
0
1
2
3
4
5
6
74 76 78 80 82 84 86 88 90 92
Lengt h
mean = 82.68
st d.dev. = 3.80
Figure 4. Metallographic cross section of a coating applied to a metal substrate (a). Superimposing a grid
of lines normal to the nominal surface (b) using a Boolean AND produces a set of lines whose
lengths can be used to measure the mean value and variation of the coating thickness (d).
The use of a random set of lines to measure the thickness of layers that are not sectioned perpendicular to their
surfaces (so that the thickness direction of the layer is not directly measurable) is also useful. In this method, the
section plane intersects different layers or portions of layers at various angles. The random lines constitute IUR
(isotropic, uniform, random) probes of the structure. The length distribution of straight lines through a layer of
thickness T can range from exactly T up to infinity, depending on the line orientation. But a plot of frequency vs.
1/Length (Figure 5) is a simple triangular function, whose mean value is 2/3 the maximum. Since this maximum is
1/T, the true layer thickness is just 1.5 times the mean value of the (1/Length) values from the line probes. The
process is thus to generate the random line probes, AND them with the image, measure the lengths and calculate
the mean value of 1/Length, multiply this by 1.5 and take the inverse, which is the mean layer thickness. Many
structures in materials (e.g., composites), biology (e.g., membranes) and other applications can be measured in
this way. This is an example of the power of the stereological approach.
Consideration of the geometry of the structure of interest (a layer) leads to the design of a probe (random lines)
and a measurement procedure (1/Length of the intersections) that yields the desired result straightforwardly.
Measurement of line lengths in the computer can be done in several ways. For straight lines at any angle the
Pythagorean distance between the end points can be calculated. For lines that may not be straight, measurement
methods (and their errors) will be discussed below.
1 / Length
1 / T
Figure 5. Distribution of 1/Intercept Length for random lines through a layer of thickness T.
Measuring areas with pixels
Probably the measurement that seems most straightforward with a digitized image consists of counting pixels. For
an array of square pixels that are either black (features) or white (background) the procedure is to count the pixels
and use that as a measurement of area. The basic stereological rule that V
V
=P
P
also states that V
V
=A
A
. The
fraction of all the pixels in the image that are black must measure the area fraction of the selected phase.
While this is true, it does not allow easy estimation of the accuracy or precision of the measurement. The problem
is that pixels are not points (they have finite area, and in most image capture processes represent an average across
that area), and the points are too close together to be independent samples of the structure, so that the N cannot
be used as a measure of counting precision as it could for counting a sparse grid of points.
For the area fraction of the image as a whole, it isn't even necessary to perform thresholding or look at the pixels
in the image. The histogram contains all of the data. Wherever the threshold levels are set to perform the
separation of features from background, the fraction of the total histogram between the thresholds is directly the
area fraction. For the image in Figure 1, this gives an area fraction of 30.51% (19995 of 65536 pixels).
For measurement of the individual features present, we do need to count the actual pixels. Most computer
measurement systems do this by treating features (sometimes called blobs) as all pixels that are in contact, defining
contact as either sharing an edge or sharing an edge or corner (called four-neighbor and eight-neighbor logic).
Except for measuring lines, either definition is usually adequate for measurement purposes since we prefer to
avoid situations in which features have dimensions as small as a single pixel. The difference between four- and
eight-neighbor rules has to do principally with the topological property of connectivity. Whichever rule is used to
define pixels in features, the opposite rule will apply to the background pixels.
There are several sources of error when pixel counting is used to measure areas in binary images. The finite area
of each pixel means that pixels around the periphery of the feature may be included in the binary image or not
depending on the fraction of each pixel that is actually covered. The same feature placed on the pixel grid in
different positions and rotations will cover a different number of pixels and thus generate different measurement
results. In addition, the thresholding operation to select which pixels to include in the feature is sensitive to noise,
which produces grey scale variations in the pixel values. This can particularly affect the edge pixels and result in
their being included or excluded. Capturing and thresholding repeated images of the same feature will not produce
perfectly reproducible results.
Notice that these sources of error are related to the edge pixels (the perimeter of the feature) and not to the total
number of pixels (the area of the feature). This means that unlike the grid point count method where the number of
points can be used to determine the estimated precision, pixel counting produces results with a precision that
depends on the feature shape. Two features with the same area will be measured with different precision
depending on the amount of perimeter each has.
0
5
10
15
20
25
30
35
C
o
u
n
t
70 72 74 76 78 80 82
Pi xel Ar ea
Figure 6. Results of 100 measurements of the area of a 10 unit diameter circle placed at random on a
square pixel grid. True area is 10
2
(/4)=78.54. Mean value of distribution above is 77.86, with
standard deviation = 1.639, skew = -1.143, kurtosis = 1.910. This variation demonstrates the
effects of the finite size of pixels for the case of many edge orientations.
Figure 6 illustrates the variability from placing a small circular feature at random on a pixel grid. Each circle has an
actual diameter of 10 units (10 pixel widths, for an ideal geometrical area of 78.54 units), but depending on
placement the number of pixels that are at least 50% covered (and hence included in the feature) will vary. The
graph shows that the results are skewed to smaller measurement values. This effect becomes less important as the
feature size increases, and sets an effective lower limit to the size of objects that can be measured in an image.
Adding noise to the image complicates the thresholding process and hence the measurement. Figure 7 shows a
series of identical circles 10 units in radius with superimposed random noise. Thresholding the image at the ideal
midpoint in the histogram (which is known in this case but would not be in general) and applying a morphological
opening (discussed later in the chapter) produces features with some pixel errors. The area results obtained by
counting pixels are low on the average because of missing internal pixels and indentations along the sides. Instead
of just counting the pixels to obtain the area, a more robust measure is the convex area obtained by fitting a 32-
sided polygon around the feature (sometimes called a taut-string perimeter or convex hull). We will see shortly
that there are a variety of ways to define feature measurements that the computer can make which can be selected
to overcome some of the limitations of the pixels in the original image.
0
5
10
15
20
25
30
250 270 290 310 330 350
Ar ea
0
5
10
15
20
25
30
250 270 290 310 330 350
ConvexArea
Area ConvexArea
Mean 307.23 321.404
Std.Dev 17.35 14.20
Skew -.96 -1.02
Kurtosis 4.17 4.08
Median 309 324
Figure 7. Measurement of circles on a noisy background showing thresholding variability. The
geometrical area of the circles is 314.16 units. The original image with added Gaussian noise (=32 grey
levels) was thresholded at a middle grey value of 128, and was processed with a morphological opening to
remove isolated pixels before measuring the area and convex area. The results are summarized in the table.
A circle includes pixels along edges that have all possible orientations with respect to the pixel grid. Some
directions are more sensitive to placement than others. For instance a feature consisting of a hollow square
(external width of 11 units, internal hole with a width of 7 units so all of the sides are 2 units wide) that is aligned
with its sides parallel to the pixel grid has no variability with placement at all. When it is rotated by 45 so that the
edges all cut diagonally across the pixels, the variability is shown in Figure 8. Notice that the mean value is close
to the actual value (good accuracy) but the variation is quite large (poor precision).
0
2.5
5
7.5
10
12.5
15
17.5
20
22.5
C
o
u
n
t
60 65 70 75 80
Ar ea
Figure 8. Results of 100 measurements of the area of a randomly placed feature shaped like a hollow
diamond with outer and inner sides of 11 and 7 units (actual geometrical area = 72). Mean value of the
distribution is 71.93, standard deviation = 4.39, skew = -4.674, kurtosis = 2.518. When the same figure
is rotated 45 degrees to align with the pixel grid the measured answer is always 72.0 exactly. This
demonstrates the effect of edge orientation on measurement precision.
We can investigate this orientation sensitivity by placing the same hollow square feature on the grid and rotating it
in 1 steps (Figure 9a). Since the feature covers various fractions of pixels, a decision of which pixels to count
must be made. If the threshold is set at 50%, the results (Figures 9b and 9c) show that the typical measured value
is low (60 instead of 72 pixels) except when the orientation is within 1 of the actual pixel grid. Setting the
threshold to 25% coverage (Figures 10a and 10b) gives a result that is biased slightly greater than the actual area,
but much closer to it. It is also interesting to look at the measured length of the perimeter (another feature
measurement that is discussed below). For the 50% thresholding (Figure 11) the results are skewed slightly low
and have some evidence of a trend with orientation angle. The trend is much more pronounced for the case of
thresholding at 25% (Figure 12).
In most cases of real images, it is not clear where the threshold actually should be set. Figure 13 shows a portion
of a grey scale metallographic microscope image of a three-phase alloy, with its histogram. This image actually
has some vignetting and is systematically darker at the corners than in the center. Leveling of the contrast using the
automatic method described in the preceding chapter (by assuming that all of the light colored features should
actually be the same) sharpens the peaks in the histogram. Measurement of the light colored phase (dendrites)
requires setting a threshold; varying its placement over a wide range makes little visual difference in the resulting
binary image, even though the area fraction varies from about 30% to about 40%. Clearly, these different values
cannot all be "correct."
0
2
4
6
8
10
12
14
16
50 55 60 65 70 75
Ar ea
50
55
60
65
70
75
0 5 10 15 20 25 30 35 40 45
Rot at ion
Figure 9. Effect of rotation on the area measurement of a hollow square, OD=11, ID=7 (actual area = 72
units) rotated in 1 degree steps on a square pixel grid (a), by counting those pixels which are at least 50%
covered. Note that the true value is only obtained for <2 degree rotation, and a value of about 60 is more
typical. (mean=60.09, =3.42, skew=1.83, kurtosis=7.46, median=60)
0
2
4
6
8
10
12
65 70 75 80
Ar ea
65
70
75
80
0 5 10 15 20 25 30 35 40 45
Rot at ion
Figure 10. Measurement results for the same rotated squares in Figure 9a, but changing the counting
criterion to include all pixels that are at least 25% covered. Mean=74.20, =2.25, skew=-1.055,
kurtosis=3.864, median=75
0
2
4
6
8
10
12
14
16
65 70 75
Per i m
66
67
68
69
70
71
72
73
74
0 5 10 15 20 25 30 35 40 45
Rot at ion
Figure 11. Results for measured perimeter from the rotated squares in Figure 9a, selecting pixels that are at
least 50% covered. The geometrical answer is 72.0; mean=70.78, =1.459, skew=-.70, kurtosis=2.76,
median=71.
0
2
4
6
8
10
12
14
16
66 68 70 72 74 76
Per i m
67
68
69
70
71
72
73
74
75
0 5 10 15 20 25 30 35 40 45
Rot at ion
Figure 12. Results for measured perimeter from the rotated squares in Figure 9a, selecting pixels that are at
least 25% covered. The mean=71.25, =1.56, skew=-.312, kurtosis=2.79, median=71.3. Although the
overall range of variation is not greatly affected, there is a definite trend of value with angle.
If there is some independent knowledge about the nature of the sample, automatic algorithms can be used to assist
with the critical threshold setting operation. In this case, the dendritic nature of the light regions suggests that they
were formed by nucleation and growth and therefore should have smooth boundaries. A thresholding operation
that seeks the setting giving the smoothest boundaries selects the value shown in Figure 14. Smooth boundaries
are one of the criteria that skilled operators also use to judge threshold settings when they are performed manually.
Others are the elimination of small features (noise), or minimal variation in area with setting (Russ, 1995b). All of
this reminds us that the so-called automatic measurement methods using a computer are only as good as the very
human input that selects methods on the basis of either prior knowledge or visual judgment.
Figure 13. Upper right corner portion of an original image of a metal alloy (a) showing corner to center
shading and the brightness histograms before (b) and after (c) leveling the image contrast.
Figure 14 (continued...)
Figure 14. Section of the metallographic image from Figure 13 (after leveling) with the results of
thresholding it at different grey levels: a) histogram with levels marked, and b) resulting binary images.
(143 = 40%, 215 = 30%, 178 as selected by method discussed in text gives 37.7% area fraction.
Measurement parameters: size
The area, as defined by the number of foreground pixels that touch each other and constitute the digital binary
image representation of an object, is one rather obvious measure of size. There are others that are accessible to
measurement using a computer. Selecting the ones that are appropriate from those offered by each particular image
analysis software package (and figuring out what the highly variable names used to describe them mean) is an
important task for the user.
Figure 15. Measures of area for a feature: Net area, filled area and convex area.
Area can be measured in three principal ways, as indicated in Figure 15.
A. The number of touching foreground pixels (it does matter in some cases whether touching is interpreted as 4-
or 8-neighbor connectivity)
B. The same as A but with the inclusion of any internal holes in the feature
C. The area inside a fitted geometrical shape. The two most common shapes are a fitted ellipse or a polygon; some
more limited systems use a rectangle, either with sides parallel to the image edges or oriented to the feature's
major axis. The polygon is typically drawn to vertices that are the minimum and maximum pixel addresses in
the feature as the image axes are rotated in steps. This produces the "taut string outline" or "convex hull" that
bridges across indentations in the periphery. The ellipse may be established in several ways, including setting
the major axis to the maximum projected length of the object and defining the minor axis to give the same area
as A or B above, or by calculating the axes to give the same moments as the pixels within the feature.
Which (if any) of these measures makes sense depends strongly on what the image and sample preparation show.
For sections through particles, the net area (A) will correspond to the particle mass, the filled area (B) to the
displaced volume, and the fitted shape (C) to results obtained from sieving methods.
Any of these area measurements can be converted to an "effective radius" or "effective diameter" by using the
relationship Area = 4 Radius
2
, but of course the underlying shape assumptions are very important. Classical
stereology calculates a distribution of sizes for spherical particles in a volume based on the measured distribution
of circle sizes on a section, and in that specific case the effective (circular) diameter is an appropriate descriptor.
Figure 16 shows an example where this is meaningful. The image is a metallographic microscope view of a
polished section through an enamel coating. The pores in this coating are spherical due to gas pressure and surface
tension, and the sections are therefore circles. The image is easily thresholded, but the pores have bright internal
spots due to light reflection. Filling holes in objects is accomplished by inverting the image (interchanging black
and white pixel values), treating the holes and background as features, discarding the feature (the background)
which touches the edges of the image, and then adding the remaining features (the holes) back into the original
image. This last step is actually done with a Boolean OR that sets each pixel to black if it is black in either the
original image or in the inverted copy. This is an example of the kinds of image processing that are performed on
binary (thresholded) images, several of which are discussed later in this chapter.
Frequency Distribution for Area(mm
2
)
From () To (<) Count N
V
(spheres)
0.0 30.0 116 5.082
30.0 60.0 3 0.141
60.0 90.0 2 0.102
90.0 120.0 4 0.221
120.0 150.0 0 0.0
150.0 180.0 1 0.067
180.0 210.0 0 0.0
210.0 240.0 1 0.084
240.0 270.0 0 0.0
270.0 300.0 0 0.0
300.0 330.0 0 0.9
330.0 360.0 0 0.
360.0 390.0 1 0.277
390.0 420.0 1 0.211
420.0 450.0 0 0
Total 129
100
10
1
0.1
0.01
Ci r cl es
Spheres
Figure 16. Metallographic microscope image of pores in an enamel coating, thresholded and with holes
filled. The measurement of circle sizes produces a distribution that can be unfolded to produce an estimate
of the size distribution of the spheres.
Measurement of the resulting circles produces a distribution that can be converted to the size distribution of the
spheres that generated them by multiplication by a matrix of values. These are calculated as discussed in Chapter
11 on shape modeling and in texts such as Underwood, 1970; DeHoff & Rhines, 1968; and Weibel, 1979; for a
sphere the calculation can be done analytically. If a sphere of given radius is sectioned uniformly, the size
distribution of the circular sections gives the probability that a circle of any particular size will result. Doing this
for a series of sphere sizes produces a matrix of values that predicts the number of circles per unit area of image
with radius in size class i, as a function of the number of spheres per unit volume with radius in size class j
N
Ai
= '
ij
N
Vj
(10.1
The inverse of this matrix, , is then used to calculate the expected size distribution of spheres from the measured
distribution of circles. The and ' matrices are published for various standard shapes such as ellipsoids
(DeHoff, 1962), cylinders, disks, and many polyhedra. In most cases the size distributions of the planar sections
through the solids for isotropic, uniform and random sectioning are not easily calculated by analytical geometry
(the sphere of course presents the simplest case, which is why it is so tempting to treat objects as being spherical).
Monte-Carlo sampling methods can be employed in these instances to estimate the distribution, as discussed in the
chapter on Geometric Modeling.
This classical approach is flawed in several ways. The underlying assumption is that all of the three-dimensional
features have the same shape, which is rarely the case. In particular, a systematic change in shape with size will
bias the results, and often occurs in real systems. In addition, the mathematics of this type of unfolding is ill-
conditioned. Small errors in the distribution of circles (which are inevitable due to the statistics of sampling and
counting) are greatly magnified in the inverse matrix multiplication. This is easy to see by realizing that large
circles can only result from sections of large spheres, while small circles can be produced by either large or small
spheres. Small errors in the number of large spheres calculated from the number of large circles will influence the
number of small circles those large spheres produce, biasing the resulting calculation of the number of small
spheres and increasing its uncertainty.
For more complicated shapes, the situation is much worse. The most likely size for a random section through a
sphere is a large circle. For a cube or other polyhedron there is a significant possibility of cutting through a corner
and getting a small feature. The distribution of the areas of sections cut by random planes varies substantially for
objects of different shape. It would in principle be possible to make a better unfolding based on measuring the
shape as well as the size of the sections, but in practice this is not done because there are the other fundamental
problems with unfolding, and new stereological techniques have evolved to deal with measurement of the number
and sizes of particles in a volume that do not depend on knowing their shapes.
There are many possible measures of size available besides area. The length and breadth of a feature can be
defined and measured in quite a few different ways. The major and minor axes of the ellipse fitted by one of the
methods mentioned above can be used. Most common as a measure of length is the maximum projected length (or
maximum Feret's diameter, or maximum caliper diameter). Testing all of the points on the periphery of the feature
to find the maximum and minimum values as the coordinate system is rotated through a series of angles, as was
used above to determine the convex hull, also identifies the maximum projected length.
It is not necessary to use a large number of angles to find the maximum. If 8 steps of 22.5 degrees are used, the
worst case error (when the actual maximum lies midway between two of the orientations tested) is only the cosine
of 11.25 degrees, or 0.981, meaning that the measured value is 1.9% low. With 16 steps the worst case angular
error is 5.625 degrees, the cosine is 0.995, and the error corresponds to 1 pixel on a feature 200 pixels wide,
which is within the error variation due to edge definition, thresholding, and object placement on the pixel grid.
It is more difficult to determine the breadth in this way. Instead of a cosine error, the maximum error depends on
the sine of the angle and can be much larger. For a very narrow and long feature, the minimum projected
dimension can be hundreds of percent high even with a large number of projections. Other estimates of breadth are
sometimes used, including the area divided by the length (which is appropriate for a rectangular feature), the
projected dimension at right angles to the longest direction, the dimension across the center of the feature at right
angles to the longest dimension, and others. It is easy to find specific shapes, even common ones such as a
square, that make each of the estimates seem silly.
Neither the length nor breadth defined in these ways makes sense for features that are curved (fibers, cross
sections of lamellae, etc.). A better measure of length is the distance along the midline of the feature, and this can
be determined by reducing the feature to its skeleton as shown in Figure 17. The skeleton is produced by an
erosion algorithm that removes pixels from the outside boundary of the feature (that is, any black pixel that
touches a white one) except when the black pixel is part of the midline of the feature. This is determined by the
touching black neighbors. It they do not all touch each other, then the central pixel is part of the skeleton and
cannot be removed. The process continues to remove pixels sequentially until there are no further changes in the
image (Pavlidis, 1980).
Figure 17. A feature and its skeleton (green midline). This provides a much more useful estimate of the
length than the maximum caliper or projected length (red arrow).
Skeletons are useful as a way to characterize shape of features, and the branching of the skeleton and counts of its
end points, branch points and loops can give important topological information, but their use here is for length.
The length of a line of pixels is difficult to measure accurately. Each individual pixel in the chain is connected to its
neighbors in either the 90 degree (edge touching) or 45 degree (corner touching) direction. The distance to the 90
degree neighbors is one pixel and that to the 45 degree pixels is 2=1.414 pixels, so summing the distance along
the line gives a straightforward estimate of the length.
But the real midline (or any other line in an image) is not well represented by the line of pixels. Only features that
are many pixels wide are represented faithfully, as discussed before. A perfectly straight line oriented at different
angles and mapped onto a pixel array will have different lengths by this method as a function of angle, with the
sum of link lengths overestimating the value except in the case of exact 45 or 90 degree orientations. The length of
a very rough line will be underestimated because it has irregularities that are smaller than the pixel dimensions.
This problem also arises in the measurement of feature perimeters, which are usually done in the same way. The
variations in measurement data in Figures 11 and 12 result from this source of error.
For features that have smooth boundaries that are not too highly curved, a fairly uniform width, and of course no
branches, the skeleton length does offer a useful measurement of feature length. Another tool, the Euclidean
Distance Map (Danielsson, 1980), can be combined with the skeleton to measure width. The EDM assigns to each
black pixel in the image a value that is its distance from the nearest white (background) pixel, using a very efficient
and fast algorithm. For the pixels along the midline that are selected by the skeleton, these are the centers of a
series of inscribed circles that fit within the feature. Averaging the value of the EDM along the skeleton gives a
straightforward measure of the average width of features that are not perfectly straight or constant in width.
Other feature measurements: shape and position
There are four basic approaches to shape measurement, none entirely satisfactory. The most common approach
uses ratios of size values that are formally dimensionless, such as
4Area/Perimeter
2
Length/Breadth
Area/Convex Area
etc.
These have the obvious advantage of being easy to calculate. Ideally they are independent of feature size, although
in practice the better resolution with which pixels can delineate large features means that trends may appear in
measured data that are not necessarily meaningful. This is particularly true for ratios that involve the perimeter
which is difficult to measure accurately as discussed above.
The main difficulty with this class of parameters is that they are not unique (many shapes that are recognized as
quite different by a human observer can have the same parameter value) and do not correspond to what humans
think of as shape. This latter point is reinforced by the fact that the names given to these parameters are arbitrarily
made up and vary widely from one system to another.
A second approach to shape measurement was mentioned before. The skeleton of a feature captures the important
topology in ways that can be easily abstracted (Russ & Russ, 1989). The difference that a human notices between
the stars in the US, Israeli and Australian flags is the number of points. The number of end points in the skeleton
(pixels which have exactly one neighbor) captures this value.
The remaining two methods for shape description concentrate on the irregularities of the feature boundary.
Harmonic analysis unrolls the boundary and performs a Fourier analysis of it (Beddow et al., 1977). The first
twenty or so coefficients in the Fourier series capture all of the details of the boundary to a precision as good as
the pixels that represent the feature, and can be used for statistical classification schemes. This approach has
enormous power to distinguish feature variations, and has been particularly widely used in geological applications
for the analysis of sediments. The computation needed to extract the coefficients is significant and few image
analysis packages offer it, but the primary barrier to the use of harmonic analysis is the difficulty that human
observers have in recognizing the distinctions that the method finds. This is clearly an example of a case in which
computer analysis takes a different direction from human vision, whereas most of the methods discussed here use
the computer to duplicate human judgment but make the measurements more accurate or easier to obtain.
The fractal dimension of boundaries is also used as a shape descriptor. Many real-world objects have surfaces that
are self-similar, exhibiting ever-increasing detail as magnification is increased (Russ, 1994b). There are important
exceptions to this behavior, principally surfaces that have smooth Euclidean shapes arising from surface tension,
membranes, or crystallographic effects. But when boundaries are rough they are frequently fractal and the
measured dimension does correlate highly with human judgment of how "rough" the boundary appears.
The principal difficulty with all of these shape measures is that they properly apply only in the two-dimensional
plane of the image, and may not be relevant to the three dimensionsal objects sampled by a section or viewed in
projection. The shapes of sections through even simple solid objects such as a cube vary from three-sided up to
six-sided, and the number of sides is correlated with the size. Given such a set of data it is not easy to realize that
all of the sections come from the same solid object, or what the object is. When variation in the shape and size of
the three-dimensional object is also present, the utility of measuring shape of the sections may be quite low.
Harmonic analysis and fractal dimension of sections does provide some information about the roughness of the
surfaces of sectioned objects. For instance, in general for a random section through an object the surface fractal
dimension is greater than the dimension of the intersection by 1.0, the same as the difference between the
topological dimensions of the line and surface (1 and 2, respectively). This is not true for outlines of projected
images of particles, which are smoother than the section or actual surface because indentations tend to be hidden
by surrounding peaks.
Measurement of the position of objects also provides stereologically useful information. Since measurements are
not usually performed in systems that have meaningful absolute coordinate systems, these measurements tend to
be relative. An example would be how far features are from each other, or from boundaries. The detection of
preferred alignment of feature axes or of boundaries is a closely related task. In most cases the most robust
measure of feature position is the coordinates of the centroid or center of mass of the feature. For a 2D object this
is the point at which it would balance if cut along its boundaries from a stiff sheet of cardboard. Of course, the
centroid of a 2D section through a 3D object does not properly represent the centroid of the 3D object, but is still
useful for characterizing some properties such as clustering.
Analysis of the distances and directions between pairs of object centers provides information on clustering,
orientation, and so forth. The distribution of nearest neighbor distances in a 2D random section provides
clustering information on 3D volumes directly; a Poisson distribution indicates a random distribution of objects,
while a mean nearest neighbor distance that is less than or greater than that for the Poisson distribution indicates
clustering of self-avoidance, respectively (Schwarz & Exner, 1983). In making these measurements, it is
necessary to stay far enough from the edges of the image so that the true nearest neighbor is always found.
The axis of orientation of a feature is typically calculated from the same ellipse axes mentioned above based either
on the longest chord (greatest distance between any two points on the periphery) or on the axis about which the
pixels have the smallest second moment. Unfortunately, it is not possible in general to use these measures on 2D
planes to understand the 3D orientations. Of course, for some specimens such as surfaces (orientation of defects,
etc.) the 2D image is quite appropriate and the measurements directly useful.
The precision of measurement of the centroid (first moment) and axis (second moment) are very good, because
they use all of the pixels within the feature and not just a few along the periphery. Determining locations with sub-
pixel accuracy and orientations to better than 1 degree is routinely possible.
Measuring the location of features with respect to boundary lines is less precise. The boundaries are usually
irregular so that they cannot be specified analytically. This means that mathematical calculation of the distance
from the centroid point to the boundary is not practical. Instead, the distance from the feature to the boundary can
be determined using the Euclidean distance map. As mentioned above, this procedure assigns to every pixel within
features a value that is the distance to the nearest background pixel. Any pixel that is a local maximum (greater
than or equal to all neighbors) is considered to be the feature center (in reality it is the center of an inscribed circle).
If Euclidean distance map of the entire area, say the grain or cell in which the features reside, is then computed,
the values of the pixels identified as "feature centers" in the first operation give the distances to the boundary.
Note that this method does not directly yield distance information for a 3D structure as the measurements are made
in the plane of the image. Using the same stereological model introduced above for measuring the thickness of
layers can solve this problem. This procedure uses averaging of 1/distance in order to estimate the mean value of
the 3D distance from features to the boundary.
Image processing to enable thresholding and measurement
There are some cases in which images can be directly thresholded and the resulting binary images measured.
Examples include metals etched to show dark lines along the grain boundaries (Figure 9 in the preceding chapter),
and fluorescence images from stained tissue. But in most cases the structures are too complex and there is no
unique set of brightness values that represent the desired structure and only that structure. In addition to the
procedures discussed in the preceding chapter to correct defects in the original image, a variety of tools are
available to enhance the delineation of features to permit thresholding, and to process the thresholded images to
permit measurement.
Figure 18 shows an example that looks initially much like the illustration in Figure 16. The sample is a slide with
red blood cells. These are easily thresholded and the holes filled (the figure shows the step-by step procedure).
Before measurement, those features that touch any edge must be discarded because their size cannot be
determined. We also discard features smaller than an arbitrary cutoff area, because we know something about the
sample and such small features cannot be the blood cells which are of interest.
Dealing with the finite size of images requires some care. Obviously, features that intersect the edge of the image
cannot be measured because there is no way to measure their actual extent. But in general, larger features are more
likely to intersect the edge and hence to be lost from the measurement process which would bias the results toward
small features. There are two general approaches to correcting for this. The one most commonly used in manual
stereology using photographs is to restrict the measurement to an inner region away from the edges, so that any
feature that has its upper left corner (for example) in the inner region can be measured in its entirety. This is not
usually practical for computer measurements as the limited size of the digitized image makes it unwelcome to
discard a wide guard region around the edges.

