Introduction to Polarized Light Microscopy

Although much neglected and undervalued as an investigative tool, polarized light

microscopy (Figure 1) provides all the benefits of brightfield microscopy and yet offers a
wealth of information, which is simply not available with any other optical microscopy
technique.

As well as providing information on absorption color and boundaries between minerals of

differing refractive indices obtainable in brightfield microscopy, polarized light microscopy can
distinguish between isotropic and anisotropic materials. The technique exploits optical
properties of anisotropy to reveal detailed information about the structure and composition of
materials, which are invaluable for identification and diagnostic purposes.

Isotropic materials, which include gases, liquids, unstressed glasses and cubic crystals,
demonstrate the same optical properties in all directions. They have only one refractive index
and no restriction on the vibration direction of light passing through them. Anisotropic
materials, in contrast, which include 90 percent of all solid substances, have optical
properties that vary with the orientation of incident light with the crystallographic axes. They
demonstrate a range of refractive indices depending both on the propagation direction of
light through the substance and on the vibrational plane coordinates. More importantly,
anisotropic materials act as beam splitters and divide light rays into two parts (as illustrated
in Figure 1). The technique of polarizing microscopy exploits the interference of the split light
rays, as they are re-united along the same optical path to extract information about these
materials.

Polarized light microscopy is perhaps best known for its geological applications--primarily for
the study of minerals in rock thin sections, but it can also be used to study many other
materials. These include both natural and industrial minerals whether refined, extracted or
manufactured, composites such as cements, ceramics, mineral fibers and polymers, and
crystalline or highly ordered biological molecules such as DNA, starch, wood and urea. The

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technique can be used both qualitatively and quantitatively and is an outstanding tool for
materials science, geology, chemistry, biology, metallurgy and even medicine.

While an understanding of the analytical techniques of polarized microscopy may be perhaps

more demanding than other forms of microscopy, it is well worth pursuing, simply for the
enhanced information that can be obtained over brightfield imaging. An awareness of the
principles of polarizing microscopy is also essential for the effective interpretation of
differential interference contrast (DIC) microscopy.

Polarized Light

The wave model of light describes light waves vibrating at right angles to the direction of
travel of light with all vibration directions being equally probable. This is "common" light. In
plane-polarized light there is only one vibration direction (Figure 1). The human eye-brain
system has no sensitivity to the vibration directions of light, and plane-polarized light can
only be detected by an intensity or color effect, for example, by reduced glare when wearing
polarized sun glasses.

The most widely used material is PolaroidTM film. Invented by Land in 1932, Polaroid film
consists of long chain polymers, treated with light absorbing dyes, and stretched so that the
chains are aligned. Light vibrating parallel with the chains is absorbed while light
perpendicular to the chains is transmitted.

There are two polarizing filters in a polarizing microscope - the polarizer and analyzer (see
Figure 1). The polarizer is situated below the specimen stage usually with its permitted
vibration direction fixed in the left-to-right, East-West direction, although this is usually
rotatable through 360 degrees. The analyzer, usually aligned North-South but again rotatable
on some microscopes, is sited above the objectives and can be moved in and out of the light
path as required. When both the analyzer and polarizer are in the optical path, their
permitted vibration directions are positioned at right angles to each other. In this
configuration, the polarizer and analyzer are said to be crossed, with no light passing
through the system and a dark field of view present in the eyepieces.

The polarizer and analyzer are the essential components of the polarizing microscope - but
other desirable features include:

• A rotating specimen stage to facilitate orientation studies with centration of the

objectives and stage with the microscope optical axis to make the center of rotation
coincide with the center of the field of view.
• Strain free objectives – stress in assembly can produce optical effects under
polarized light, a factor that could complicate observations.

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 • An eyepiece fitted with a cross wire graticule to mark the center of the field of view.
Often, the cross wire graticule is substituted for a photomicrography graticule that
assists in focusing the specimen and composing images with a set of frames
bounding the area of the viewfield to be captured either digitally or onto film.
• A Bertrand lens – to enable easy examination of the objective rear focal plane, to
allow accurate adjustment of the illuminating aperture diaphragm and to view
interference figures, as presented in Figure 2.
• A slot to allow the insertion of compensators/retardation plates between the
polarizers, which are used to enhance optical path differences in the specimen. In
most modern microscope designs, this slot is placed either in the microscope
nosepiece or an intermediate tube positioned between the body and eyepiece tubes.
Compensation plates inserted into the slot are then situated between the specimen
and the analyzer.