Figure 18. Microscope image (a) of red blood cells; (b) the result of thresholding; (c) holes identified as
features that do not touch the edges of the inverted image; (d) OR combination of the holes with the
original image to produce filled features (with those that are small or touch edges eliminated); (e)
watershed segmentation to separate two overlapped features
Instead, it is equivalent to measure all of the features that do not intersect the edge but count them so as to
compensate for the greater likelihood of losing large features. Each measured feature is counted as WxWy/(Wx-
Fx)(Wy-Fy) where Wx and Wy are the image dimensions and Fx and Fy are the projected widths of the feature
in the horizontal and vertical directions. For small features this is 1.0 but for large features the effective count
becomes greater than 1.0 and corrects for the probable loss of similar size features that intersect a randomly placed
image boundary. Of course, if the image is not random in placement this is not appropriate. Human operators
must avoid selecting the placement of their measuring frame to reduce or eliminate having features touch edges (or
for any other reason) as it inevitably introduces uncorrectable bias into the measurements.
Two of the features overlap in the image in Figure 18, and a watershed segmentation is used to separate them.
This procedure first calculates the Euclidean distance map of the features in the thresholded binary image, and then
by proceeding downhill from each local maximum finds the watershed lines between the features that mark
presumed boundaries.
Measuring the area of each feature and using that to calculate an effective circular diameter, as was done for the
pores in Figure 16, is not appropriate here. The overlap that is corrected by the watershed leaves each feature
slightly too small. Instead, measuring the maximum external dimension and using that as a diameter gives a
reasonably robust measure of size and can be used to determine the mean size and variance. In this case, the
sample is not a section through a set of spheres in a volume, but a projected image of disks that can be assumed to
be lying flat on the slide. Hence no further processing of the size distribution is required.
Figure 19 shows an SEM image of a two-phase ceramic. The grains are distinguished in brightness by atomic
number variations (the light phase is zirconia and the dark phase alumina), and the boundaries between the grains
by relief produced by polishing and thermally etching the surface. It is not possible to directly threshold the grain
boundaries to delineate the grains. The Laplacian edge sharpening procedure from the previous chapter does not
solve this problem. In general second derivative operators are better at locating noise and points in the image,
rather than linear edges. A first derivative is very good at locating steps or edges. A typical edge finding kernel is
used in the figure. In order to find edges running in any orientation, two derivatives, one for vertical steps and the
other for horizontal ones, are calculated. Then the magnitude of the gradient (regardless of direction) is calculated
as the square root of the sum of squares of the two directional derivatives, for every pixel in the image. This
operator is the Sobel, one of the most commonly used edge-finding procedures.
Figure 19. SEM image of an alumina-zirconia ceramic, and the grain boundaries delineated by a Sobel operator.
With the boundaries outlined, the same procedures for determing the grain size as discussed in the preceding
chapter can be used. However, it is easier with the computer to measure slightly different parameters than would
normally be determined using the same grid-based schemes used in manual measurement procedures. Instead of
counting the number of intercepts that a line grid makes with the boundaries to determine P
L
, we can measure the
total length of the boundaries after thresholding and skeletonizing, which gives the length of boundary per unit
area of image L
A
. Both of these quantities are related to the surface area per unit volume S
V
.
S
V
= 2 P
L
= 4/ L
A
(10.2
Counting grains per unit area, which is the other approach to estimating a "grain size" parameter, gives a value that
is not a measure of the grain size, but actually of the length of triple line where three grains meet in the sample
volume. But counting the grains in the image automatically is often difficult because even a tiny break in one of the
grain boundaries will cause the computer algorithm doing the counting to join the two grains and get a count that is
biased low. It is usually difficult to assure that all of the grain boundaries are uniformly etched or otherwise
delineated and this results is a significant error. Watershed segmentation is a possible answer to this problem but
is a relatively slow operation in the computer, and also tends to introduce bias because it will subdivide grains that
are not convex in shape.
However, the triple points in the 2D image (where three grain boundaries join) are the intersections of the triple
lines in the 3D volume with the plane of the image, and these can be counted and are usually well defined by
etching. A triple point can be identified in the skeleton as any pixel with more than two adjacent black neighbors.
The number per unit area N
A
is related to the length of triple line per unit volume as
L
V
= 2 N
A
(10.3
It is possible using the same computer algorithm applied in preceding examples to count the touching pixels in
each grain and convert this to an area. Plotting the distribution of areas seems at first like a measure of the size of
the grains but in fact it is not. Just as for the spherical pores in Figure 16, the section areas do not represent the
three dimensional sizes of the grains, and in fact do not even sample the grains in an unbiased way. It is much
more likely that the random section plane will cut through a large grain than a small one. We dealt with this in the
case of pores by using an matrix to unfold the sphere size distribution from the circle size distribution. But we
cannot do that for the grains because they do not have a simple spherical shape (indeed, they can not since spheres
do not pack together to fill space).
There are shapes that have been proposed for grain and cell structures that fill space (such as the
tetrakaidodecahedron) but this still implicitly makes the assumption that all of the grains have the same shape. This
is clearly not true, and in fact in general the smaller grains will be quite different in shape that the larger ones, with
a smaller number of contact faces and hence more acute angles at the corners. This has been demonstrated by
experiments in which grain structures are literally taken apart to reveal the constituent grains. The dependence of
shape upon size changes the distribution of the intersection areas dramatically and makes unfolding impractical.
The parameters that do describe the grain structure (S
V
and L
V
) in an unbiased way seem more limited than an
actual distribution of the three-dimensional sizes, but they are actually more useful since they really describe the
structure and are not dependent on questionable assumptions.
Figure 20 shows another example of grain boundaries, this one a metallographic microscope image of etched
aluminum. The boundaries are not delineated in this image. Instead, the grains have different grey values resulting
from chance orientations of the crystallographic structure. It is necessary to use an edge-finding procedure to
replace the brightness differences of the grains with dark lines at the boundaries. The operator shown is the
variance. The brightness values of pixels within each placement of a small circular neighborhood are used to
compute a variance (sum of squares of differences from the mean) and this value is used as the brightness of the
central pixel in the neighborhood as a new image is formed. The result is a very small variance within each grain,
regardless of the absolute grey value, and a large variance shown by the dark lines at the boundaries where a
change in brightness occurs.
This image can be thresholded and the boundaries skeletonized as shown in the figure. It can then be measured
either using by a grid overlay and counting intersections (either manually or automatically), or by measuring total
boundary length to get S
V
, or counting grains or triple points to get L
V
. Both give values for ASTM grain size as
discussed in the previous chapter. Note as mentioned above that the grain count would be biased low by breaks in
the network of lines along the boundaries which allow grains to join in the computer count, but that these breaks
have a negligible effect on the determination of L
A
for the boundary lines, or N
A
for the triple points.
Figure 20. Metallographic image of etched alumina (a), the application of a variance operator (b) and the
skeletonized binary image of the boundaries (c). A manual counting operation to determine S
V
using a grid
as shown in (d) gives less precision than measuring the line length in (c).
Image processing to extract measurable information
Some information in the original image may be more easily accessed for measurement if processing steps are
applied first to enhance and isolate the important data. An example of this is the use of a gradient or edge detector
to isolate the edges, as discussed above. If the orientation of the edges is of interest, for example to determine the
degree of preferred orientation present in a structure, then some additional steps can be used. Figure 21 shows the
major steps in the procedure.
The Sobel operator determines the magnitude of the gradient of brightness for each pixel in the image by
combining two orthogonal first derivatives to get the square root of the sum of squares as a non-directional
measure of gradient magnitude (Figure 21b). Combining the same two derivatives to get the angle whose tangent
is their ratio gives the orientation of the gradient vector (Figure 21c). The magnitude and direction of the gradient
can be combined with the original grey scale image as shown in Figure 21d to illustrate the process.
Thresholding the Sobel magnitude values identifies the pixels that lie along the major edges in the structure (Figure
21e). Selecting only those pixels and forming a histogram of the angle values for them produces a distribution
(Figure 21f) showing the orientation of edges (which are perpendicular to the maximum gradient). Because the
angle values have been assigned to the 256 value grey scale or color values available for the image, this graph
shows two peaks, 180 degrees (128 grey scale values) apart for each set of edges in the original image.
Figure 21. SEM image of an integrated circuit (a). The Sobel operator gives the magnitude of the
brightness gradient (b) and can also create an image of the direction of the gradient vector (c).
These can be combined with the original image in color planes (d). Thresholding the gradient
magnitude (e) and forming a histogram of the pixel values from the direction direction image (f)
provides a direct measure of the orientation of edges in the image.
Processing is also useful as a precursor to thresholding structures for measurement when the original image does
not have unique grey scale values for the structures or phases present. Figure 22 shows a typical example. The
curds and whey protein are visually distinguishable because the former have a smooth appearance and the latter are
highly textured, but both cover substantially the same range of grey scale values.
Once the human has recognized that the structures have texture as their defining difference, then the use of a
texture operator to process the image is appropriate. There are a variety of texture extraction processes available,
many developed originally for remote sensing (satellite and aerial photography) to distinguish crops, forest,
bodies of water, etc. They generally operate by comparing the brightness differences between pixels as a function
of their separation distance (Peleg et al., 1984). A very simple one is used in Figure 22b; the difference in grey
scale value between the brightest and darkest pixel in each 5 pixel wide circular neighborhood. Since the visually
smooth areas of the image have low pixel-to-pixel brightness variations while the highly textured regions have
large differences, this range operator produces a new grey scale image in which the smooth areas are dark and the
textured ones bright. Thresholding this gives a representation of the phase structure of the sample, which is then
measured conventionally to determine volume fraction, boundary area, mean intercept length, and the other
stereological parameters of interest.
Figure 22. Light micrograph of a thin section of curds and whey. The visually smooth areas (curds)
have the same range of brightness values as the textured regions (whey protein). The application
of a range operator (b) creates a new image that assigns different values to the pixel brightness
based on the difference in values for the neighboring pixels. The derived image can be
thresholded to delineate the structures for measurement.
More elaborate texture operators compare brightness differences over a range of distances. Plotting the maximum
difference in brightness between pixels in circular neighborhoods centered around each pixel as a function of the
diameter of the neighborhood produces a plot from which a local fractal dimension can be obtained. If this value is
used to generate a new image, it often allows thresholding images in which subtle differences in texture are
visually distinguishable. This is particularly suitable for images of surfaces, such as SEM images of fractures.
Human vision seems to have evolved the ability to distinguish surface roughness by the magnitude of the fractal
dimension produced by the roughness (and visible in the scattered light or secondary electron images).
Grey scale processing of the images is not the only way to segment images based on a local texture; sometimes it
is possible to use the thresholded binary image. Figure 23 shows a light micrograph of etched steel. The lamellar
pearlite structure contributes much of the strength of the material and the single phase bright ferrite regions give
the metal ductility. The volume fraction of the two structures is directly related to the carbon content of the steel,
but the size distribution of the pearlite regions (measured for instance by a mean lineal intercept) depends upon
prior heat treatment.
Figure 23. Metallographic microscope image of etched carbon steel (a), with the dark iron carbide particles
and platelets thresholded(b). Application of a closing (dilation followed by erosion) to merge together the
platelets, followed by an opening (erosion followed by dilation) to remove the isolated carbide particles)
produces a binary representation (c) of the pearlite (lamellar) regions. Superimposing the outline on the
original (d) shows the regions selected for measurement.
Thresholding the dark etched iron carbide (Figure 23b) does not identify the structural constituents of interest.
Applying dilation and erosion to the binary image first fills in the gaps between the lamellae and then erases the
isolated spots in the pearlite (Figure 23c). Superimposing the outlines of these regions back onto the original
image (Figure 23d) shows that the major structural components of the steel have been isolated and can be
measured in the usual ways. This is an example of morphological operations (Serra, 1982; Coster & Chermant,
1985; Dougherty & Astola, 1994) applied to binary images which add or remove pixels based on the presence of
specified patterns of neighboring pixels.
Combining multiple images
In many cases, it is necessary to acquire several different images of the same area to fully represent the
information needed to define the structure. A familiar example of this is X-ray maps acquired with the scanning
electron microscope. Each map shows the spatial distribution of a single element, but it may require several to
delineate the phase structures of interest. Figure 24 shows an example using a mineral specimen. The individual
X-ray maps for iron and silicon (Figures 24a and b) must be combined to define the phase of interest.
Since the original images are not grey scale, but have been recorded as a series of discrete pulses, they are quite
noisy in appearance. A smoothing operation using a Gaussian filter reduces the noise and produces smooth phase
boundaries that can be thresholded (Figures 24c and d). The size of the smoothing operator should ideally be set
to correspond to the range of the electrons in the sample, since this is the size of the region from which X-rays are
emitted and hence represents the distance by which a recorded pulse may vary from the actual location of the atom
that produced it. Boolean logic can then be employed to combine the two images, for example in Figure 24e to
define the regions that contain silicon and not iron. Once delineated, which in some cases may require many more
elemental images, the phases can be measured stereologically.
Figure 25 shows a case in which combination of several grey scale images is performed. The images show a pile
of small rocks. The situation is similar to many SEM images of particulates. Because of the complex surface
geometry of this sample, no single picture makes it possible to delineate the individual particles. Shadows are
different on the different sides of the particles, so using an edge-finding process does not suffice. Recording a
series of images with the camera and specimen in the same position but with the light source rotated to different
positions produces the series of images shown in Figure 25a. Subtracting these in pairs to produce the "north
minus south", "east minus west" , etc., images accentuates the shadows on corresponding sides of the pictures
(Figure 25b), but assures that each side of the particles is well illuminated in at least one pair. Combining these
pictures to keep the brightest value at each pixel location eliminates the shadows and makes the edges uniformly
bright around each particle (Figure 25c). Applying an edge-enhancement operator to this image delineates the
particle boundaries.
Figure 24. SEM X-ray maps of a mineral
specimen showing the spatial distribution of
iron (left) and silicon (right). Smoothing
operation using a Gaussian filter and
thresholding produces binary maps of the
areas with concentrations of the elements.
Boolean logic combines these to define the
regions that contain silicon and not iron.
Processing this image is actually not the major problem for characterization. There is no robust stereological model
that allows measurement for such a pile of rocks. When particulates are dispersed through a transparent volume
and viewed in projection, so that some particles are partially or entirely hidden by others, a simple first order
correction for the missing (invisible) particles can be made. Normally, the size distribution is shown as a
histogram with number of particles in each size class per unit volume of sample, where the volume is the area of
the image times the thickness of the viewed section. For each large particle, the hidden volume can be estimated as
the area of the particle times half the section thickness (because on the average each particle will be half way
through the section). Any smaller particles in this region would be missed, so the effect is to reduce the volume
for all smaller particle classes. This increases the number per unit volume value and shifts upwards the histogram
for smaller sizes. The process is repeated for each size class.
Figure 25. Macroscopic image of a pile of rocks: a) montage showing portions of the same image area
with the light source moved to north, east, south and west locations; b) montage showing image
differences between opposite pairs; c) montage showing the brightest pixel value at each location
from the difference images, and the result of applying a Sobel edge operator.
For a "pile of rocks" or other similar structures, the only suitable model is based on a "dead leaves" random
generation of an image. This is done by placing features taken from a known distribution of size and shape using a
random number generator, and gradually building up an image. This has been used for known shapes such as
spheres and fibers. Special attention to the orientation with which individual features are added to the
conglomerate is needed, because in real cases forces of gravity, surface tension, magnetic fields, etc. may or may
not be important.
The generated image is then compared to the real one, and parameters such as the histogram of intercept lengths or
the partial areas of features revealed on the surface determined and compared. If the simulation agrees with the
actual images, it offers some evidence that the structure may be the same as the model used for the simulation. But
since several quite different models may produce images that are very similar and difficult to distinguish
statistically without a great deal of measured data, this is not a particularly robust analytical method. It is usually
preferable to find other specimen preparation techniques, such as dispersal of the particulates over a surface so
they are not superimposed, or dispersal through a volume which can be sectioned.
Multiple images also arise in other situations, such as polarized light images from a petrographic microscope and
multiple wavelengths in fluorescence microscopy. It may also happen that the multiple images may all be derived
from a single original color or grey scale image by different processing, for example to isolate regions that are
bright and have a high texture.
Figure 26. Color fluorescence image showing two different stains (a), and only those the red-stained cells
that contain a green-stained nucleus (b) as selected using a feature-based AND operation.
Boolean logic used to combine multiple images is generally done on a pixel-by-pixel basis. This allows defining
pixels of interest depending on whether they are on or off in the various images using AND (both pixels are on),
OR (either pixel is on), Exclusive-OR (one but not both pixels are on) and NOT (changing a criterion from on to
off). These conditions can be combined in flexible ways, but do not address applications in which features in one
image plane are marked by indicators in a second. Figure 26 shows an example. Two different stains have been
used, one to mark cells and the other the chromatin in nuclei. Using the green nuclei as markers, the entire cells
containing them can be selected for measurement. This method is called a feature-based AND rather than a pixel-
based comparison (Russ, 1993). It keeps all connected pixels in a feature in one image if any of the pixels are
matched by ones in the second.
Summary
The kinds of image processing operations can be summarized as follows:
a) Ones that operate on a single image based on the brightness values of neighborhood pixels. This includes
arithmetic combinations such as multiplicative kernels as well as ones that rank the pixel values and keep the
maximum, minimum or median value. They are used for smoothing, sharpening, edge enhancement and texture
extraction.
b) Ones that operate in Fourier space to select specific frequencies and orientations in the image that correspond to
wanted or unwanted information and allow the latter to be efficiently removed. Measurements of regular
spacings can be made in the Fourier image more conveniently than in the spatial domain. There are also other
alternate spaces, such as Hough space, that are convenient for identifying lines, circles or other specific shapes
and alignments of features.
c) Ones that operate on two images to combine the grey scale pixel values. This includes arithmetic operations
(add, subtract, etc.) as well as comparisons (keep the brighter or darker value). They are used in leveling
contrast, removing unwanted signals and combining multiple views.
d) Ones that operate on single binary images based on the local pattern of neighboring pixels. These are usually
called morphological operators (erosion, dilation, etc.). They add or remove pixels from features to smooth
shapes, fill in gaps, etc. They can also be used to extract basic information about feature shape (outlines or
skeleton)
e) Ones that are based on the Euclidean distance map, in which each pixel within the features in a binary image is
given a grey scale value equal to its distance from the nearest background pixel. These allow segmentation of
touching convex features (or their rapid counting), and measuring of the distance of features from irregular
boundaries.
f) Ones that combine two binary images using Boolean logic. These allow flexible criteria to be used to combine
multiple representations of the same area, either at the pixel or feature level.
Many image analysis systems offer most of these capabilities. It remains a significant challenge for the user to
employ them to best effect. This generally means that the user must first understand what is in the image and how
she or he is able to visually recognize it. Depending on whether the discrimination is based on color, texture,
relationship to neighbors, etc., the choice of appropriate processing must be made. Few real problems are solved
by a single step operation; sequences of grey scale and binary image processes are often needed to isolate the
desired structures for measurement.
This demands a considerable effort by the user to understand the available tools and what they can be used for. In
most cases in which only a few measurements are to be made on complex images, it is more efficient to use the
computer-assisted manual stereology techniques discussed in the previous chapter. But when hundreds of
measurements are needed to permit statistical analysis, and the data are to be accumulated over a long period of
time so that human variability becomes a concern (for either one or multiple operators), the design of an
appropriate image processing method to delineate the structures of interest for measurement becomes important
and worthwhile.
Chapter 11 - Geometric Modeling
Stereology is at heart a geometric science, using the principles of geometric probability to estimate measures of
three-dimensional structures from measurements that can be accessed in lower dimensions using planes, lines and
points as probes. There are modern texts in geometrical probability (Kendall & Moran, 1986; Matheron, 1975;
Santalo, 1976). However, one of the first areas of study in this field (long predating the use of the name
"stereology") was the calculation of the probabilities of intersection of various probes with objects of specified
shape. This has roots back to the Buffon needle problem (18th century) and also involves Bertand's paradox, with
its consideration of what a random probe really entails.
Quite a bit of work during the middle part of this century dealt with the relationship between the size distribution
of objects in 3D with the size distribution of the linear or planar intercepts that a set of isotropic, uniform and
random probes would produce (see for example Wicksell, 1925; Cruz-Orive, 1976). These values are needed for
the unfolding of the size distribution of the intercepts to estimate the size distribution of the 3D objects. As
discussed elsewhere the trend in recent decades has been away from this approach (Gundersen, 1986) because of
the ill-conditioned nature of the mathematics used in unfolding (its sensitivity to small variations in the measured
size distribution producing much larger variations in the calculated one), and because it makes some flawed
assumptions about the nature of the specimen. Basically it assumes that the shape of the 3D objects is known (and
has some relatively simple geometric form) and that all of the objects are the same in shape. The most common
shape used for unfolding is a sphere, because of the mathematical simplicity that it yields. However, there are few
real samples in which all of the objects of interest are actually perfect spheres. Even small deviations from this
shape, especially deviations that are systematic with size, introduce substantial bias into the final results.
Nevertheless it is useful to understand how the process for determining the relationship between object shape and
the size distribution of intercepts works. It is unlikely that a working stereologist will need to develop such a set of
data individually (and sets of such data have been published for many geometrical shapes such as cylinders,
ellipsoids, and polyhedra). But the basic method helps to educate the mind into the relationship between three-
dimensional objects and the intercepts which planes and lines make with them, and this is an important step in
developing an understanding and even an intuition for visualizing the three dimensional objects that are revealed in
traditional two dimensional microscopy.
This chapter reviews the tools used for making these calculations and estimations. There are two avenues by
which to approach the determinations: integration of analytic expressions, and random sampling. The former will
initially be more familiar, as an outgrowth of normal analytic geometry and calculus, but the latter is ultimately be
more powerful and useful for dealing with many of the problems encountered with real three dimensional features.
Methods: Analytic and Sampling
As an introductory example, consider the problem of determining the area of a circle of unit radius. Figure 1
shows the familiar analytical approach. The circle is broken into strips of width dx, whose length if expressed as a
function of x is
L 2 1 x
2
(11.1
Then the area of the circle is determined by integration, giving
Chapter 11 - Geometric Modeling Page # 225
Area 2 1 x
2
1
+1
dx
(11.2
which can be directly integrated to give
2
sin
1
(1)
2
sin
1
(1)
2

'

;

2
(
2
)
(11.3
Figure 1. Drawing illustrating the integration of the area of a circle
The other approach to this problem is to draw a circle of unit radius inside a square (of side 2, and area 4). Hang
the paper on a tree, back off some suitable distance, and shoot it with a shotgun. Figure 2 shows an example of
typical results that you might observe. Now count the holes that are inside the circle, and those that are inside the
square. The fraction that are inside the circle divided by the total number of holes should be /4.
Figure 2. The "shotgun" method of measuring the circle's area.
This is a sampling method. It carries the implicit assumption of a perfectly random shotgun, in the sense that the
probability of holes (points) appearing within any particular unit area of the target (plane) is equal. This is in
agreement with our whole idea of randomness. And, of course, the precision of the result depends on counting
statistics: we will have to shoot quite a lot of holes in the target and count them to arrive at a good answer.
This is easier to do with a computer simulation than a real shotgun. Most computer systems incorporate some type
of random number generator, usually a complex software routine that multiplies, adds and divides numbers to
obtain difference values that mimic true random numbers. It is not our purpose here to prescribe tests of how good
these "pseudo random number" generators are. (Some are quite good, producing millions or hundreds of millions
of values that do not repeat and pass elaborate tests for uniformity and unpredictability; others are barely good
enough for their intended purpose, usually controlling the motion of alien space invaders on the computer screen.)
Assuming the availability of a random number function, and some high-level computer language in which to
work, a program to shoot the holes and count them might look like the one in Listing 1 (see Appendix). This
program (and the others in the Appendix) can be translated quite straightforwardly into any dialect of Basic,
Fortran, Pascal, C, etc. It uses a function RND that returns a real number value in the range 0 <= value < 1 with a
uniform random distribution. This means that we are in effect looking not at the entire target, but at one-quarter of
it (the center is at X=0,Y=0). Each generated point is checked, and counted if it is inside the circle. The program
prints out the area estimate periodically, in the example every thousand points, and continues until stopped. A
typical run of the program produced the example data shown in Figure 3. Other runs, using a different set of
random numbers, would be different, at least in the early stages. But given enough trials, the value approaches the
correct one.
Figure 3. Typical result of running the program to measure circle area.
Because of the use of random numbers to measure probability, this sampling approach is called the Monte-Carlo
method (after the famous gambling casino). It finds many applications in sampling of processes where the
individual rules and physical principles are well known, but the overall process is complex and the steps are not
easily combined in analytical expressions that can be summed or integrated, such as scattering or electrons,
photons or alpha-particles.
In the example here, it is easy to see that the method does eventually approach the "right" answer, but that it takes
a lot more work than the straightforward integration of the circle. However, given some other much more complex
shape bounded by lines that could individually be described by relationships in X and Y, but for which the integral
cannot so readily be evaluated, the Monte-Carlo approach could be a useful alternative way to find the area.
Measuring the area of the British Isles for example would simply require hanging up the map and blasting away
with our shotgun. We will find this approach especially useful for three-dimensional objects.
If the sampling pattern were not random, but instead the program had two loops that varied X and Y through a
regular pattern, for instance
FOR Y = 0 TO 1 STEP 0.02
FOR X = 0 TO 1 STEP 0.02
. . .
then 2500 values would be sampled, with a good result. This systematic sampling is in effect a numerical
integration, if the number of steps is large enough (the step size is small enough). But with systematic sampling,
you have the same problems in establishing the limits as for analytic integration. Furthermore, there is no valid
answer at all until you have completed the program, and no way to improve the answer by running for a longer
time (if you repeat it, you will get exactly the same answer). The random sampling method produces a reasonable
answer (whose accuracy can be estimated statistically) in a short time, and it will continue to improve with running
time.
Sphere Intercepts
The frequency distribution of the lengths of linear intercepts passing through a sphere is also a problem that can be
solved analytically, as indicated in Figure 4. Figure 4a shows many lines passing vertically through the sphere.
Because of its symmetry, it is not necessary to rotate the sphere or consider many directions. If the vertical lines
correspond to shots fired at the target (the flat surface beneath it) then the sampling condition is satisfied and we
need only to determine the intercept length of each line.
The length of each intercept line passing vertically through a sphere is uniquely related to its distance from the
center (Figure 4b). A cross section (Figure 4c) shows that this is 2(1r
2
)
1/2
), and the number of lines at each
radius is proportional to the area of the circular strip shown on the plane, which is 2 r dr. The result is a
frequency curve for the intercept length L whose shape is just the straight line that was shown before.
d
2
L dL
(11.4
The Monte-Carlo sampling approach to this is similar to that used before for the area of a circle. Because of the
symmetry of the sphere, we can work just in one quadrant. X,Y values are generated by the RND function, and
for each line that hits the sphere, the length is calculated. These lengths are summed in an array of 20 counters that
become the frequency vs. length histogram. The program could be written as shown in Listing 2 in the Appendix.
Figure 4 (continued).
Figure 4. Intercept lengths in a sphere (a). The number of lines with the same length is proportional to the
area of a circular segment on the plane (b) and the length is a function of the distance of the line from the
center (c).
As before, a fairly large number of trials are required to obtain a good estimate of the shape of the distribution
curve. Figure 5 shows three results from typical runs of the program, with 100, 1000 and 10,000 trials. The latter
is a fairly good fit to the expected straight line. The uncertainty in each bin is directly predictable from the number
of counts, as described in the chapter on statistics.
Figure 5. Probability curves for intercept lengths in a sphere generated by a Monte-Carlo program.
Intercept Lengths in Other Bodies
It is possible to perform both the integration and Monte-Carlo operations very simply for a sphere, because of the
high degree of symmetry. For other shapes this becomes more difficult. For example, the cube shown in Figure 6
has a radically different distribution of intercept lengths than the sphere. It is very unlikely to find a short intercept
for the sphere (as shown in the frequency histogram) because the line must pass very close to the edge of the
sphere. But for a cube, the edges and corners provide numerous opportunities for short intercept lengths.
However, if we restricted the test lines to ones vertical in the drawing, no short intercepts would be observed (nor
would any long ones; all vertical intercepts would have exactly the same length). Unlike the case for the sphere, it
is necessary to consider all possible orientations of the lines with respect to the figure. This will require more
random numbers.
Figure 6. A cube with illustrative intercept lines.
It is quite straightforward to determine a histogram of frequency versus intercept length in a circle (2-dimensional)
or sphere (3-dimensional) using the Monte-Carlo approach, even though both these problems can also be solved
analytically. For other shapes, the Monte-Carlo method offers many practical advantages, including that it is not
difficult to program in even a small computer. However, care is required so that the intersecting lines are properly
randomized in space with respect to the feature, and have uniform probability of passing through all regions and in
all directions.
In two dimensions, consider the case of determining intercept lines in a square. The square may be specified as
having a unit side length and corners at (0.5, 0.5). Consider the following ways of specifying lines, and
whether they will meet the requirements for random sampling (all of the random numbers indicated by RND
below are considered to vary from 0 to 1, with uniform probability density, as produced by most computer
subroutines that generate pseudo-random numbers):
1) generate one number Y = RND 0.5, and use it to locate a horizontal line across the square. This will
uniformly sample space (if the square is subdivided into a checkerboard of smaller squares, and the number of
lines passing through each is counted, the numbers will be the same within counting statistics). However, all of
the intercept lengths through the square will have length exactly 1.0 because the directions are not properly
randomized.
2) generate one number THETA = RND, and use it to orient a line passing though the origin (center of the
square). This will uniformly sample orientations, but not positions. The counts in the sampling squares near the
center will be much higher than those near the periphery. Short intercept lengths will not be generated, and this
will consequently not produce a true frequency histogram.
3) generate three random numbers: X = RND 0.5, Y = RND 0.5, and THETA = RND. The line is defined
as passing through the point X,Y with slope THETA. It is less obvious that this also produces biased data, but
it, too, favors the sampling squares near the center at the expense of those near the periphery and produces a
biased set of intercept lengths. So do other combinations such as using four random numbers for X1,Y1 and
X2,Y2 (two points to define the line).
To reveal the bias in these (and other) possible methods, it is instructive to write a small program to perform the
necessary steps. This is strongly recommended to the practitioner, as the 2-dimensional geometry and simple
shape of the square make things much easier than some of the 3-dimensional problems that may be encountered.
Set up an array to count squares (e.g., 10x10) through which the lines pass. Then use various methods to
generate the lines, sum the counts, and also construct a histogram of intercept lengths. It is also useful to keep
track of the histogram of line lengths within the circumscribed unit circle around the square, since the shape of that
distribution is known and departures will reflect some fault in the randomness of the line generation routine.
When this is done for the methods described above, arrays like those shown in Figures 7 and 8 indicate improper
sampling. Both of these methods, and the others mentioned, also produce quite wrong histograms of intercept
length for both the cube and sphere.
112 110 122 143 147 163 121 114 97 103
123 170 163 181 195 175 185 153 138 124
134 191 228 242 216 220 188 189 176 157
159 181 213 259 260 255 255 247 175 130
147 172 201 253 285 287 273 254 200 166
156 175 211 259 288 305 286 258 224 151
139 183 230 252 283 265 271 270 209 172
127 165 230 252 261 230 244 193 189 160
123 158 166 190 209 207 208 179 141 125
115 126 118 127 145 159 160 132 124 118
Figure 7. Array of counts for 10x10 grid using four random numbers to generate X
1
= RND 0.5,Y
1
=
RND 0.5 and X
2
= RND 0.5,Y
2
= RND 0.5 points to define the line. Note the center weighting.
The graph shows the counts as an isometric display.
177 109 59 48 39 37 37 50 63 185
197 184 173 167 142 132 138 152 161 168
235 232 216 203 213 206 239 279 297 241
249 244 282 367 312 301 333 302 266 240
257 280 318 383 423 419 341 288 260 225
220 250 298 353 422 458 406 313 240 239
196 233 280 322 360 356 361 342 274 246
197 234 225 245 222 245 399 269 242 222
201 233 181 141 128 134 145 209 196 218
181 92 51 37 35 33 38 50 101 197
Figure 8. Array of counts for 10x10 grid using two random numbers to generate points along left and
right edges of square. In addition to center weighting note the sparse counts along top and bottom edges.
The graph shows the counts as an isometric display.
A proper method for generating randomized lines is:
Generate R=0.5 RND and THETA = RND. This defines a point within the unit circle, and a vector from the
origin to that point. Pass the line for intercept measurement through the point, perpendicular to the line. (Note
that generating an intercept line from two points on the circumscribed circle using two angles THETA = RND
does not produce random lines, although for a sphere it does.)
An equivalent way to consider the problem is to imagine that instead of the square being fixed, and the lines
oriented at all angles, the square is rotated inside the circle (requiring one random number to specify the orientation
angle). Then the lines can all be parallel, just as for the earlier method of obtaining intercepts through the circle.
This also requires one random number (the position along the axis). With these methods, or others which are
equivalent once rotation of coordinates is carried out, proper sampling of the grids is achieved.
The result is a histogram of intercept lengths in the square as shown in Figure 9. This shows a flat shelf for short
lengths, where the line passes through two adjacent sides of the square. The peak corresponds to the length of the
square's edge, and the probability then drops off rapidly to the maximum intercept (the diagonal, which is equal to
the circle diameter).
The program used to generate the data for the histogram is shown in Listing 3 in the Appendix (it uses the first of
the randomizing methods described above). In this program, the array CT is defined (to build a 20 point histogram
in this example). DG is the radius of the circumscribed circle, or half the square's diagonal; P2 is 2. The random
function is used to generate a point, from which the slope (M) and intercept (B) of the line through this point
perpendicular to the vector from the origin is calculated (the equation of the line is y = Mx+B). The intersection of
this point with the left side of the square, or if it does not pass through the side, the intersection with the top or
bottom is determined as one end of the line, and the process repeated for the other end. The line length is obtained
from the Pythagorean theorem, and for lines that intersect the square produces an integer from the ratio of this
length to the circle diameter which is used to build the histogram.
Figure 9. Histogram of intercept lengths in a square, generated by Monte-Carlo program.
Intercept lengths in three dimensions
Progressing to three dimensions, things become more complicated, and there are many more ways to bias the
sampling. An extension of the methods described above for the circle and square can be used for a sphere and
cube, as follows (refer to Figure 10 for the nomenclature used for a spherical coordinate system).
Figure 10. Spherical coordinates as described in the text.
1) Generate a random vector from the origin. This requires a radius R which is just the circle radius times a
random number, and two angles. Using the nomenclature of the figure above, the angle can be generated as
2RND, but the angle cannot be just (/2)RND. If this were done, the vectors that pointed nearly vertically
would be much denser in space than those that were nearly horizontal, because for each vertical angle there
would be the same number of vectors, and the circumference of the "latitude" circles decreases toward the pole.
The proper uniform sampling of orientations requires that be generated as the angle whose sine is a random
number from 0 to 1 (producing angles from 0 to /2 as desired). Through the point at the end of the vector
defined by these two angles and radius, pass a plane perpendicular to the vector. Then within this plane,
generate a random line by the method described above (angle and radius from the initial point to define another
point, and a line through that point perpendicular to the vector from the initial point). This is less cumbersome
than it sounds if proper use of matrix arithmetic is used to deal with the vectors.
2) Locate two random points on the circumscribed sphere. As in the method above, the points are defined by two
angles, and , which are generated as = 2 RND, = arc sin (RND). Connect these points with a line.
As before, alternate methods where the cube rotates while the line orientation stays fixed in space can be used
instead (but the same method for determining the tilt angle is required). Conceptually it may be helpful to visualize
the process as one of embedding the cube inside a sphere which is then rotated while the intersection lines pass
through it vertically, as shown in Figure 11. Once the line has been determined, it is straightforward to find the
intersections with the cube faces and obtain the intercept length. In the program shown as Listing 4 in the
Appendix, this is done by setting up a matrix equation and solving it. Simpler methods can be used for the cube,
but this more general approach is desirable when we next turn our attention to less easy shapes.
Figure 11. Linear intercepts in a cube can be determined by embedding the cube in a sphere which is then
oriented randomly and vertical lines passed through it.
This program dimensions an array for the histogram and line and defines the equations for each of the six faces on
the cube (equations such as 1X+0Y+0Z=0.5). Then a random point on the sphere is generated. For convenience,
the elevation angle E used here is the complement of the angle F discussed above, and the complicated arctan
function is used to get the arc sine, which many languages do not provide. The point X1,Y1,Z1 (and the second
point X2,Y2,Z2 ) define the intersections of the random line on the sphere.
Coefficients in the A matrix define this line. Each face is checked for intersections with the line, by placing the
face coordinates into the matrix. PC is a counter used to find two intersections of the line with faces. The
determinant of the matrix (if zero, skip to another face, since the current one is exactly parallel to the line)
calculates the coordinates (X,Y,Z) of the intersection of the line with the face plane, which are checked to see if
the intersection lies within the face of the cube. For the first such intersection, the coordinates are saved and the
counter PC incremented. When the second point is found, the distance between them (the intercept length) is
calculated and converted it to an integer for the histogram address, and one count added. The process repeats for
the selected number of intersection lines. Some output of the data as a graph is needed to make the results
accessible.
Figure 12. Linear intercepts in other solids: a)
tetrahedron; b) many-sided polyhedron; c)
cylinder; d) torus; e) arbitrary non-convex
shape (a banana).
Several additional features can be added to this type of program to serve as useful checks on whether gross errors
are present in the various parts of the program. First, the sphere intercept length can also be used to build a
histogram (this is just the distance between the points X1,Y1,Z1 and X2,Y2,Z2) which should agree with the
expected frequency histogram for a sphere. Also, by summing the lengths of all the lines in both the sphere and
cube, the volume fraction V
V
and the surface area per unit volume S
V
of the cube in the sphere can be obtained
and compared to the known correct values. The mean intercept length in the feature can also be obtained, which
should equal 4 V/S for the cube. It is also relatively simple to add a counter for each face of the cube, and compare
the number of intersections of lines with each face (if random orientations are correctly used, these values should
be the same within counting variability).
Before looking at the results from this program, It is worth pointing out that it may be relatively easily extended to
other shapes, even non-regular or concave ones. As shown in Figure 12, for polyhedra the smaller the number of
faces the easier it is to produce short intercepts where the lines pass through adjacent faces. The number of faces
will vary, and so some of the dimensions will change, and it may be worthwhile to directly compute the equations
of each face plane from vertex points. The most significant change is in the test to see if the intersection point of
the line with each face plane lies within the face. For the general polyhedron, the faces are polygons. The simplest
way to proceed is to divide the face into triangles formed by two adjacent vertices and the intersection point, and
sum the triangle areas. If this value exceeds the known area of the face, then the point lies outside the face
(provision for finite numerical accuracy within the computer must be made in the equality test). Many curved
surfaces can also be described by analytical geometry, but complex objects (e.g. those defined by splines) require
more difficult techniques to locate the intersection points. For convex objects some lines will create more than one
intercept.
Figure 13. Frequency histograms for intercept lengths through various regular polyhedra,
compared to that for a sphere, generated by Monte-Carlo program using 50 intervals
for length, and about 40,000 intercept lines.
Applying this program to a series of regular polyhedra produces the results shown in Figure 13. The results are
interesting in several respects. For instance, note that whereas for the sphere, the most likely intercept length is the
maximum (equal to the sphere diameter), large values are quite unlikely for the polyhedra because they requires a
perfect vertex-to-vertex alignment (and is impossible for the tetrahedron because even this distance is less than the
sphere diameter). Also, the peaks present in the frequency histogram correspond to the distance between parallel
faces (again, there are none for the tetrahedron), while the sloping but linear shelves correspond to intersections
through adjacent faces.
The mean intercept lengths in the various solids are obtained from the distributions. They are listed below as
fractions of the diameter of the circumscribed sphere.
Solid Mean Intercept
Tetrahedron 0.232
Cube 0.393
Octahedron 0.399
Icosahedron 0.538
Sphere 0.667
Intersections of planes with objects
Not all distribution curves are for intercept lengths, of course. It is also possible to model using geometric
probability (or to measure on real samples) the areas of intersection profiles cut by planar sections through any
shape feature. As usual, this is easiest for a sphere. Figure 14a shows that any intersection of a plane with a
sphere produces a circle. Because of the symmetry, rotation is not needed and a series of parallel planes (Figure
14b) can generate the family of circles whose radii are calculable from the position of the plane, analogous to the
case for the line intercept.
a b
Figure 14. Intersection of a plane with a sphere in any orientation produces a circle (a).
A family of parallel planes (b) produces a distribution of circle sizes.
For a cube, the intersection can have 3, 4, 5 or 6 sides (Figure 15). It is evident that the cube's corners produce a
large number of small area intersections, and that there are ways to cut areas quite a bit larger than the most
probable slice (which is nearly equal to the area of a square face of the cube). Figure 16 shows a plot (Hull &
Houk, 1958) of the area of profiles cut through spheres and cubes. Generating similar distributions for planar
intersections with other shapes is a straightforward if rather lengthy exercise in geometric probability. It also
reveals that humans are not good intuitive judges of intersections of planes with solids. Figure 17 shows a torus,
familiar to many researchers in the form of a bagel. A few of the less obvious cuts which produce sections
unexpected to casual observers are shown. Considering the difficulty in predicting the shape of section cuts from
known objects, it should not be surprising that the reverse process of imagining the unknown 3D object that may
have given rise to observed planar sections easily leads to errors.
a b
c d
Figure 15. Planes intersection a cube can produce intersections with: a) 3, b) 4; c) 5; d) 6 sides.
For other shapes with even less symmetry, the analytical approach becomes completely impractical. Even Monte-
Carlo approaches are time-consuming, because of the problems of calculating the intersection lines and points, and
it may take a very large number of trials to get enough counting statistics in the low-probability regions of the
frequency histogram to adequately define it (particularly when the shape of the curve is to be used to deconvolute
distributions from many different feature sizes).
0.000
0.025
0.050
0.075
0.100
0.125
F
r
e
q
u
e
n
c
y
0
.
0
0
0
.
2
5
0
.
5
0
0
.
7
5
1
.
0
0
Area/Max Area
Cube
Sphere
Figure 16. Intercept area distributions for a sphere and cube.
Figure 17. Some of the less familiar planar cuts through a bagel (torus), which can produce one or two
convex or concave intersections.
Bertrand's Paradox
There is a problem which has been mentioned briefly in the previous examples, but which often becomes
important when dealing with ways to orient complex shapes using random numbers. It is easy to bias (or, in
mathematical terminology, to constrain) the orientations so that the random numbers do not produce a random
sampling of the space inside the object. In a trivial example, if we only rotated the cube around one axis, we
would not see all intercept lengths; but if we rotate it uniformly around all three space angles, the proper results are
obtained.
A classic example of the problem of (im)proper sampling is stated in a famous paradox presented by Bertrand in
1899. The problem to be solved is stated as follows: What is the probability that a random line passing through a
circle will have an intercept length greater than the side of the inscribed equilateral triangle in the circle?
Figure 18. Illustrations for Bertrand's Paradox.
Using the three drawings in Figure 18 (and working from left to right), here are three arguments that can be
presented to quickly arrive at an answer.
1. Since the circle has perfect symmetry, and any point on the periphery is the same as any other, we will consider
without loss of generality, lines that enter the circle at one corner of the triangle, but at any angle. Since the total
range of the angle theta is from 0 to 180 degrees, and since the triangle subtends an angle of 60 degrees (and
any line lying within the shaded region clearly has an intercept length greater than the length of the side of the
triangle), the probability that the intercept line length is greater than the side of the triangle is just 60/180, or
one-third.
2. Since each line passing through the circle produces a line segment which must have a center point, which will
lie within the circle, we can reduce the problem to determining how many of the line segments have midpoints
that lie within the shaded circle inscribed in the triangle (these lines will have lengths longer than the side of the
triangle, while any line whose midpoint is outside the circle will have a shorter length). Since the area of the
small circle is just one-fourth the area of the large one, the probability that the intercept line length is greater
than the side of the triangle is one-fourth.
3. Since the circle is symmetric, we lose no generality in considering only vertical lines. Any vertical line which
passes through the circle but passes outside of the smaller circle inscribed in the triangle will have a length
shorter than the side of the triangle, so the probability that the intercept line length is greater than the side of the
triangle is the ratio of diameters of the circles, or one-half.
These three arguments are all, at least on the surface, plausible. But they produce three different answers. Perhaps
we should choose one-third, since it is in the middle and hence "safer" than the two extreme answers!
No, the correct answer is one-half. The other two arguments are invalid because they impose constraints on the
random lines. Lines spaced at equal angle steps all passing through one point on the circle's periphery do not
uniformly sample it, but are closer together at small and large angles. This under-represents the number of lines
that lie within the shaded triangle, and hence produces too low an answer. Likewise the lines whose midpoints lie
within the shaded inner circle would be correct if only one angular orientation were considered (it would then be
equivalent to the third argument), but when all angles are considered it undercounts the lines that pass outside the
shaded circle. It can be very tricky to find subtle forms of this kind of bias. They may arise in either the analytical
integration or the random sampling methods.
The method used to generate random orientation of lines in space for the Monte-Carlo routines described above
must avoid this problem. The trick is to use = 2 RND and = arc sin (RND), using the terminology of Figure
10. This avoids clustering many vectors around the Z axis, and gives uniform sampling.
The Buffon Needle Problem
A good example of the role of angles in geometrical probability is the classical problem known as Buffon's Needle
(Buffon, 1777): If a needle of length L is dropped at random onto a grid of parallel lines with spacing S, what is
the probability that it crosses a line? This is another example of a problem easy enough to be solved by either
analytical means or random sampling. First, we will look at the procedure of integration.
Figure 19. Geometry for the Buffon needle problem
Figure 19 shows the situation. Two variables, y and theta, are involved; theta is the angle of the needle with
respect to the line direction, and y is the distance from the midpoint of the needle to a line. It is only necessary to
consider the angle range from 0 to /2, because of symmetry. At any angle, the needle subtends a distance of L
sin perpendicular to the lines, so if y is less than half this, there will be an intersection. Hence, the probability of
intersection is, as was stated before
L sin
S
d
0
d
0
L
S
cos
2

_
,
+ cos 0 ( )