Polarizing microscopy can be used both with reflected and transmitted light. Reflected light is
useful for the study of opaque materials such as mineral oxides and sulphides, metals and
silicon wafers (Figure 3). Reflected light techniques require a dedicated set of objectives that
have not been corrected for viewing through the coverslip, and those for polarizing work
should, again, be stress free.

Illustrated in Figure 3 is a series of reflected polarized light photomicrographs of typical

specimens imaged utilizing this technique. On the left (Figure 3(a)) is a digital image
revealing surface features of a microprocessor integrated circuit. Birefringent elements
employed in the fabrication of the circuit are clearly visible in the image, which displays a
portion of the chip's arithmetic logic unit. The polished surface of a ceramic superconducting
tape (Yttrium-1,2,3) is presented in Figure 3(b), which shows birefringent crystalline areas
with interference colors interspersed in a matrix of isotropic binder. Metallic thin films are also
visible with reflected polarized light. Figure 3(c) illustrates blisters that form imperfections in
an otherwise confluent thin film of copper (about 0.1 micron thick) sandwiched over a
nickel/sodium chloride substrate to form a metallic superlattice assembly.

Careful specimen preparation is essential for good results. The method chosen will depend
on the type of material studied. In geological applications the standard thickness for rock thin
sections is 25-30 micrometers. Specimens can be ground down with diamond impregnated
wheels and then hand finished to the correct thickness using abrasive powders of
successively decreasing grit size. Softer materials can be prepared in a manner similar to
biological samples using a microtome. Slices between one and 40 micrometers thick are
used for transmitted light observations. These should be strain-free and free from any knife
marks. Specimens are mounted between the slide and the coverslip using a mounting
medium whose composition will depend on the chemical and physical nature of the

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specimen. This is particularly significant in the study of synthetic polymers where some
media can chemically react with and cause structural changes to the material being studied.

Making Use of Anisotropy

Different levels of information can be obtained in plane-polarized light (analyzer out of the
optical path) or with crossed polarizers (analyzer inserted into the optical path). Observations
in plane-polarized light reveal details of the optical relief of the specimen, which is
manifested in the "visibility" of boundaries, and increases with the increase of refractive index
across them. Differences in the refractive indices of the mounting adhesive and the
specimen determine the extent to which light is scattered as it emerges from the uneven
specimen surface. Materials with high relief, which appear to stand out from the image, have
refractive indices, which are appreciably different from that of the mountant. Immersion
refractometry is used to measure substances having unknown refractive indices by
comparison with oils of known refractive index.

Examinations of transparent or translucent materials in plane-polarized light will be similar to

those seen in natural light until the specimen is rotated about the optical axis of the
microscope. Then observers may see changes in the brightness and/or the color of the
material being examined. This pleochroism, that is, variation of absorption color with
vibration direction of the light, depends on the orientation of the material in the light path and
is a characteristic of anisotropic materials only. An example of a material showing
pleochroism is crocidolite, more commonly known as blue asbestos. This effect helps in its
identification.

Polarization colors result from the interference of the two components of light split by the
anisotropic specimen and may be regarded as white light minus those colors that are
interfering destructively. Figure 2 illustrates conoscopic images of uniaxial and biaxial
crystals observed at the objective rear focal plane. Interference patterns are formed by light
rays traveling along different axes of the crystal being observed. Uniaxial crystals (Figures
2(a) and 2(b)) display an interference pattern consisting of two intersecting black bars
(termed isogyres) that form a Maltese cross-like pattern. When illuminated with white
(polarized) light, birefringent specimens produce circular distributions of interference colors
(Figure 2), with the inner circles, called isochromes, consisting of increasingly lower order
colors (see the Michel-Levy interference color chart, Figure 4). A common center for both the
black cross and the isochromes is termed the melatope, which denotes the origin of the light

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rays traveling along the optical axis of the crystal. Biaxial crystals display two melatopes
(Figure 2(c)) and a far more complex pattern of interference rings.

The two components of light travel at different speeds through the specimen and have
different refractive indices, or refringences. Birefringence is the numerical difference
between these refringences. The faster beam emerges first from the specimen with an
optical path difference (OPD), which may be regarded as a "winning margin" over the slower
one. The analyzer recombines only components of the two beams traveling in the same
direction and vibrating in the same plane. The polarizer ensures that the two beams have the
same amplitude at the time of recombination for maximum contrast.

There is constructive and destructive interference of light in the analyzer, depending on the
OPD on the specimen and the wavelength of the light, which can be determined from the
order of polarization color(s). This relies on the properties of the specimen, including the
thickness difference between the refractive index and the birefringence of the two beams,
which has a maximum value dependent on the specimen and on the direction of travel of
light through the specimen. Optical path differences can be used to extract valuable "tilt"
information from the specimen.