1
]
1
2

_
,

2L
S
(11.5
A Monte-Carlo approach to solving this problem is shown as Listing 5 in the Appendix. L and S have been
arbitrarily set to 1, so the result for number / count should just be /2.
One particular sequence of running this program ten times, for 1000 trials each, gave a mean and standard
deviation of 1.5727 0.0254, which is quite respectable. There is a probably apocryphal story about an eastern
European mathematician who claimed that he dropped a needle 5 cm. long onto lines 6 cm. apart, and counted 226
crossing events in 355 trials. He used this to estimate , as 2 x 355 / 266 = 3.1415929 (correct through the first 6
decimal places).
We can evaluate the (im)probability of the truth in this story. Dropping the needle one more time, regardless of the
outcome, would have seriously degraded the "accuracy" of the result! This tale may serve as a caution to always
consider the statistical precision of results generated by a sampling method such as Monte-Carlo simulation, as
well as the statistics of measured data to which it is to be compared.
Appendix - Geomet ri c Model i ng
Listing 1: Random points in a unit square used to measure circle area.
Li sti ng 1
Integer: Increment = 1000;
Integer: Number = 0;
Integer: Count = 0;
Repeat
{ X = RND;
Y = RND;
IF (X*X + Y*Y) < 1
THEN Count = Count + 1;
Number = Number + 1;
IF Float(Number/Increment) = Integer Number/Increment)
THEN PRINT Number, Float(4*Count/Number);
}
Until Stopped;
Listing 2: Intercept lengths in a sphere.
Li sti ng 2
Integer Count[1..20] = 0;
Integer Number, Bin, i;
Float X, Y, rsquared, Icept;
Input ("Number of trials= ",Number);
for (i = 1; i <= Number; i++)
{ X = RND;
Y = RND;
rsquared = X*X + Y*Y;
if rsquared < 1 then
{ Icept = sqrt (1 - rsquared);
Bin = Integer (20*Icept) + 1;
Count[Bin] = Count[Bin] + 1;
}
}
for (Bin = 1; Bin <= 20; Bin++)
PRINT Bin, Count[Bin];
Listing 3: Intercept lengths in a square.
Li sti ng 3
Integer CT[20] = 0;
Integer N, NU, L;
Float M, B, TH, R, XL, YL, XR, YR;
Const DG = SQR(2)/2;
Const P2 = 8*ATN(1);
Input ("Number of Lines: ",NU);
for (N = 1; N <= NU; N++)
{ R = DG * RND; /* two random numbers */
TH = P2 * RND;
M = -1/tan(TH);
B = 0.5 + R * sin(TH) - M * (0.5 + R * cos(TH));
/* get the coordinates of the ends of the line */
XL = 0;
YL = B;
if ((YL>1) or (YL<0)) then
{ if (YL>1)
then YL=1
else YL=0;
XL=(YL-B)/M;
}
XR = 1;
YR = M + B;
if ((YR>1) or (YR<0)) then
{ if (YR>1)
then YR=1
else YR=0;
XR=(YR-B)/M
}
LEN = sqrt((XR-XL)*(XR-XL)+(YR-YL)*(YR-YL));
if (LEN>0) then
{ L = integer(0.5+10*LEN/DG);
CT[L] = CT[L]+1;
}
}
Listing 4: Intercept lengths in a cube.
Li sti ng 4
Float F[6,4] = 1,0,0,0.5
1,0,0,-0.5
0,1,0,0.5
0,1,0,-0.5
0,0,1,0.5
0,0,1,-0.5; // the six faces of the cube
Float A[3,4]; // used to calculate line/face intersection
Integer LX[50]; // for histogram
Const P2 = 8 * atan(1.0); // 2
Const R = sqrt(3.0)/2.0; // radius of circumscribed sphere
Input (Number of Lines:,NU);
for (n = 1; n <= NU; N++)
{ TH = P2 * RND;
E = -1 + 2 * RND;
E = atan (E/sqrt(1 - E*E));
X1 = R * cos(E) * cos(TH);
Y1 = R * cos(E) * sin(TH);
Z1 = R * sin(E); // one random point on the sphere
TH = P2 * RND;
E = -1 + 2 * RND;
E = atan (E/sqrt(1 - E*E));
X2 = R * cos(E) * cos(TH);
Y2 = R * cos(E) * sin(TH);
Z2 = R * sin(E); // second random point on sphere
A[1,1]=Y1*Z1-Y2*Z1;
A[1,2]=Z1*X2-Z2*X1;
A[1,3]=X1*Y2-X2*Y1;
A[1,4]=0;
A[2,1]=Y1*Z2-Y2*Z1;
A[2,2]=Z1*X2-Z2*X1+Z2-Z1;
A[2,3]=X1*Y2-X2*Y1+Y1-Y2;
A[2,4]=Y1*Z2-Y2*Z1; // define the line in A matrix
PC=0;
for (j=1; j<=6; j++) // check faces for intersection
{ for (k=1; k<=4; k++)
A[3,K]=F[J,K];
DE= A(1,1)*(A(2,2)*A(3,3)-A(2,3)*A(3,2))
+A(1,2)*(A(2,3)*A(3,1)-A(2,1)*A(3,3))
+A(1,3)*(A(2,1)*A(3,2)-A(2,2)*A(3,1));
if (DE<>0) then // zero means parallel to the face
{ X= (A(1,4)*(A(2,2)*A(3,3)-A(2,3)*A(3,2))
+A(1,2)*(A(2,3)*A(3,4)-A(2,4)*A(3,3))
+A(1,3)*(A(2,4)*A(3,2)-A(2,2)*A(3,4)))/DE;
Y= (A(1,1)*(A(2,4)*A(3,3)-A(2,3)*A(3,4))
+A(1,4)*(A(2,3)*A(3,4)-A(2,4)*A(3,3))
+A(1,3)*(A(2,1)*A(3,4)-A(2,4)*A(3,1)))/DE;
Z= (A(1,1)*(A(2,2)*A(3,4)-A(2,4)*A(3,2))
+A(1,2)*(A(2,4)*A(3,1)-A(2,1)*A(3,4))
+A(1,4)*(A(2,1)*A(3,2)-A(2,2)*A(3,1)))/DE;
// intersection point of line with face
if ((abs(X)<=0.5)
and (abs(Y)<=0.5)
and (abs(z)<=0.5))
then // see if intersection is inside cube
{ if (PC=0)
then PC=1;
X0=X;
Y0=Y;
Z0=Z;
}
else
{ LE=SQR((X-X0)*(X-X0)+(Y-Y0)*(Y-Y0)+
(Z-Z0)*(Z-Z0));
// distance between points = length
H=integer(25*LE/R);
LX[H]=LX[H]+1; // increment histogram
}
}// if de
} // for j
} // for n
Listing 5: The Buffon needle problem.
Li sti ng 5
Integer Number, j, Count;
Float Y, Theta, Vert;
Const HalfPi = 2 * Atan(1); // /2
Input ("How many trials: ",Number);
for (j = 1; j<=Number; j++)
{ Y= RND;
Theta = HalfPi * RND;
Vert = Sin (Theta) / 2;
if ((Y - Vert < 0) OR (Y + Vert > 1))
then Count++;
}
PRINT Number/Count
Chapter 12 - Unfolding Size Distributions
In the preceding chapter, methods were discussed for predicting the distribution of intercepts that a random probe
would make with objects in a three-dimensional space. This kind of modeling can be done using either line or
plane probes. With line probes the length of the intersection can be measured, and with plane probes the area as
well as the shape of the intersection can in principle be determined. This approach requires making several
assumptions: the size and shape of the objects being sampled must be known, and the probes must be isotropic,
uniform and random with respect to the objects.
In actual practice, the experiment is carried out in the opposite direction. A real specimen is probed with lines or
planes, and the data on intercept lengths, areas and perhaps shape information are recorded. Then the goal is to
calculate the (unknown) sizes of the objects that must have been present in order to produce those results. Note
that it is not possible to determine the size of any single object this way, from the intersection that the probe makes
with it. It is only in the statistical aggregate that a distribution of objects can be inferred.
And even then it is far from easy to do so. There are several aspects to the problem, which will be discussed
below. First, mathematically the inverse problem is ill-conditioned. The small statistical variation in measured data
(which arises because a limited number of measurements are made, even if they are an unbiased sample of the
specimen) grows to a much larger variation in the calculated answer because of the ways that these variations
propagate through the calculation.
Second, a critical assumption must be made for the method to be applied at all: we must assume that we know
what the shape of the objects is, and usually that they are either all of the same shape or have a very simple
distribution of shapes. The most popular shape assumption is that of a sphere, because its symmetry makes the
mathematics easy and also relaxes the requirements for isotropic sampling.
But in fact not very many structures are actually spheres, and even a small variation from the assumed shape can
introduce quite a large bias in the calculated results. To make matters worse, many real specimens contain objects
that have a wide variety of shapes, and the shape variation is often a function of size. This presents major
problems for unfolding techniques.
In spite of these difficulties, the unfolding approach was the mainstay of classical stereology for decades and is
still used in spite of its limitations in many situations. Certainly there are specimens for which a reasonable shape
assumption can be made - spheres for bubbles, cubes for some crystalline materials (e.g., tungsten carbide),
cylinders for fiber composites, and so on. And the use of computer-based measurement systems makes it possible
to collect enough data that the statistical variations are small, limiting the extent of the errors introduced by the
inverse solution method.
Linear intercepts in spheres.
The preceding chapter developed the length distribution of lines intersecting spheres. The frequency distribution
for a sphere of diameter D is simply a straight line, and when measured intercept lengths are plotted in a
conventional histogram form we would expect to see a result as shown in Figure 1. The presentation of data in
Chapter 12 - Unfolding Size Distributions Page # 246
histogram form is the most common way to deal with measurement data of all types. The bin width is typically
chosen to permit each bin to accumulate enough counts for good statistical precision, and enough bins to show the
shape of the distribution. In most cases from 10 to 15 bins are linearly spaced in size up to the largest value
obtained in measurement.
Figure 1. Frequency distribution of linear intercepts in a sphere of diameter D,
shown as a continuous plot and as a discrete histogram.
One consequence of the number of bins used is the ability to measure spheres (or other objects) whose sizes vary
over that range of dimensions. Ten or 15 bins allows determining spheres in that number of sizes. If the size range
of the spheres is much larger, say 50:1, and information on the smallest is actually needed, then a histogram with
at least that many bins is required. This greatly increases the number of measurements that must be made to obtain
reasonable counting precision.
When a mixture of sphere sizes is present, the measured distribution represents a summation of the contribution of
the different sizes each in proportion to the abundance of the corresponding size. As shown in Figure 2, the result
is that the histogram bins corresponding to smaller intercept lengths contain counts from intersections with many
different size spheres, while the largest bin contains counts only from the largest spheres.
This offers a straightforward graphical way to unfold the data (Lord & Willis, 1951). As shown in Figure 3,
knowing that the largest bin contains counts only from the largest spheres and that those spheres should also
generate a proportionate number of shorter intercepts allows subtracting the expected number of shorter intercepts
from each of the other (smaller) bins. This process can then be repeated for the next bin, using the number of
counts remaining, and so on.
Figure 2. Frequency distribution of linear intercepts from a mixture of sphere sizes,
shown as a continuous plot and a discrete histogram.
Figure 3. Unfolding the linear intercept distribution from a mixture of sphere sizes.
Lines project the histogram bin heights onto a vertical axis and the increments given
the relative proportion of each sphere size.
Notice that the number of counts in the smaller size bins (and hence the estimated number of smaller size spheres)
is obtained by successive subtractions. Subtraction is a particularly poor thing to do with counting data, since the
difference between two numbers has a standard deviation that is the sum of the deviations of the two numbers
whose difference was taken. This means that in effect the uncertainty of the estimated number of smaller size
spheres grows rapidly and depends strongly on the number of larger size spheres and the precision with which
they are determined.
Hence the need to obtain large numbers of interception counts, since the standard deviation of any counting data is
simply the square root of the number counted. In other words if you count one hundred events the one-sigma
uncertainty is 100 = 10 out of 100 or 10%, and to reduce this to 1% precision would require counting 10,000
events.
Plane intersections
Linear intercepts were primarily used when manual measurements were required to obtain data, because they
could be performed by drawing (random) lines on images of sections and measuring the length of the
intersections. With modern computer-based instruments, it is usually easier to measure the intersection areas made
by the sampling plane with the objects. It is very difficult to use plane probes that must be isotropic, uniform and
random (IUR) in a real object because once a single plane section has been taken, other planes that might intersect
that one cannot be generated. However, for specimens which are themselves IUR any plane is as good as another,
and examination of plane sections is a very common approach.
Figure 4. Different size circles are produced by sectioning of a sphere.
Every section through a sphere is a circle as shown schematically in Figure 4. Figure 5 shows an image of pores
in an enamel coating on steel. The pores may be expected to be spherical as they result from the evolution of gas
bubbles in the firing of the enamel, and all of the intersections are observed to be circular. The distribution of the
sizes of circles can be calculated analytically. The probability of obtaining a circle of radius dd (corresponding
to counts that will fall into one bin of a histogram) from a sphere of diameter D is equal to the vertical thickness of
a slice of the sphere with that diameter. The shape of the distribution is
P(d
circle
) = d / (D (D
2
- d
2
)
1/2
) (12.1
as shown in Figure 6.
Figure 5. Example of sectioning of spheres: bubbles in a fired enamel coating.
Figure 6. The distribution of planar intercepts through a sphere is proportional to the
vertical thickness z of a slice of the sphere covering a range of circle sizes r.
As for the linear intercept case, if there are several different sizes of spheres present in the sample a particular size
of intercept circle could result from the intersection of the plane with several different sphere sizes. The resulting
measurement histogram would show the superposition of data from the different sizes in proportion to their
relative abundance and to their size (it is more likely for the plane to strike a large sphere than a small one).
This distribution might be unfolded sequentially and graphically as shown before for the linear intercepts, since
the largest circles could only come from the largest spheres. Then as before, a corresponding number of intercepts
would be subtracted from each smaller bin and the process repeated until all sizes are accounted for. This is not a
very practical approach because of the tendency for statistical variation to concentrate in the smaller size ranges,
and instead a simultaneous solution is generally used.
For the case of spheres, this method was first described by Saltykov (1967) and has been subsequently refined by
many others, with an excellent summary paper by Cruz-Orive (1976) that includes tables of coefficients suitable
for general use. The method is simply to solve a set of simultaneous equations in which the independent variables
are the number of circles of various sizes (measured on the images and recorded as a histogram) using the
coefficient matrix, to obtain the numbers of spheres of each size present in the material.
The matrix is determined by calculating as discussed above, from geometric probability, the frequency distribution
for the number of circles in each size class due to three dimensional objects of each size. This can be written as
N
Ai
= '
ij
N
Vj
(12.2
where the subscript i ranges over all size classes of circles in the measured histogram and the subscript k covers
the corresponding sizes of spheres in the (unknown) distribution of three-dimensional objects. The equations are
more readily solved by using the inverse matrix , so that
N
Vj
= (1/)
ij
N
Ai
(12.3
where is the size of the bin used in the histogram. A typical matrix of coefficients is shown in Table 1.
Figure 7. Images of graphite nodules in cast iron.
As an example of the use of this method, Figure 7 shows images of nodular graphite in a cast iron. The sections
are all at least approximately circular and we will make the (typical) assumption that the nodules are spherical.
Figure 8a shows a distribution of circle sizes obtained from many fields of view covering the cross section of the
enamel. Converting these N
A
data using the coefficients in Table 1 produces the plot of sphere sizes shown in
Figure 8b.
Figure 8. The distribution of measured circle sizes from the cast iron in Figure 7 (left),
and the calculated distribution of sphere sizes that generated it (right).
Other shapes
The "sphere unfolding" method is easily programmed into a spreadsheet, and is often misused when the measured
objects are not truly spheres. Of course, similar matrices for ' can be computed for other shapes of objects, and
inverted to produce matrices. In fact, this has been done for an extensive set of convex shapes including
polyhedra, cylinders, etc. (see for example Wasn & Warren, 1990). It is well to remember, however, that the
results are only as useful as the assumption that the three-dimensional shape of the objects is known and is the
same for all objects present.
Another shape model that has been applied to the "nearly spherical" shapes that arise in biological materials in
particular (where membranes tend to produce smooth boundaries as opposed to polyhedra) is that of ellipsoids.
These may either be prolate (generated by revolving an ellipse around its major axis) or oblate (generated by
revolving the ellipse around its minor axis), and can be used to approximate a variety of convex shapes.
All of the sections made by a plane probe passing through an ellipsoid produce elliptical profiles (Figure 9). These
can be characterized by to dimensions, for instance the lengths of the major and minor axes. DeHoff (1962)
presented a method for unfolding the size distribution of the ellipsoids, which are assumed to have a constant
shape (axial ratio q) but varying size (major axis length), from the size distribution of the ellipses. This is a
modification of the sphere method in which
N
Vj
= (K(q)/)
ij
N
Ai
(12.4
is the size increment in the histogram, and the additional term K(q) is a shape dependent factor that depends on
the axial ratio of the ellipsoid q and whether the ellipsoids are prolate or oblate; this factor shown in Figure 10 for
both cases.
Figure 9. Ellipses formed by planar sections with prolate (blue) and oblate (green) ellipsoids.
a
b
Figure 10. K shape factors for prolate (a) and oblate (b) ellipsoids as a function
of the axial ratio q of the generating ellipse.
Note that it is necessary to decide independently whether the generating objects are oblate or prolate, since either
can produce the same elliptical profiles on the plane section image. If this is not known a priori, it can sometimes
be determined by examining the sections themselves. If the generating particles are prolate, then the diameters of
the most equiaxed sections will be close in size to the width of those with the highest aspect ratio, while if the
particles are oblate then the most equiaxed sections will be close to the length of those with the highest aspect ratio
(Figure 11). In either case, the axial ratio q is taken as the aspect ratio of the most elongated profiles and used to
obtain k from the graph.
Figure 11. Elliptical profiles from prolate and oblate ellipsoids.
It is in principle possible to extend the unfolding method of analysis to convex features that do not all have the
same shape. The profiles observed in the plane section can be measured to determine both a size and shape (e.g.
an area and eccentricity) and a two-dimensional histogram of counts constructed (Figure 12). From these data,
calculation of both the size and shape of the generating ellipsoids or other objects should be possible. The
calculation would be of the form
N
Vij
=
ijkl

N
Akl
(12.5
where the subscripts i,j cover the size and shape of the three-dimensional objects and k,l cover the size and shape
of the intersection profiles. Calculating the four-dimensional a' matrix and inverting it to obtain a is a
straightforward extension of the method shown before, although selecting the correct three-dimensional shape and
its variation is not trivial. The major difficulty with this approach is a statistical one. The number of counts
recorded in many of the bins in the two-dimensional N
A
histogram will be sparse, especially for the more extreme
sizes and shapes. These are important to the solution of the equations and propagate a substantial error into the
final result.
Shape information from the profiles can be useful even when a single shape of generating object is present. Ohser
& Nippe (1997) have shown that measuring the shapes of intersections with cubic particles (which are polygons
with from 3 to 6 sides) can significantly improve the determination of the size distribution of the generating cubes.
Figure 12. An example of a two-dimensional histogram of size and shape
of intersections made by a plane probe.
Simpler methods
The unfolding of linear intercepts is one of the simplest methods available, in use long before modern computers
made the solution of more complicated sets of equations easy. It can be performed using other distributions than
the linear one that applies to spheres, of course. If the actual shape of the objects present is known, the exact
distribution could be generated as discussed in the preceding chapter and used for the unfolding. A relatively
compact and general approach has been proposed for the case of other convex shapes by several investigators
(Weibel & Gomez, 1962; DeHoff, 1964). Using the usual nomenclature of N
V
(number per unit volume), N
A
(number per unit area), V
V
(volume fraction), P
L
(intersections per length of line) and P
P
(fraction of points
counted), the relationships proposed are
N
V
= (K/b) N
A
3/2
/ V
V

1/2
(12.6
and
N
V
= 2 N
A
P
L
/ P
P
(12.7
Note than both procedures require determining the total volume fraction of the particles (V
V
or P
P
) as well as the
number of objects per unit area of section plane, and one requires determining the mean linear intercept (the
inverse of P
L
). The parameter K takes into account the variation of the size of features from their mean, and is
often assumed to have a value between 1.0 and 1.1 for many real structures that constitute a single population
(e.g., cell organelles). Figure 13 shows K as a function of the coefficient of variation of the sizes of the objects.
Notice that the size distribution of the intercept lengths is not used here. Instead, the fact that the mean value of the
intercept length of a line probe through a sphere is just (/4) times the sphere diameter is used (and adjusted for
other shape objects). and are shape factors that depend upon an assumed shape for the three-dimensional
objects. They vary only slightly over a wide range of convex shapes, as indicated in Table 2 and Figure 14. This
method represents a very low cost way to estimate the number of objects present in three dimensions based on
straightforward measurements taken on plane sections. However, it is presently giving way to more exact and
unbiased techniques described under the topic of the "new" stereology.
Figure 13. The shape distribution parameter K as a function of the
relative standard deviation of the mean diameter.
Table 2. Shape coefficients for particle counting
Shape
Sphere 6.0 1.38
Prolate Ellipsoid 2:1 7.048 1.58
Oblate Ellipsoid 2:1 7.094 1.55
Cube 9.0 1.84
Octahedron 8.613 1.86
Icosahedron 6.912 1.55
Dodekahedron 6.221 1.55
Tetrakaidekahedron 7.115 1.55
Figure 14. The shape coefficient for ellipsoids and cylinders of varying axial ratio.
Lamellae
Linear probes are also useful for measurement of other types of structures. One of the advantages of linear probes
is the ease with which they can be produced with uniform, random and isotropic orientations (which as noted
above is very difficult for plane probes). Drawing random lines onto a plane section can be used to measure
intercept lengths, either manually or using a computer.
Figure 15. A plane section through a layered structure produces an apparent layer
thickness that is generally larger than the true perpendicular dimension.
Lamellar structures occur in many situations, including eutectic or other layered structures in materials,
sedimentary layers in rocks, membranes in tissue, and so on. Because these structures will not in most cases lie in
a single known orientation perpendicular to the section plane, the apparent spacing of the layers will be larger than
the true perpendicular spacing (Figure 15). There is no guarantee that on any particular section plane the true
spacing will be revealed.
Figure 16. Plots of the intercept length distribution for linear intercepts through a layer.
Random (IUR) linear intercepts through a layer of true thickness t produce a distribution of intercept lengths as
shown in Figure 16. If the data are replotted as a histogram of 1/, the inverse of the measured intercept length, a
much simpler distribution is obtained (Gundersen, 1978). The mean value of this triangular distribution is just
(2/3) (1/t) so the true layer thickness can be calculated as
t = 3/2 (1/
) (12.8
where
is the mean measured intercept length. Furthermore, if the layers are not all the same thickness but have
a range of thickness values, the distribution can be unfolded using the same graphical technique as shown above
for linear intercepts through spheres.
Chapter 13 - Anisotropy and Gradients
Much of the discussion of stereological measurement in other chapters has emphasized the importance of
isotropic, uniform and random sampling of the structure. The techniques by which isotropic sampling can be
accomplished using vertical sections and cycloids, for example, can be applied even to materials that are not
themselves isotropic. In fact, there are very few real samples for which isotropy or uniformity can be assumed.
Biological specimens usually have complex directional relationships between local structure and the organism,
geological strata preserve orientation information even when tilted, folded or faulted, plants know which way
gravity points and where the sun shines, and materials bear the marks of their solidification and processing
history, and even (except for a few precious shuttle-grown crystals) gravity.
One of the important ways to study such materials is to quantify the non-uniformity, preferred orientation,
clustering and gradients that may be present. Non-uniformity means the variation in measures of structure such as
volume fraction, specific surface area, and particle size, shape or spacing, with position. Anisotropy refers to
variations with respect to orientation. There may also be more complex relationships in which the anisotropy
varies with position, and so forth. Usually a very specific experimental design based on considerable a priori
knowledge of the structure is needed to measure and to describe such combinations of variation.
Grain structures in rolled metals
There are relatively straightforward techniques that can be used to measure the basic parameters that describe the
individual variations. For example, a common measure of preferred orientation that has been applied to metal
structures produced by rolling (in which the metal is reduced in thickness and elongated in the rolling direction) is
to examine a vertical surface, and to measure the mean intercept length as a function of orientation. Overlaying a
template of radial lines on the structure and manual counting of intersections of the lines with grain boundaries
gives the basic P
L
data, which can then be plotted as a rose plot to show the degree of preferred orientation
graphically. The ratio of maximum intercept length (in the rolling direction) to minimum intercept length (in the
vertical direction) is simply taken from the inverse of P
L
.
Figure 1 shows plots of P
L
obtained by this method. For a set of parallel lines, the distribution of lengths is
simply a cosine function (Figure 1a). For a square grid of lines, the distribution becomes more equiaxed but is still
not isotropic (Figure 1b). For a typical real structure with preferred orientation, the distribution may be more
complex (Figure 1c) but is always rotationally symmetric. The degree of anisotropy may be measured by the
departure from a perfect circle.
Of course, if the grain boundary structure is well delineated, manual counting may not be required. Thresholding
the image to obtain the grains, combining that with the line templates to produce a series of line segments, and
then measuring the length of the lines as a function of angle is one automatic method that can be used to determine
the same information. This use of templates and Boolean combination with binary (thresholded) images has been
discussed in several other chapters as a substitute for purely manual techniques. Figure 2 illustrates the procedure:
the original light micrograph (Figure 2a) of the structure (a low carbon steel whose thickness has been reduced
50% by cold rolling) is processed in the computer so that the grains and grain boundaries can be thresholded
(Figure 2b). The image of the grains is then ANDed with a set of radial lines to produce line segments (Figure 2c).
Chapter 13 - Anisotropy and Gradients Page # 260
Plotting the average length of the lines as a function of orientation (Figure 2d) reveals the preferred orientation.
Either the ratio of maximum to minimum dimension of the plot, or the deviation from a perfect isotropic circle, can
be used to quantify the degree of anisotropy.
a b
c
Figure 1. Rose plots of P
L
as a function of
direction for a) a set of horizontal parallel
lines; b) a grid of orthogonal lines; c) a typical
anisotropic structure.
For more complicated structures such as polymer composites containing many phases this automatic delineation is
difficult to achieve and so manual recognition and counting may be more efficient. Particularly for biological
specimens complex image processing is needed to isolate and define structures of interest, and so the manual
methods are often preferred.
a b
c d
Figure 2. Measurement using a template of lines: a) original micrograph of 50% cold rold steel;
b) processed image thresholded to delineate grains; c) grains ANDed with a set of uniform radial lines;
d) rose plot of the average intercept line length (reciprocal of P
L
) as a function of angle.
As indicated in Figures 3 and 4, this approach of measuring intercept length or P
L
as a function of direction does
not tell the whole story. The grains are also squashed in the lateral direction by rolling and the vertical section that
lies in the rolling direction does not reveal this. It is unusual to sample the three orthogonal surfaces for
measurement, but necessary if the full information on anisotropy is desired. Measurements in three orthogonal
directions using linear probes and the simple determination of intercept length can still be used to calculate a
measure of the degree of anisotropy.
Figure 3. Micrographs taken on three orthogonal planes in a rolled steel, showing the anisotropy of the grains.

Figure 4. Longitudinal and transverse sections through muscle fiber, showing the anisotropy of the structure.
At least two planes are necessary because no single plane, and sometimes not even two planes, can distinguish
between the possible kinds of anisotropy. Figure 5 shows schematic examples of equiaxed, needle-like and plate-
like structures. Each of the structures share some identical faces. In the relatively straightforward cases of rolled or
drawn metals and fiber or laminar composites it is possible a priori to judge which directions are useful as natural
axes to orient the data. In other applications such as biological tissue this is much more difficult and the axes may
change from place to place or even from specimen to specimen.
a b
c
Figure 5. Examples of some simple kinds of
preferred orientation: a) equiaxed; b) needles;
c) plates.
It is possible to learn much from measurements made in two directions, parallel and perpendicular to the axis of
preferred orientation, if the preferred orientation in three dimensions is already known either from more complete
studies and multiple sections, or from an understanding of the process by which the specimens are created. Even
in a single plane, human vision is quite efficient at determining a principal orientation direction so that two
measurements (in that direction and in the perpendicular direction) can be made.
In the chapter on modeling, the Buffon needle problem was used as an example of geometric probability. The
results of that calculation are directly applicable here. The intercept line length per unit area for uniformly random
lines is L
A
= /2 P
L
if the measurement is made using test lines that are parallel to the orientation axis. If the test
lines are oriented perpendicular to the orientation axis the value of P
L
will be greater, and the value of L
A
(total
line length per unit area of image) is obtained by subtracting P
L
for the parallel lines. Combining these
relationships gives
L
A
= P
L
perpendicular
+ 0.571 P
L
parallel
(13.1
for the total line length per unit area. So by performing two counts of P
L
, one in the principal direction of
elongation and the other perpendicular to it, the total line length per unit area can be determined. The degree of
preferred orientation is then give by (Underwood, 1970)
12
=
P
L
perp
P
L
par
P
L
perp
+ 0.571 P
L
par
(13.2
The subscript 12 indicates that we are dealing with lines (1 dimensional) on planes (2 dimensional). The value of
is a dimensionless ratio, often expressed as a percentage.
To determine the preferred orientation of lines is space (e.g., dislocations in metal, fibers in a composite) a similar
technique can be applied. Two perpendicular planes are examined and the number of points where the three-
dimensional lines intersect the planes are counted. Then the degree of preferred orientation is
13
=
P
A
perp
P
A
par
P
A
perp
+ P
A
par
(13.3
For surfaces in space, the specific area of the surface S
V
is estimated by counting P
L
on sections that are parallel
and perpendicular to the orientation axis. For systems that are arrays of parallel, needle-like grains or fibers that
appear equiaxed on a plane perpendicular to the orientation axis the degree of orientation is given by
23
=
P
L
perp
P
L
par
P
L
perp
+ 0.273P
L
par
(13.4
Arrays of plate-like grains give rise to a more complicated situation, where there are three combinations of
directions and hence three directions in which to count P
L
(called perpendicular, parallel and transverse), and three
terms. This is equivalent to the generalized Buffon needle problem in three dimensions. The overall degree of
anisotropy can be estimated as the square root of the sum of squares of the three terms, in cases where a single
descriptive parameter is needed.
23a
=
P
L
perp
P
L
trans
P
L
perp
+ 0.429 P
L
par
+0.571 P
L
trans
23b
=
1.571 (P
L
trans
P
L
par
)
P
L
perp
+ 0.429 P
L
par
+0.571 P
L
trans
(13.5
23c
=
P
L
perp
1.571 P
L
par
+ 0.571 P
L
trans
P
L
perp
+ 0.429 P
L
par
+0.571 P
L
trans
Note that these terms include much less detail than the rose plot, as they compare intercept lengths in only two or
three directions (which are assumed to be the important directions for the structure). All of these coefficients can
be useful to describe the degree of preferred orientation for quantitative comparison in a given class of materials,
but they do not really "describe" the nature of the orientation and they depend upon finding the correct natural
coordinate system for the polished faces.
Boundary orientation
Computer-based image analysis has tools that make it possible to measure the orientation of boundary lines
directly. The derivative of brightness in the horizontal and vertical directions can be calculated for every pixel in
the image by using local neighborhood operators. For example, multiplying the adjacent pixels in a 3x3
neighborhood by the integers
1 0 -1
2 0 -2
1 0 -1
produces the derivative in the horizontal direction, and a similar set of numbers rotated by 90 degrees produces the
vertical derivative. If these are then combined as
Pixel =
255

arctan
B
y
B
x

(13.6
a new image is produced in which every pixel has a grey scale value that represents an angle, the direction of the
maximum local gradient. Using the same local derivatives to get the magnitude of the local brightness gradient as
Pixel =
B
x

2
+
B
y

2
(13.7
allows selecting just the pixels that actually correspond to edges (this is called the Sobel operator, and is discussed
in the chapter on image processing). Then a histogram of the pixel brightness values provides a direct measure of
the preferred orientation of the boundaries. Because of the limitations of performing this operation on a discrete
grid of pixels in a 3x3 neighborhood in which some pixels are farther from the center than others, the results are
not perfect. Measuring the boundaries of circles as shown in Figure 6 shows the directional bias, so that
measurements on real structures can be normalized.
This is a relatively fast technique for measurement of real structures. Figure 7 shows the results for the same
image used above for intercept length measurements (Figure 2a). The histogram of the pixels shows the 45 and 90
degree "spikes" mentioned above, but they do not affect the ability to analyze the results. These indicate a strong
degree of preferred orientation in which most of the boundary length is horizontal and little is vertical.
a b
c
Figure 6: Measurement of edge orientation: a) image of ideal circles; b) the direction results for the boundaries
(color coded); c) histogram of direction values, showing bias favoring 45 and 90 degree directions.
a b
Figure 7. Boundary orientation measurement on the 50% cold rolled steel image in Figure 2a: a) color coding of
gradient direction for boundary pixels; b) histogram of values.
It is important to note that the measurement of intercept length or feature axis (discussed below) is not necessarily
the same thing as measuring boundary orientation. The graph in Figure 2d shows about a 2:1 ratio for the aspect
ratio of the rose plot, and this is in agreement with the fact that the steel was rolled to half of its original thickness
(50% reduction). The histogram in Figure 7b shows a much greater ratio of the peaks at 0 and 180 degrees to the
valleys in between. In part this is due to the fact that the valley covers a wide range of angles (boundaries between
the ends of grains are not precisely vertical). It also reflects the fact that grain shapes are not regular.
Figure 8 shows two extreme examples of feature shapes for which boundary orientation produces a very different
result than a consideration of orientation based on the longest dimension (or the moment axis introduced below).
Both objects are visually oriented at about 45 degrees to the left of vertical. The feature in Figure 8a has
boundaries that are primarily oriented at 90 degrees to the main feature direction, but the one in Figure 8b has
boundaries that are primarily oriented in the same direction as the feature. Measuring feature or grain orientation
with intercept lengths or other methods that depend on the interior of the grain produces different results and has a
different meaning than measuring the orientation of the boundaries. It is important to decide beforehand which has
meaning in any particular experimental application.
a b
Figure 8. Two features whose major axis orientation and boundary orientation have different relationships.
In principle the measurement of boundary orientation can be performed in three dimensions the same as in two. It
is not enough to simply measure the pixels in images on several sections, however. Instead, a true 3D imaging
technique that generates arrays of cubic voxels must be used. Magnetic resonance imaging and computed
tomography are capable of doing this, and so in some situations is confocal light microscopy. Then the same local
neighborhood operators, extended to taking derivatives in the three X, Y, and Z directions, can be used to
measure the orientation of the gradient in three dimensions and used to construct histograms for interpretation.
Gradients and neighbor relationships
Nonuniformity in structures (and hence in images, provided the section planes are properly oriented) can take an
endless variety of forms. Human perception is very good at detecting some of them, such as alignments and
orientation of features, and quite insensitive to others, such as variations in size. Figures 9 and 10 show a few
examples of gradients. The first case (Figure 9a) illustrates a variation in area fraction, which can happen in
materials due to compositional differences arising from diffusion, heat treating, welding, etc. If the direction in
which the gradient exists is known, it can often be measured simply by plotting the average pixel brightness.
Figure 9b shows a plot averaged over a band about 40 pixels wide, which in spite of the local bumps and dips
indicates the gradual overall change in area fraction.
a b
Figure 9. Concentration gradient in a tool steel due to carburization (a),
and a plot of average brightness in the gradient direction (b).
a b
Figure 10. Examples of gradients: a) size; b) orientation.
Gradients such as the variation in size with position (Figure 10a) or orientation with position (Figure 10b) are
much more difficult to quantify. Generally it requires measuring each feature to determine the appropriate size and
shape parameters and the centroid location. The latter may be in some global coordinate system taken from the
microscope stage or the location of specimens in the material being sampled, depending on the scale. Then the data
file containing all of the individual measurements can be subjected to various statistical tests to obtain the desired
quantitative description. When mixed gradients (changes in several parameters of size and shape) or nonlinear
directions of variation are present, the situation becomes extremely complex, and requires knowing a great deal in
advance about the specimen.
a b
c
Figure 11. Examples of clustering (a), self-
avoidance(b) and a random distribution of
features (c).
One of the most interesting location-related variables in structures is the distance between neighboring features.
Figure 11 shows three example cases, corresponding to a random distribution of features, uniform spacing of
features, and clustering of features. Many physical situations correspond to one of these. Random distributions
result when every instance is independent of all others. A section through a random three-dimension structure will
show a random distribution of features on the section plane. Clustering results when structures are attracted to one
another by some force (gravity causes clustering of galaxies, surface tension causes clustering of dust on fluids,
human nature causes people to live in cities). Spacing or self-avoidance also arises from physical conditions; for
example the precipitation of particles in metals typically produces self avoidance because forming one particle
depletes the surrounding region of the element in the particles and makes it less likely that a second particle will
form nearby.
To have a quantitative measure of the tendency of structures to clustering or self avoidance, Exner (Schwarz &
Exner, 1983)) has proposed measuring the nearest neighbor distance. For a random structure, this produces a
Poisson distribution, with a few particles having quite close neighbors and others much large spacings. In fact,
the random distribution is sometimes called a Poisson random process. In a clustered structure, as shown in
Figure 12, the nearest neighbor is usually much closer, and conversely in the self-avoiding structure the mean
nearest neighbor distance is much greater.
a b
c
Figure 12. Histograms of the measured nearest
neighbor distances for the features in the images in
Figure 11.
For a Poisson distribution, the mean is the only parameter needed to fully characterize the data. If the number of
features per unit area is known, the mean value that a random distribution will produce is therefore known. It
should be simply
Mean Distance =
0.5
N
A
(13.8
which has the proper units of distance. For any Poisson distribution, the standard deviation is simply the square
root of the mean. If the mean nearest neighbor distance and standard deviation value actually measured from an
image is different from the mean value, we can conclude that the specimen is not random and decide whether it
shows clustering or self-avoidance. The chapter on statistical analysis presented the t-test, a simple tool that can be
used to compare the values and make a determination, with explicit confidence levels. Table 1 shows the values
for each of the images in Figure 11.
Table 1
Nearest neighbor data from Figures 11 and 12.
Image: Clustered Self-Avoiding Random
Mean Nearest Neighbor
Distance (pixels) 7.41 20.81 13.56
Standard Deviation 6.16 0.01 6.33
Predicted (equation 7) 12.86 11.40 13.06
Standard Deviation 3.59 3.38 3.61
Nearest neighbors can also be used to identify anisotropy. Figure 13 shows an illustration of using the nearest
neighbor direction to produce a rose plot of orientation. In the example, most of the nearest neighbors lie in either
the northeast or southwest direction, indicating a strong preferred orientation. This method has proven particularly
useful in finding and correcting the compression of samples produced by microtoming, which can result in
distortions of 10-20%. Stretching the image back until the rose plot becomes circular (provided the structure is
isotropic) restores the correct dimensions.
a b
Figure 13. Illustration of preferred orientation revealed by a rose plot of the number
of nearest neighbors as a function of the orientation angle at which they occur.
Distances and irregular gradients
Measuring the distance of features from each other is generally carried out after the measurements have been taken
on each feature. The locations of the centroids of the features can then be compared, pairwise, to find the closest
neighbor and the distance or direction used for the statistical comparisons discussed above. The coordinates of
features are generally in some external coordinate system, based on the pixel location within the image and
perhaps the stage micrometer coordinates or the location from which the sample was taken in the original
specimen. But it is often useful to measure the distances of features from some other structure present in the
specimen. This could be the surface, but in most cases is likely to be an irregular feature such as a grain boundary,
some other type of particle, etc. In biological specimens it may be a membrane or cell wall, or another organelle.
In a real world it might be the distance from the nearest road.
Calculating this distance in the traditional analytical geometry way would be at least very difficult, since the target
boundary is not likely to have a simple algebraic function to describe its shape. Also, for irregular boundaries
there may be several different perpendicular distances and finding the appropriate shortest one adds more
complexity.
Fortunately, there is a relatively straightforward and very fast way for the computer to make this measurement by
processing the original image. It is based on the Euclidean Distance Map (EDM), which assigns a value to each
pixel in the image based on its straight line distance from a defined structure regardless of shape or irregularities.
The algorithm for constructing the EDM (Danielsson, 1980) requires only two passes over the image pixels, and
no algebra, taking only a fraction of a second on the computer. As shown in Figure 14 this produces an image
(color coded for clarity) in which all pixels with the same value lie at the same distance from the boundary.
Combining this image with an image of the features of interest assigns the distance values to the features.
Measuring each feature's color value then gives the desired information.
In order to use the data in Figure 14e to detect gradients with respect to distance from the boundary, an additional
step is needed. The shape of the interior region within the boundary is important because there is more space near
the boundary than far away. This can also be measured with the distance map. A simple histogram of all of the
pixels within the region gives the number of pixels at each distance from the boundary. This can be used to
normalize the histogram of feature distances, to reveal gradients that place many features near the boundary or
perhaps show depletion there.
a b
c d
e
Figure 14. Illustration of using the
Euclidean distance map to measure distance
from an irregular boundary: a) test image
showing boundary and features; b) the
interior of the boundary with colors
assigned according to distance (the
Euclidean distance map); c) the features; d)
colors from figure b assigned to the features
from figure c; e) histogram of distances
obtained by tallying the colors of features in
figure d.
Alignment
Human vision is particularly good at finding alignments of features along straight lines (even if they really aren't
based on any physical reason, which is why we see constellations in the night sky). There is a computer technique
that can be used to locate these alignments automatically, called the Hough transform (Hough, 1962). Figure 15
shows the basic steps involved. Each point in the original image is mapped into Hough space as a sinusoid. The
(,) coordinates of this space are the polar coordinates of a line in the original spatial domain. Each point on the
sinusoid represents one of the possible lines that could be drawn through the corresponding point in the original
spatial image. Since all of the sinusoids in Hough space are added together, a maximum value results where many
of them cross over. In Figure 15b, color coding has been used to show this peak. The peak corresponds to the
coordinates of the line that passes through the points in the original image, and shown in Figure 15c. This method
can also be applied to fitting circles and other shapes (Duda & Hart, 1972), but the details go beyond the intended
scope of this text.
a b
c
Figure 15. Illustration of the linear Hough
transform. Each point in the original spatial
domain image (a) generates a sinusoid in
Hough (,) space (b) corresponding to
allpossible lines through the point. The
crossover point in this space identifies a single
straight line in the spatial domain image (c) that
passes through the points.
Chapter 14 - Finite Section Thickness
Projected images
The classic applications of stereology and the principal rules which have been derived and applied are specific to
the case of planar sections through three-dimensional solids. This is more-or-less the situation in metallographic
microscopy, where polished sections cut through the material are examined. It is not so appropriate for many other
fields. Light microscopy in biological and medical applications usually looks through a slice of material, for
instance cut with a microtome, whose thickness is not negligible. Microscopy of particulates also looks through a
thick layer, in which the matrix is transparent air. Confocal light microscopy images an optical section from within
material, whose thickness is finite and depends on the numerical aperture of the optics, and whose boundaries are
not sharp. Even electron microscopy, which uses much thinner sections than light microscopy, deals with finite
sections (the lateral resolution of the images is improved along with the thinner sections so that features much
smaller than the section thickness are detected). And in scanning electron microscopy although surfaces are
imaged to negligible depth (at least with secondary electrons, if not backscattered electrons or X-rays), the
surfaces are rarely flat.
The introduction of finite sections and viewing of images projected through them raises some significant problems
for stereology. In cases where the section thickness is much less than the dimensions of any features or details of
interest it may be possible to ignore the differences and treat the images as though they were ideal sections.
Indeed, this is the assumption that is commonly made in applying stereological rules to microscope images,
although its appropriateness is rarely evaluated.
Viewing images that are projections through a very thick section makes some kinds of measurements quite simple.
The outer dimensions of features are seen directly, so that no unfolding is required to ascertain the actual particle
size distribution based on the sizes of sections. There are still some important underlying assumptions, of course.
Most basic is that the dispersion is quite sparse so that particles are visible and do not obscure one another. The
specific case of particles deposited onto a substrate meets this test, since the particles are generally present as a
single layer. The original "thickness" of that layer corresponding to a volume of material from which the particles
have settled onto the substrate is not known, but it is usually possible to control the deposition conditions to
prevent the deposit from becoming so dense that particles touch or overlap one another.
A second assumption is that the particles have uniformly random orientations with respect to the viewing
direction. This may or may not be true when viewing through an actual matrix, but is always suspect when
particles are deposited on a substrate since physical forces can act to reorient the particles when they contact the
surface. These forces range from gravity (for large particles) to electrostatic or magnetic attraction, surface tension
in the fluid as it is removed, and others. The general result is that particles may align with their long axis parallel to
the surface or to each other, and may tend to clump together. This presents an image that is not random and makes
the determination of three-dimensional particle sizes suspect. Special preparation techniques can sometimes be
employed to produce well dispersed samples with unmodified particle orientations. One example is the use of a
camphor-napthalene eutectic wax for dispersing cement powders (Thaulow & White, 1971). The wax supports
the particles and is spread onto a slide as a substrate. It is then evaporated away allowing the particles to settle onto
Chapter 14 - Finite Section Thickness Page # 276
the slide. Obviously this is a specialized method, but similar approaches may be practical for a variety of
applications.
When particulates are viewed in projection, the external diameter or projected area gives a direct measure of size.
This of course is only true for convex features. Any interior details such as surface pits or voids are hidden, and
important topological characteristics may be hidden as well. Consider for example a distribution of toroids -
Cheerios
for instance. As shown in Figure 1, some of the projected images will show the presence of the central
hole but most will not. If it is known a priori that all of the features are the same, the multiple projections make it
possible to determine the three-dimensional shape. In fact this technique has been used to reconstruct the shape of
virus particles from transmission electron microscope pictures, by combining multiple projections of presumably
identical particles using tomographic reconstruction. But when a mixture of shapes is present, the assessment of
the actual shapes and relative populations is difficult or impossible.
Figure 1. Illustration of a projected view of toroids: a) surface image which reveals the shapes;
b) projected image in which most images do not indicate the three dimensional shape.
Even if the assumption of random orientation can be met, many projected images do present the viewer with some
overlaps. Figure 2 illustrates this problem. Small particles are more likely to hide behind larger ones, and
overlapping projections of particles reduce the number and alter the size of the features in the projected image. In
the example, the spatial distribution is not isotropically random. In one direction (Figure 2b) the particles are
nearly all separately visible, while in another (Figure 2c) they are heavily overlapped. In most cases, of course, it
is not possible to select a particular viewing direction or to compare the results in different directions. Figure 3
shows a real example, a section viewed in the TEM containing latex spheres. The apparent clusters of particles are
in fact simply superpositions of images along the particular projection direction and the particles are actually
separated in three dimensions.
As noted above it is more likely for small particles to be hidden by large ones than the converse. A statistical
correction for the systematic undercounting of small features is possible. The size distribution of the visible
features is constructed. Normally such a distribution would report the number per unit volume, where the volume
is the area of the viewed image times the thickness of the section. However, for small features the actual volume
being viewed is less. The projections of large features block the view of a portion of the area, and this must be
subtracted from the volume measured when counting the smaller features that could hide within that shadow as
shown in Figure 4. This correction can be made for each bin in the distribution, increasing the number per unit
volume of the smaller particles.
a
b c
Figure 2. Illustration of particles in a thick section, which overlap in a projected image:
a) perspective view; b) projection along the Z axis; c) projection along the Y axis.
There are actually two variants of this correction. When the projected image is a shadow or binary image, the
volume covered by the large feature extends completely through the section thickness so that the sampled volume
is reduced by the projected area times the thickness. When the image shows the object surfaces (Figure 5), small
features lying in front of larger ones can be seen, but particles lying behind larger ones cannot. The hidden volume
is reduced by one half since on the average the large particles lie in the middle of the section.
Figure 3. TEM image of latex spheres, which appear to form clusters
but are actually separate in three dimensions.
Figure 4. Schematic showing the hidden shadow region above and below a large particle. As discussed in
the text, the area of the feature may be reduced if partially protruding objects can be distinguished.
In either case, it is clear that particles are hidden if they are entirely within the shadow area. But if their centers lie
within a band equal in thickness to the radius, they will partially protrude and may be detected depending on the
criteria for visibility. Often, particles whose centers lie outside the shadow but intersect the projection of the large
particle can be seen and measured.
a b
Figure 5. For a surface image, small particles above a larger one are visible and can be measured while
those below are completely or partially hidden: a) perspective view; b) vertical image.
Consequently, it is an application-specific decision whether the area of the large feature is the proper value to use
for the correction, or whether a region smaller or larger by the radius of the smaller feature for which the
correction is to be made, and whether the hidden volume is equal to this area times the thickness or half the
thickness of the section.
Bias in stereological measurements
Finite section thickness introduces two opposite sources of bias into conventional stereological measurements.
These have been recognized for a long time (Holmes, 1927), and various correction procedures have been derived
(Cruz-Orive, 1983). For the most part, however, these are not used widely. This is because they require knowing
the section thickness, making some assumptions about feature shape and uniformity (also isotropy of orientation),
and/or performing more measurement work to obtain results. Determining the thickness of a microtomed section
accurately is difficult, and for many materials specimens techniques used to prepare TEM sections such as
electrochemical thinning or ion erosion produce sections whose thickness varies dramatically and imaging is
actually performed on wedge-shaped regions around the edges of holes.
Figure 6. Diagram of the cross section of a thick section in which the opaque phase
appears to cover more area because of overprojection.
Figure 7. Diagram of the cross section of a surface with polishing relief, in which the harder
phase projects from the surface and appears to cover more area because of overprojection.
For the case in Figure 6, we have a finite section with one transparent and one opaque phase. Note that this is
exactly equivalent to the case of a polished metallographic section with finite polishing relief due to one phase
being harder than the other (Figure 7). In each case, the opaque or harder phase appears to cover a greater fraction
of the viewed area than it should, because of overprojection. The apparent area fraction A
A
is greater than the
actual volume fraction V
V
by an amount that depends on the thickness of the section t and the amount of surface
area per unit volume S
V
. (Cahn & Nutting, 1959)
A
A
= V
V
+
1
4
S
V
t (14.1
Detemining S
V
also requires a correction for the section thickness, which depends on the number of features
present and their mean curvature. In terms of practical measurement quantities A
A
(the apparent area fraction,
which is typically measured by counting a point fraction P
P
), B
A
(the phase boundary length per unit area,
which is typically measured by counting linear intercept points P
L
) and the number of features per unit area N
A
,
the true stereological structure parameters are
V
V
= P
P
t
1
2
P
L
N
A
t H
t + H