Superimposed on the polarization color information is an intensity component. As the

specimen is rotated relative to the polarizers, the intensity of the polarization colors varies
cyclically, from zero (extinction) up to a maximum after 45 degrees and back down to zero
after a 90-degree rotation. That is why a rotating stage and centration are provided, which
are critical on a polarizing microscope. Centration of the objective and stage ensures that the
center of the stage rotation coincides with the center of the field of view, a great
convenience, as anyone who has tried to manage without it will know.

Whenever the specimen is in extinction, the permitted vibration directions of light passing
through are parallel with those of either the polarizer or analyzer. This can be related to
geometrical features of the specimen, such as fiber length, film extrusion direction, and
crystal faces. In crossed polarizers, isotropic materials can be easily distinguished from
anisotropic materials as they remain permanently in extinction (remain dark) when the stage
is rotated through 360 degrees.

To help in the identification of fast and slow beams, or to improve contrast when polarization
colors are of low order, such as dark grey, accessory plates can be inserted in the optical
path. These will cause color changes in the specimen, which can be interpreted with the help
of a polarization color chart (Michel-Levy chart; see Figure 4). These charts show the
polarization colors provided by optical path differences from 0 to 1800-3100 nanometers
together with birefringence and thickness values. The wave plate produces its own optical
path difference. When the light passes first through the specimen and then the accessory
plate, the OPDs of the wave plate and the specimen are either added together or subtracted
from one another in the way that "winning margins" of two races run in succession are
calculated. They are added when the slow vibration directions of the specimen and
accessory plate are parallel, and subtracted when the fast vibration direction of the specimen
coincides with the slow vibration direction of the accessory plate. If the slow and fast
directions are known for the accessory plate (they are usually marked on the mount of
commercially available plates), then those of the specimen can be deduced. Since these
directions are characteristic for different media, they are well worth finding out and are
essential for orientation and stress studies.

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The strengths of polarizing microscopy can best be illustrated by examining particular case
studies and their associated images. All images illustrated in this section were recorded with
a Nikon Eclipse E600 microscope equipped with polarizing accessories, a research grade
microscope designed for analytical investigations.

Identification of Asbestos Fibers

Asbestos is a generic name for a group of naturally occurring mineral fibers, which have
been widely used, for example, in insulating materials, brake pads and to reinforce concrete.
They can be harmful to health when inhaled and it is important that their presence in the
environment be easily identified. Samples are commonly screened using scanning electron
microscopy and x-ray microanalysis, but polarizing microscopy provides a quicker and easier
alternative that can be utilized to distinguish between asbestos and other fibers and between
the major types asbestos – chrysotile, crocidolite and amosite. From a health care point of
view, it is believed that the amphibole asbestos varieties (crocidolite and amosite) are more
harmful than the serpentine, chrysotile.

Plane-polarized light provides information about gross fiber morphology, color, pleochroism
and refractive index. Glass fibers will be unaffected by rotation under plane-polarized light
while asbestos fibers will display some pleochroism. Chrysotile asbestos fibrils may appear
crinkled, like permed or damaged hair, under plane-polarized light, whereas crocidolite and
amosite asbestos are straight or slightly curved. Chrysotile has a refractive index of about
1.550, amosite 1.692 and crocidolite, 1.695.

With the use of crossed polars it is possible to deduce the permitted vibration direction of the
light as it passes through the specimen, and with the whole wave plate, a determination of
the slow and fast vibration directions (Figure 5). Under crossed polars, chrysotile shows pale
interference colors - low order whites (Figure 5(a)). When a full wave plate is added (530-560
nanometers), the colors are transformed. Aligned Northeast-Southwest, the wave plate is
additive and gives blue and yellow in the fiber (Figure 5(b)). When aligned Northwest-
Southeast (Figure 5(c)) the plate is subtracting to give a paler yellow fiber with no blue. From
this it is possible to deduce that the slow vibration direction is parallel with the long axis of
the fiber. Amosite is similar in this respect.

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Crocidolite displays blue colors, pleochroism and murky brown polarization colors and has its
fast vibration direction parallel with its length. In summary, identification of the three asbestos
fiber types depends on shape, refractive indices, pleochroism, birefringence, and fast and
slow vibration directions.

Uncovering the History of Rock Formation

Phyllite - An examination of geological thin sections using polarizing microscopy, as well as

providing information on component minerals, can reveal a great deal about how the rock
was formed. Phyllite, a metamorphic rock, clearly shows the alignment of crystals under the
effects of heat and stress. Small-scale folds are visible in the plane-polarized image (Figure
6(a)) and more clearly defined under crossed polars (Figure 6(b)) with and without the wave
plate. The crossed polars image reveals that there are several minerals present--quartz in
grey and whites and micas in higher order colors. The alignment of the micas is clearly
apparent. Addition of the wave plate (Figure 6(c)) improves contrast for clear definition in the
image.