(14.2
S
V
= 2 P
L
4 N
A
t H
t + H
(14.3
The parameter t is the section thickness and H is the average mean caliper diameter of the particles or phase
regions in the section. It is usually assumed that H is much less than t , corresponding to the case in which
particles are small compared to the section thickness, but this is not essential. Of course, if H is not small
compared to t it is not likely that H will be seen or measurable in the section images and will have to be
determined separately from other experiments. The model also assumes that the phase regions or particles are
convex and do not overlap each other (the section thickness is very small, or the dispersion is very sparse and V
V
is small).
If measurements on two sections of thickness t
1
and t
2
are performed, a further simplification is possible and we
obtain
V
V
=
t
1
A
A
1
t
2
A
A
2
t
1
t
2
(14.4
S
V
=
4
t
2
B
A
1
t
1
B
A
2
t
1
t
2
(14.5
In actual practice, determining the thickness values with sufficient precision to divide by the difference (t
1
t
2
) is
extremely difficult to accomplish.
R. E. Miles (1976) derived a complete set of stereological equations for volume density, surface density, and
curvature based on measurements on the projected image of a section of thickness t which are more general than
the preceding equations. They apply to the case of an aggregate of opaque, isotropic, random convex particles in a
transparent matrix. The required measurements can be obtained using a point count using a grid, and intersection
count using a grid, and a tangent count using a sweeping test line. Data must be taken on paired sections and the
estimated values depend on the differences between the pairs of measurements (requiring a large number of counts
for adequate precision) and the accuracy of the thickness measurements.
The above discussion deals only with the problem of overprojection, features appearing too large in the projected
image. It is restricted to convex phase regions and cannot deal with particles hiding behind others. An additional
effect may also be present, usually referred to as the "end cap" problem. Consider the illustration in Figure 8. The
spherical particles are of three types: ones that lie entirely within the section thickness, ones that project out (and
are truncated) but whose centers lie inside the section thickness, and ones whose centers lie outside the section.
The latter group have only their polar caps in the section. In many cases, the contrast with which the opaque phase
is visible in the projected image is such that these thin end caps may not be seen, or they may literally fall out of
the section.
Figure 8. Diagram of a cross section of a thick section containing spherical particles that lie entirely
within the section, ones that protrude out but have their centers inside, and ones with their
centers outside the section but protrude into it producing end caps.
In terms of the number of features seen per unit area of image, the contribution of the first two types of particles is
a function of the section thickness

t while that of the third group is a function of the mean particle diameter D ,
where N
V
is the true number density per unit volume.
N
A
= N
V
t + N
V
D (14.6
Making corrections for the presence of end caps or for the fact that some may be missing requires making many
assumptions. Weibel (1980) derives a family of curves to correct V
V
and S
V
measurements for invisible end
caps as a function of the diameter at which they disappear. In most cases, it is more useful to consider the
thickness at which they disappear since the visibility of the opaque phase is usually related to this dimension. For
the assumption of spherical particles, the two dimensions are of course related.
The same method can be extended to the sphere unfolding method discussed in Chapter 12. This is typically done
by assuming either that the caps disappear at a constant latitude angle in all size classes, or that they disappear at a
fixed thickness due to insufficient image contrast. Of course, the assumption of spherical particle shape is already
a basic part of the unfolding method. If the shape of the distribution can also be assumed (e.g., normal or log-
normal), then an iterative calculation of the size distribution of the circles produced by sectioning spheres can be
used to determine the mean and variance of the sphere size distibution (Nauman & Cavanaugh, 1998).
Measurements within sections
With all of the difficulties and assumptions involved in applying classical stereology to thick sections, it is
surprising to find that some very straightforward measurements are possible. One is the length of linear structures,
regardless of their complexity, connectedness or branching. These may be separate discrete lines such as fibers or
dislocations, or branching structures such as roots of plants (Figure 9) or neurons in stained tissue. Linear
structures are also formed by the intersection of planar ones, such as the triple line that forms a network in grain
structures where the surfaces of three grains meet. This network is important as a path for diffusion and other
processes, but is not often visualized as a three-dimensional linear structure.
Figure 9. A projected image of the roots of a plant. The branching structure can be considered
to occupy a volume in which the transparent matrix is air.
The projected image of a linear structure is of course a set of lines, and we will make the assumption that they are
all visible and the magnification and section thickness are appropriate to allow the lines to be resolved and viewed.
But of course since the lines are embedded in a three-dimensional volume and can run in any direction, the length
of the lines may be much greater than the length of the projections. Without making any assumption about the
orientation of the lines, it is possible to measure the actual value with little effort.
One of the rules of classical stereology is that a plane probe can be used to intersect lines, and that the expected
value of the length of the lines per unit volume L
V
is equal to 2 N
A
where N
A
is the number of intersections that
the lines make with the plane. These intersections appear as points, of course, and sometimes they can be used
directly for classical measurement. For instance, etching a piece of semiconductor to produce pits where
dislocations intersect a polished surface makes it possible to count them. The factor two arises because an
isotropic distribution of lines would intersect the surface at many different angles. So the classical measurement of
L
V
from the value of N
A
on a section surface is dependent on the assumption of isotropy.
This highlights the usual difficulty with plane probes - it is very difficult to make them isotropic in three-
dimensional space, if the specimen itself is not isotropic. Fortunately with thick sections there is a way to
accomplish this. As shown in Figure 10, a line drawn on the projected image represents a surface extending
through the thick section. If the line is straight the plane is flat; if the line is curved the surface is a cylindrical one.
The area of the surface is the product of the length of the line times the section thickness. Counting points where
the linear structure of interest crosses the surface can be accomplished by counting the intersections of the
projection of the linear structure with the projection of the surface, which is the drawn line.
Figure 10. The relationship between a line drawn on a projected image and the surface
that line represents in the original thick section.
Drawing a uniform set of straight lines on the projected image does not produce a set of random planes in three
dimensions, so the problem of anisotropic sampling is still present. All such surfaces share a common direction
through the thickness of the section, and so are far from isotropic. However, the vertical section method discussed
in other chapters can be used here as well. Select one direction in the plane of the section as the nominal "vertical"
direction, cut several sections that include that direction (experiments show that as few as three sections cut 60
degrees apart provide a reasonably sample set), and then draw cycloids on projected images of each section as
shown in Figure 11.
Note that the vertical direction is shown on the image and the orientation of the cycloids with respect to that
direction is not the same as when cycloids are drawn on a planar vertical section. the cycloidal surfaces have more
of their area parallel to the vertical direction than perpendicular to it, since every thick section cut to include the
same vertical section will have the same planes perpendicular to that direction, which would be oversampled by
planes corresponding to isotropic lines drawn on the projection.
Counting the intersections of the linear structure with the cycloids allows an easy and straightforward
measurement of the total length of the structure. Sometimes this is performed manually and sometimes by using
image processing to combine the image of the grid with the linear structure (a Boolean AND) to mark the
crossings for automatic counting. Both methods have been discussed in other chapters.
Figure 11. Transmission electron microscope image of nerve axons with a superimposed grid of cycloids.
In some cases it is not even necessary to cut multiple thick sections. In the case of the root in Figure 9, the entire
volume of the root can be treated as a (very) thick section with a transparent (air) matrix. Rotating the entire
structure to three orientations about 60 degrees apart provides the isotropic sampling around some selected vertical
direction; in this case the plant stem would provide a logical reference. Superimposing a cycloid grid on each of
the three projected images and counting the intersections can be used to measure the total length of the structure.
As a practical demonstration of this method, it is convenient to take a wire or other structure that can later be
measured and bend it into a complex three-dimensional shape, place it on a grid (using a transparency and
overhead projector is a good classroom demonstration method), and count the intersections in each of three
rotational orientations. As shown in the example of Figure 12, this produces a number of counts that can be
combined with the known length of the lines and the area of the grid.
If the volume occupied by the wire is assumed to be a cube whose face is the area of the grid, then the key
relationships can be written in terms of the length per unit volume of the linear structure L
V
, the length of the grid
line G, the number of counts N (averaged over the three orientations of the specimen), and t the size of the grid
(and the assumed section thickness). Equation (14.7) is simply the classical relationship between L
V
and the
number of intersections, and equation (14.8) gives the actual length L in terms of the number of counts and the
length of grid line per unit area (a known constant for any test grid).
L
V
=
2 N
G t
(14.7
L =
2 N
G
A
(14.8
Figure 12. Projected image of a wire tangle superimposed on a cycloid grid, with intersections marked.
In the example of Figure 12, the number of intersections is 23. The total length of grid line is 68.054 cm in an area
of 7.5 x 7.5 cm, so G
A
= 1.209 cm-1. That produces an estimate of the wire length of 19 cm. The uncertainty in
this estimate comes from the number of counts (
23
23
is about 20%, or 194 cm (the actual length of the wire
was 20 cm.). Repeating the measurement with other placements of the grid and other orientations around the same
vertical direction would produce more counts and thus improve the statistical precision of the measurement.
Chapter 15 - Three-dimensional imaging
Limitations of stereology
Stereological analysis provides quantitative measures of several kinds of structural information. Whether
determined manually or by computer measurement, most of these require only simple counting or measurement
operations and give unbiased parameters (provided that IUR conditions on the point, line and plane probes are
met). Measures of phase volume and surface area are reasonably straightforward. Distributions of feature size and
shape, and alignments and neighbor distances, can be determined in many cases. However, the data that are
provided by stereological measurement are sometimes difficult to interpret in terms of the appearance of a
microstructure as it is understood by a microscopist.
While the global metric parameters of a microstructure can be measured with the proper stereological probes and
techniques, and with more difficulty the feature-specific ones can be estimated, topological ones are more difficult
to access. These include several fundamental properties that are often of great interest to microscopist, such as the
number of objects per unit volume. Counting on a single plane of intersection can estimate this for the case of
convex features of a known size, or with some difficulty for the case of a distribution of such features of a known
shape. Counting with two or more planes (the disector, Sterio, 1984)) can determine the number per unit volume
of convex features of arbitrary size and shape.
But the general case of objects of arbitrary shape, and networks of complex connectivity, cannot be fully
described by data obtained by stereological measurement. This is essentially a topological problem, and is related
to the desire to "see" the actual structure rather than some representative numbers. Humans are much more aware
of topological differences in structure than in metric ones.
Serial methods for acquiring 3D image data
The method longest used for acquiring 3D image data is serial sectioning for either the light or electron
microscope. This is a difficult technique, because the sectioning process often introduces distortion (e.g.,
compression in one direction). Also, the individual slices are imaged separately, and the images must be aligned to
build a 3D representation of the structure (Johnson & Capowski, 1985). This is an extremely critical and difficult
step. The slices are usually much farther apart in the Z direction than the image resolution in the X, Y plane of
each image (and indeed may be nonuniformly spaced and may not even be parallel, complications that few
reconstruction programs can accommodate).
This alignment problem is best solved by introducing some fiduciary marks into the sample before it is sectioned,
for instance by embedding fibers or drilling holes. These provide an unequivocal guide to the alignment, which
attempting to match features from one section to another does not (and can lead to substantial errors). For
sequential polishing methods used for opaque specimens such as metallographic samples, hardness indentations
provide a similar facility. They also allow a direct measure of the spacing between images, by measuring the
change in size of the indentation before and after polishing.
Chapter 15 - Three-Dimensional Imaging Page # 288
For objects whose matrix is transparent to light, optical sectioning allows imaging planes within the specimen
nondestructively. Of course, this solves the section alignment problem since the images are automatically in
registration. It also allows collecting essentially continuous information so that there is no gap between successive
image planes. The conventional light microscope suffers significant loss of contrast and resolution due to the light
from a plane deep in the sample passing through the portions of the sample above it. This applies to the case of
reflected light viewing or fluorescence imaging. For transmission imaging the entire thickness of the sample
contributes to the blurring.
Deconvolution of the blurring is possible using an iterative computational procedure that calculates the point
spread function introduced by each layer of the specimen on the affected layer. As shown in Figure 1 this allows
sharpening image detail and recovering image contrast. However, it does little to improve the depth of field of the
optics and the resolution in the Z direction remains relatively poor.
Figure 1. Immunofluorescence cell image (a) showing green microtubles, red mitochondrial protein, and
blue nucleus. Deconvolution of the point spread due to specimen thickness (b) performed using software
by Vaytek (Fairfield, Iowa), who provided the image.
The confocal microscope solves both problems at once. By rejecting light scattered from points other than the
focal position, contrast, resolution and depth of field are all optimized. Scanning the point across the specimen
optically in the X, Y, plane and shifting the specimen vertically in the Z direction can build up a 3D data set in
which the depth resolution is 2-3 times poorer than the lateral resolution, but still good enough to delineate many
structures of interest. There are still some difficulties with computer processing and measurement when the voxels
(the volume elements which are the 3D analogs to pixels in a plane image) are not cubic, and these are discussed
below. Confocal microscopy has primarily been used with reflected light and fluorescence microscopy, but in
principle can also be extended to transmission imaging.
Inversion to obtain 3D data
In confocal microscopy, or even in conventional optical sectioning, light is focused onto one plane within the
sample. The light ideally enters through a rather large cone (high numerical aperture objective lens), so that the
light that records each point of the image has passed along many different paths through the portion of the
specimen matrix that is not at the focal point. This should cause the variations in the remainder of the sample to
average out, and only the information from the point of focus to remain.
Figure 2. The principle of inverse reconstruction. Information from many different views through a
complex structure is used to determine the location of objects within the volume.
This idea of looking at a particular point from many directions lies at the heart of inverse methods used to
reconstruct images of microstructure (Figure 2). They are often called tomographic or computed tomographic (CT)
methods, and the most familiar of them is the X-ray CAT scan (computed axial tomography, because the most
common geometry is a series of views taken from different radial positions around a central axis) used in medical
diagnosis (Kak & Slaney, 1988). This method actually reconstructs an image of one plane through the subject,
and many successive but independent planes are images to produce an actual 3D volume of data. Other projection
geometries are more commonly used in industrial tomography and in various kinds of microscopy, some of which
directly reconstruct the 3D array of voxels (Barnes et al., 1990).
There are two principal methods for performing the reconstruction: filtered back projection (Herman, 1980) in
which the information from each viewing direction is projected back through the voxel array that represents the
specimen volume, with the summation of multiple views producing the final image, and algebraic methods
(Gordon, 1974) that solve a family of simultaneous linear equations that sum the contribution of each voxel to
each of the projections. The first method is fast and particularly suitable for medical imaging, because speed is
important and the variation in density of geometry of the sample is quite limited, and because image contrast is
important to reveal local variations for visual examination, but measurement of dimension and density are not
generally required. The second method is more flexible to deal with the unusual and asymmetrical geometries
often encountered in microscopy applications, and the desire for more quantitatively accurate images from a
relatively small number of projections.
Tomographic reconstruction requires some form of penetrating radiation that can travel through the specimen
volume, being absorbed or scattered along the way as it interacts with the structure. There are many situations in
which this is possible. X-rays are used not only in the medical CAT scan, but also for microscopy. A point source
of X-rays passing through a small specimen produces a magnified conical projection of the microstructure.
Rotating the specimen (preferably about several axes) to produce a small number of views (e.g., 12-20) allows
reconstruction of the microstructure with detail based on variation of the absorption cross section for the X-rays,
which is usually a measure of local specimen density. However, using a tunable X-ray source such as a
synchrotron, it is possible to measure the distribution of a specific element inside solid samples (Ham, 1993) with
a resolution on the order of 1 m.
Electron tomography (Frank, 1992) in the transmission electron microscope typically collects images as the
specimen is tilted and rotated. Constraints upon the possible range of tilts (due to the stage mechanism and
thickness of the sample) cause the reconstruction to have poorer resolution in the Z direction than in X and Y, and
it is important to avoid orientations that cause electron diffraction to occur as this is not dealt with in the
reconstruction.
Light is used tomographically in some cases, such as underwater imaging methods developed for antisubmarine
detection. It is also used to measure the variation in index of refraction with radius in fibers. Sound waves are
used for medical imaging as well as seismic imaging and some acoustic microscopy. In the latter case, frequencies
in the hundreds or thousands of megahertz provide resolution of a few m. Seismic tomography is complicated
greatly by the fact that the paths of the sound waves through the specimen are not straight lines, but in fact depend
upon the structure so that both the attenuation and path must be part of the reconstruction calculation. However,
using pressure and shear waves generated by many earthquakes and detected by a worldwide network of
seismographs, detailed maps of rock density and fault lines within the earth have been computed.
Magnetic resonance imaging (MRI) used in medical diagnosis also has curved paths for the radio waves that
interact with the hydrogen atom spins in a magnetic field. The use of very high field gradients has allowed
experimental spatial resolutions of a few m for this technique. Many other signals have been used, including
neutrons (to study the structure of composite materials) and gamma rays (photons with the same energies as X-
rays but from isotope sources, which are suitable for use in some industrial applications and for down-borehole
imaging in geological exploration).
In all these cases, the reconstruction methods are broadly similar but the details of the microstructure that interact
with the signal and thus are imaged vary greatly. Some techniques respond to changes in density or composition,
others to structural defects, others to physical properties such modulus of elasticity or index of refraction. In the
ideal case, the image that is reconstructed is a cubic array of voxels. Depending on the application, the size of the
voxels can range from nanometers (electron microscopy) to kilometers (seismic imaging). A significant amount of
computing is generally required, and the number of voxels that can be reconstructed is quite small and gives
resolution that is poor compared to the number of pixels in a two-dimensional image. Processing is generally
needed to minimize noise and artefacts. This means that tomography is not a "live" viewing technique, and that the
resolution of the images is limited
Stereoscopy as a 3D technique
Tomography usually employs a fairly large number of projections taken from as widely distributed a set of
viewpoints as possible. Medical imaging of a single slice through the body may use more than 100 views along
radial directions spaced on a few degrees apart, while some conical projection methods for 3D reconstruction use
as few as a dozen projections in directions spread over the full solid angle of a sphere. By contrast, stereoscopy
uses only two views that are only slightly different, corresponding to the two points of view of human vision. The
advantage is that humans possess the 3D reconstruction software to merge these two views into a 3D structural
representation (Marr & Poggio, 1976).
It is important not to confuse stereoscopy with stereology. In both, the root stereo is from the Greek and refers to
three-dimensional structure. Stereology is defined as the study of that structure based on geometric principles and
using two dimensional images. Stereoscopy is the recording or viewing of the structure, and it generally taken to
mean the two-eye viewing of structures in a way that reveals them to the brain of the observer. This does not
exclude the possibility of making measurements based on those two images (Boyde, 1973).
Figure 3. The principle of stereoscopic measurement. Measurement of the parallax or apparent
displacement of features in two views separated by a known angle () allows calculation
of the vertical displacement of the features.
As shown in Figure 3, the parallax between two points viewed in left- and right-eye images gives a direct measure
of their vertical separation, provided that the scale of the pictures and the angle between the two views is known.
Because the angle is typically small (5-15 degrees) so that the views can be successfully fused by the human
visual system, small uncertainties in the angle strongly affect the precision of the Z calculation. Finite precision in
the lateral measurements that give the parallax measurement, which ultimately depend on the lateral resolution or
pixel size in the images, produce uncertainties in the Z measurement about an order of magnitude worse than the
X, Y measurements.
Figure 4. SEM image of a three-dimensional clump of yeast cells. Stereoscopy can measure the outer
dimensions of the clump but cannot reveal internal details such as the individual cells.
Stereoscopy (and for that matter stereology) do not offer any way to determine the number of features in a clump
as shown in Figure 4. This SEM image of yeast is certainly not densely packed so that the number might be
estimated from the outer dimensions, nor are all of the particles the same size nor are those visible on the outside
necessarily representative of the interior ones. Stereoscopy can provide the height of the clump from two images,
but the invisible interior is not accessible.
Viewing stereo images does not require making measurements, of course. It is the relative position of objects
which humans judge based on whether the parallax increases or decreases, and this is determined by the vergence
(motion of the eyes in their sockets) needed to bring points of interest to the fovea in the eyes for detailed viewing.
This means that relative position is judged one feature at a time, rather than a distance map being computed for the
entire image at once.
When stereo pair images are used for applications such as aerial mapping of ground topography or SEM mapping
of relief on integrated circuits, the computer works entirely differently than human vision (Medioni & Nevatia,
1985). It attempts to match points between the two images, typically using a cross correlation technique that looks
for similarities in the local variation of grey (or color) values from pixel to pixel. Some systems rely on human
matching of points in the two images; this can be quite time consuming when thousands of points need to be
identified and marked. Matched points then have their parallax or horizontal offset values converted to elevation,
and a range image is produced in which the elevation of each point on the surface is represented by a grey scale
value.
These techniques are suitable for surface modeling and in fact are widely used for generating topographic maps of
the earth's surface, but they are much more difficult to apply to volumetric measurement. Matching of points
between the two views is complicated when the left-to-right order in which they appear can change, and the
contrast may vary or points may even disappear because of the presence of other objects in the line of sight.
Stereoscopic images of volumes can be used for discrete measurements of z spacings between objects, but
producing a complete voxel array with the objects properly arranged is not possible with only two views - it
requires the full tomographic approach.
Figure 5. Stereoscopic display of a processed voxel array. The left and right images can be viewed to see
the internal structure of a human brain as imaged with MRI, and processed with a 3D gradient operator to
reveal regions where there is a large change in concentration of water.
However, stereoscopic images are very useful and widely employed to communicate voxel-based data back to the
human user. From a volumetric data set, two stereoscopic projections can be generated quickly and displayed for
the user to view. This is sometimes done by putting the images side by side (Figure 5) and allowing the user to
relax his or her eyes to fuse them, or by putting the images into the red and green color planes of a display and
using colored glasses to deliver each image to one eye.
The example display in Figure 5 is a processed image of the human brain. The original data were obtained by
magnetic resonance imaging, which shows the concentration of protons (and hence of water molecules) in tissue.
The images were assembled into a voxel array, and processed using a gradient operator as discussed below. The
two projections 9 degrees apart were generated for viewing. Other schemes in which LCD lenses on glasses
rapidly turn opaque and transparent and the computer display alternately flashes up the left and right images, or
lens and mirror systems oscillate to assist the viewer, are also used.
The majority of people can see and correctly interpret stereo images using one or more of these methods, but a
significant minority cannot, for a variety of reasons. Many of the people who cannot see stereo images get the
same depth information by sequential viewing of images from a moving point of view, for example by moving
their head sideways and judging the relative displacement of objects in the field of view to estimate their relative
distance. The relationship is the same as for the parallax. Displays that rotate the voxel array as a function of time
and show a projection draw on this ability of the human visual system to judge distance. Almost everyone
correctly interprets such rotating displays.
Visualization
When a three-dimensional data set has been obtained, whether by serial sectioning, tomographic reconstruction or
some other method, the first desire is usually to view the voxel array so as to visualize the volume and the
arrangements of objects within it. The example in Figure 5 shows one mode than is often used, namely stereo
presentation of two projections. This relies on the ability of the human to fuse those images and understand the
spatial relationships which they encode. As mentioned above, the second common technique is to use motion to
convey the same kind of parallax or offset information. This may either be done by providing a continuous display
of rotation of the volume to be studied, or an interactive capability to "turn it over" on the screen and view it from
any angle (usually with a mouse or trackball, or whatever user interface the computer provides).
Producing the projected display through the voxel array can be done in several different ways, to show all of the
voxels present, or just the surfaces (defined as the location where voxel value - density for example - changes
abruptly), or just a selected surface cut through the volume. We will examine these in detail below. But in all
cases, the amount of calculation is significant and takes some time. So does addressing the voxels that contribute
to each point on the displayed image. If the array of data is large, it is likely to be stored on disk rather than in
memory, which adds substantially to the time to create a display. For these reasons, many programs are used to
create a sequence of images that are then displayed one after the other to create the illusion of motion. This can be
very effective, but of course since it must be calculated beforehand it is not so useful for exploring an unknown
sample, but rather is primarily used to communicate results that the primary scientist has already discovered to
someone else.
Computer graphics allow three principal modes of display (Kriete, 1992): transparent or volumetric, surface
renderings, and sections. The volumetric display is the only one that actually shows all of the information present.
For any particular viewing orientation of the voxel array, a series of lines through the array and perpendicular to
the projected plane image are used to ray-trace the image. This is most often done by placing an extended light
source behind the sample and summing the absorption of light by the contents of each voxel. This makes the tacit
assumption that the voxels values represent density, which for many imaging modalities is not actually the case,
but at least it provides a way to visualize the structure. It is also possible to do a ray tracing with other light
sources, to use colored light and selective color absorption for different structures.
The chief difficulty with volumetric displays is that they are basically foreign to human observers. There are few
natural situations in which this kind of image arises - looking at bits of fruit (which must themselves be
transparent) in Jell-O can be imagined as an example. In most cases, we cannot see through objects; instead we
see their surfaces. Even if the matrix is transparent or partially visible, the objects we are interested in are usually
opaque (consider fish swimming in a pond, for example). For this reason, most volumetric display programs also
include the capability to add incident light to reflect from surfaces, and to vary the color or density assigned to
voxels in a nonlinear way so that surfaces become visible in the generated image.
Figure 6. Several views of magnetic resonance images of a hog heart from a movie sequence of its
rotation. The data set was provided by B. Knosp, R. Frank, M. Marcus, R. Weiss, Univ. of Iowa Image
Analysis Facility, and the images were generated using software from Vital Images (Fairfield, Iowa).
Figure 6 shows an example. The data are magnetic resonance images of a hog heart, and the color and density of
the heart muscle and the vasculature (which can be distinguished by their different water content) have been
adjusted to make the muscle partially transparent and the blood vessels opaque. Arbitrary colors have been
assigned to the different voxel values to correspond to the different structures. The sequence of images from
which the selected frames have been taken shows the heart rotating so that all sides are visible. Note however that
no single frame shows all information about the exterior or interior of the object. The observer must build up a
mental image of the overall structure from the sequence of viewed images, which happens to be something that
people are good at. Movies in which the point of view changes, or the opacity of the various voxel values is
altered, or the light source is moved, or a section plane through the data is shifted, all offer effective tools for
communicating results. They do not happen to fit into print media, but video and computer projectors are
becoming common at scientific meetings and network distribution and publishing will play an important role in
their dissemination.
Figure 7. The same data set as Figure 6 with the opacity of the voxels with values
corresponding to muscle tissue increased. This shows the exterior form of the heart.
Changing the nonlinear relationship between the values of voxels in the array and their color and opacity used in
generating the image allows the transparency of the different structures to be varied so that different portions of the
structure can be examined. Figure 7 shows the same data set as Figure 6 but with the opacity of the heart muscle
tissue increased so that the interior details are hidden but the external form can be displayed. Again, because of the
number of variables at hand and the time needed to produce the images, this is used primarily to produce final
images for publishing or communicating results rather than exploring unknown data sets.
The surface rendering produced by changing voxel contributions in volumetric display programs is not as realistic
as can be produced by programs that are dedicated to surface displays. By constructing an array of facets (usually
triangles) between points on the surface defined by the outlines of features in successive planes in the 3D array,
and calculating the scattering of light from an assumed source location from the facet as a function of its
orientation, visually compelling displays of surfaces can be constructed.
Figure 8. Views of serial section slices through the cochlea from the inner ear of a bat. The surface
voxels are shaded to indicate their orientation based on the offset from the voxels above.
The data set was provided by A. Keating, Duke Univ., and the reconstruction
performed using Slicer (Fortner Research., Sterling, VA).
Figure 8 shows data from more than 100 serial section slices through the cochlea from the inner ear of a bat. The
program used to generate the surface display simply shades each individual surface voxel according to the offset
from the voxels above, so that the surface detail is minimally indicated but not truly rendered. This program also
allows rotating the object, but only in 90 degree steps, and does not allow varying the location of the light source.
The same data set is shown in Figure 9 using a more flexible reconstruction program that draws outlines for each
plane, fills in triangles between the points in adjacent planes, calculates the light scattering from that triangle for
any arbitrary placement of the light source, and allows free rotation of the object for viewing (it also generates
movie sequences of the rotation for later viewing).
Surface rendering provides the best communication of spatial relationships and object shape information to the
human viewer, because we are adapted to properly interpret such displays by our real-world experiences. The
displays hide a great deal of potentially useful information - only one side of each object can be seen at a time,
some objects may be obscured by others in front of them, and of course no internal structure can be seen. But this
reduction of information makes available for study that which remains, whereas the volumetric displays contain so
much data that we cannot usually comprehend it all.
Figure 9. The same data set as Figure 8 reconstructed by converting each image plane to a contour outline
and connecting points in each outline to ones in the adjacent outlines forming a series of facets which are
then shaded according to their orientation. The entire array can be freely rotated. Reconstruction
performed using MacStereology (Ranfurly MicroSystems, Oxford UK).
There is a third display mode that is easily interpretable by humans, but (or perhaps because) it is rather limited in
the amount of information provided. The three-dimensional array of voxels is either obtained sequentially, one
plane at a time, or produced by an inverse computation that simultaneously calculates them all. Showing the
appearance of any arbitrary plane through this array is a direct analog to the sectioning process that is often used in
sample preparation and imaging. The amount of computation is minimal, requiring only addressing the appropriate
voxels and perhaps interpolating between neighbors.
As shown in Figure 10a, this allows viewing microstructure revealed on planes in any orientation, not just those
parallel to the X, Y, Z axes. Sometimes for structures that are highly oriented and nonrandom, it is possible to
find a section plane that reveals important structural information. But such unique planes violate most of the
assumptions of stereology (IUR sections) and do not pretend to sample the entire structure.
Arbitrary section displays do not reveal very much about most specimens, except for allowing stereological
measurements to test the isotropy of the structure. In the example of Figure 10, which is an ion microscope image
of a two-phase metal structure, the volume fraction of each phase, the mean intercept lengths, and the surface area
per unit volume of the interface can easily be determined from any plane. Three-dimensional imaging is not
required for these stereological calculations. The key topological piece of information about the structure is not
evident in section images. Reconstructing the 3D surface structure (the figure show two different methods for
doing so) reveals that both phases are connected structures of great complexity. This is the kind of topological
information that requires 3D imaging and analysis to access. But the amount of added information, while key to
understanding the structure, requires only visualization, not measurement. Measurement is often performed more
straightforwardly with less effort and more accuracy on two-dimensional images than on three-dimensional ones,
as discussed below.
Figure 10. Ion microscopic images of a two-phase Fe-Cr alloy (data provided by M. K. Miller, Oak Ridge
National Labs): a) arbitrary sections through the voxel array do not show that the regions for each phase are
connected; b) display of the voxels on the surface of one phase; c) outlines of the phase regions; d) rendered
surface image produced by placing facets between the outlines of (c).
In summary, visualization of three-dimensional data sets typically involves too much data to put all on the screen
at once. Selection by discarding parts that are not of interest (e.g., the matrix) or selecting just those parts that are
most easily interpreted (e.g., the surfaces) helps produce interpretable displays. These are often used to create
movies of sequences (e.g., a moving section plane, changing transparency, or rotating point of view) to convey to
others what we have first learned by difficult study. Such pictures are in much demand, but it isn't clear how well
they convey information to the inexperienced, or how they will be efficiently disseminated in a world still
dominated by print media.
Processing
Image processing in three dimensions is based on the same algorithms as used in two dimensions. Most of these
algorithms generalize directly from 2 to 3D. Fourier transforms are separable into operations on rows and columns
in a matrix, the Euclidean distance map has the same meaning and procedure, and so forth. The important change
is that neighborhood operations involve a lot of voxels. In a 2D array, each pixel has 8 neighbors touching edges
or corners. In 3D this becomes 26 voxels, touching faces, edges and corners. For most processing, a spherical
neighborhood is an optimal shape. Constructed on an array of cubic voxels, such a 5-voxel wide neighborhood
involves 57 voxels (Figure 11). Addressing the array to obtain the values for these neighbors adds significant
overhead to the calculation process.
Figure 11. The 57 voxels that form an approximation to a spherical neighborhood
with a diameter of 5 voxels in a cubic array.
A cubic array is not the ideal one for mathematical purposes. Just as in a 2D image with square pixels the problem
of distinguishing the corner and edge neighbors can be avoided by using a grid of hexagonal pixels for greater
isotropy, so in three dimensions a "face-centered cubic" lattice of voxels is maximally isotropic and symmetrical.
But this is not used in practice because the acquisition processes do not lend themselves to it, and computer
addressing and display would be further complicated. Cubic voxels give the best compromise for practical use.
But many acquisition methods instead produce voxels that are not cubic, but as noted before have much different
resolution between planes than within the plane. This causes difficulties for processing since the voxels should be
weighted or included in the neighborhood in proportion to their distance from the center. The usual approaches to
deal with this are to construct tables for the particular voxel spacing present in a given data set, or to re-sample the
data (usually discarding some resolution in one or two directions and interpolating in the others) to construct a
cubic array.
Because of the size of the arrays and the size of the neighborhoods, processing of three-dimensional images is
considerably slower and needs more computer horsepower and memory than processing of two-dimensional
images. The purposes are similar, however. As discussed in earlier chapters, these include correction of defects,
visual enhancement, and assistance for segmentation. Figure 5 shows an example, in which the gradient of voxel
value is calculated by a three-dimensional Sobel operator (combining the magnitude of first derivatives in three
orthogonal directions to obtain the square root of the sum of squares at each pixel) to generate a display of
brightness values that highlights the location of structures in the brain.
Measurement
Global parameters such as volume fraction and surface area can of course be measured directly from 3D arrays by
counting voxels. In the case of surfaces, the area associated with each one depends on the neighbor configuration.
It is not necessarily the case that this type of direct measurement is either more precise nor more accurate than
estimating the results stereologically from 2D images. For one thing, the resolution of the voxel arrays is generally
much poorer. A 256*256*256 cube requires 16 Megabytes of ram (even if only one byte is required for each
voxel). A cube 1000 voxels in each direction would require a Gigabyte. However the data are obtained, by a serial
sectioning technique or volumetric reconstruction, such a large array is unworkable with the current generation of
instrumentation and computers. The size of voxels limits the resolution of the image, and so details are lost in the
definition of structures and surfaces which limits their precise measurement. And accuracy in representing the
overall structure to be characterized generally depends upon adequate sampling. The difficulty in performing 3D
imaging tends to limit the number of samples that are taken. Measurement of global parameters is best performed
by taking many 2D sections that are uniformly spread throughout the structure of interest, carrying out only rapid
and simple measurements on each.
The measurement of feature-specific values suffers from the same limitation. With resolution limited, the range of
sizes of features that can be measured is quite restricted. The size of the voxels increases the uncertainty in the size
of each feature, and the result is often no better than can be determined much more efficiently from 2D images.
Figure 12 shows an example. The structure is a loosely sintered ceramic with roughly spherical particles, imaged
by X-ray microtomography. The figure shows various sections through the structure, and the surfaces of the
particles. Figure 13 shows the measurement results, based on counting the voxels in the 3D array and also by
measuring the circles in 2D sections and using the unfolding method to determine the sizes of the spheres that
must have produced them. Even with the same resolution in the 2D images as in the 3D array, the results are
similar. It would be relatively easy to increase the resolution of 2D section images to obtain a more precise
measurement of sizes.
Counting voxels to determine volume is analogous to counting pixels in 2D to determine area. Other parameters
are not quite so easily constructed. The convex hull in 3D consists of a polygon, ideally with a large number of
sides. In practice, cubes and octahedrons are used. The length (maximum distance between any two points in a
feature) is determined by finding a maximum projection as axes are rotated, and since this need be done only in
fairly crude steps (as discussed in the chapter on measurement of 2D images) this generalizes well to 3D. But the
minimum projected dimension requires much more work.
The midline length of an irregular object can be based on the skeleton. Actually there are two different skeletons
defined in 3D (Halford & Preston, 1984; Lobregt et al., 1980), one consisting of surfaces and a different one of
lines; both require quite a bit of work to extract. Line lengths can be summed as a series of chain links of length 1,
2 and 3 (depending on how the voxels lie adjacent to each other), but the poor resolution tends to bias the
resulting length value (too long for straight lines, too short for irregular ones). Shape parameters in 3D are
typically very ad-hoc. In principle the use of spherical harmonics, similar to the harmonic shape analysis in 2D,
can be used to define the shape. However, the resolution of most images is marginal for this purpose, and the
presence of re-entrant shapes can occur with real features and frustrate this approach.
Figure 12. X-ray microtomographic
reconstruction of sintered particles in a ceramic
(the data set is artificially stretched by a factor of
2 in the vertical direction): a) sections along
arbitrary planes; b) sections along a set of
parallel planes; c) display of the surface voxels
of the spherical particles.
<=1.0 10.0=> Circle Equiv.Diam.( m)
N
u
m
b
e
r
/
U
n
i
t

A
r
e
a
a
<=0.0 Sphere Equiv.Diam ( m) 10.0=>
N
u
m
b
e
r
/
U
n
i
t

V
o
l
u
m
e
b
<=0.0 10.0=> Sphere Equiv.Diam ( m)
N
u
m
b
e
r
/
U
n
i
t

V
o
l
u
m
e
c
Figure 13. Comparison of 2D and 3D
measurement of size of spherical particles in
structure shown in Figure 12: a) size distribution
of circles in 2D plane sections; b) estimated size
distribution of spheres by unfolding the circle
data in (a) - note the negative values; c) directly
measured size distribution of spheres from 3D
voxel array.
Figure 14. Volumetric reconstruction of Golgi-stained neurons (data provided by Vital Images,
Fairfield, Iowa). The two different orientations show the topological appearance of the network,
but the limited resolution of this 256x256x256 array does not actually define a continuous
network nor reveal many of the smaller structures.
Figure 14 shows an example of the power and limitation of 3D imaging. The Golgi-stained neurons can be
displayed volumetrically and the structure rotated to allow viewing of the branching pattern. However, it is
probably not desired and would in any case be very difficult to achieve a quantitative measure of the branching. In
fact, the comparatively poor resolution (as compared to 2D images) means that the linear structures are not
necessarily fully connected in the voxel image, and that some of the finer structures are missing altogether. This
does not impede the ability of the viewer to judge the relative complexity of different structures in an qualitative
sense, but it does restrict the quantitative uses of the images.
The simple conclusion must be that few metric properties of structures or features are efficiently determined using
3D imaging. The resolution of the arrays is not high enough to define the structures well enough, and the amount
of computation required for anything beyond voxel counting is too high. Metric properties are best determined
using plane section images and stereological relationships. On the other hand, topological properties do require 3D
imaging. However, these are not usually measured but simply viewed. Most of the use of 3D imaging is for visual
confirmation of structural understanding that has been obtained and measured in other ways using classical
stereology.
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Practical Stereology

=0.16. If the set of fields included in the count is

. This area fraction is

= 0.28. Statistically valid

Probe population: Points in two dimensional space

, are computed, along with

by applying equation (4.5).

= 30/70 = 0.43 and V

0.46. The fraction

. Add the and counts, (P

). Next, take the ratio [P

=< A > ( Nh) (4.9

= 2 0.10 = 0.20 (m/m

= 2 0.21 = 0.42 (m/m

(occ) = 2 0.08 = 0.16 (m/m

, the total area of this collection of particles. The

, the area fraction occupied

Chapter 7 - Procedures for IUR sampling Page # 128

0.10 0.05 0.025 0.010

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