Oolite - Oolite, a light gray rock composed of siliceous oolites cemented in compact silica, is
formed in the sea. The mineral's name is derived from its structural similarity to fish roe -
caviar! It forms in the sea when sand grains are rolled by gentle currents over beds of
calcium carbonate or other minerals. These minerals build up around the sand grains and
subsequent cementation transforms the grains into coherent rock. The thin sections show
the original quartz nuclei (Figure 7(a-c)) on which the build up of carbonate mineral occurred.

In plane-polarized light (Figure 7(a)), the quartz is virtually invisible having the same
refractive index as the cement, while the carbonate mineral with a different refractive index
shows high contrast. The crossed polarizer image (Figure 7(b)) shows quartz grains in grays

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and whites and the calcium carbonate in the characteristic biscuit colored, high order whites.
The groups of quartz grains in some of the cores reveal that these are polycrystalline and
are metamorphic quartzite particles. When a full-wave retardation plate is inserted into the
optical path (Figure 7(c)), optical path differences become apparent in the specimen, and
contrast is enhanced.

Natural and Synthetic Polymers

During the solidification of polymer melts there may be some organization of the polymer
chains, a process that is often dependent upon the annealing conditions. When nucleation
occurs, the synthetic polymer chains often arrange themselves tangentially and the solidified
regions grow radially. These can be seen in crossed polarized illumination as white regions
with the black extinction crosses. When these spherulites impinge, their boundaries become
polygonal. This can be clearly seen in crossed polars but not under plane-polarized light.

The addition of the whole wave plate (Figure 8(a)) confirms the tangential arrangement of
the polymer chains. The banding occurring in these spherulites indicates slow cooling of the
melt allowing the polymer chains to grow out in spirals. This information on thermal history is
almost impossible to collect by any other technique. Nucleation in polymer melts can take
place as the result of accidental contamination or contact with a nucleating surface and can
lead to substantial weakening of the product. Identification of nucleation can be a valuable
aid for quality control.

Other polymers may not be birefringent (evidenced by the polycarbonate specimen

illustrated in Figure 8(b)), and do not display substantial secondary or tertiary structure. In
other cases, both biological and synthetic polymers can undergo a series of lyotropic or
thermotropic liquid crystalline phase transitions, which can often be observed and recorded
in a polarized light microscope. Figure 8(c) illustrates a birefringent columnar-hexatic liquid
crystalline phase exhibited by DNA at very high concentrations (exceeding 300
milligrams/milliliter).

Nylon Fibers - Observations under plane-polarized light (Figure 9(a)) reveal refractive index
differences between the fiber and the mountant and the presence of opacifying titanium
dioxide particles. The image under crossed polars (Figure 9(b)) shows third order
polarization colors and their distribution across the fibers indicates that this is a cylindrical
and not a lobate fiber useful in predicting mechanical strength. The use of the quartz wedge
(Figure 9(c)) enables the determination of optical path differences for birefringence
measurements.

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In summary, polarizing microscopy provides a vast amount of information about the
composition and three-dimensional structure of a variety of samples. Virtually unlimited in its
scope, the technique can reveal information about thermal history and the stresses and
strains to which a specimen was subjected during formation. Useful in manufacturing and
research, polarizing microscopy is a relatively inexpensive and accessible investigative and
quality control tool, which can provide information unavailable with any other technique

Polarized Light Microscopy

Microscope Alignment

In polarized light microscopy, proper alignment of the various optical and mechanical
components is a critical step that must be conducted prior to undertaking quantitative
analysis between crossed polarizers alone, or in combination with retardation plates and
compensators. Several essential components must be correctly positioned with respect to
both the microscope optical axis and to other mechanical and optical components. The
series of alignment steps outlined below are intended for general use with polarized light
microscopes and should be applicable to both student and research-level instruments.

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The basic optical and mechanical components of a polarized light microscope are illustrated
in Figure 1. At a minimum, these microscopes must be equipped with two linear polarizing
elements. One of the polarizers (termed the Polarizer in Figure 1) is placed beneath the
stage inside or below the condenser, or covering the microscope light source exit port in the
base. The second polarizer (termed the Analyzer in Figure 1) is installed above the objective
rear aperture, either in a nosepiece slot or within an intermediate tube positioned between
the frame and the observation tubes. Many polarized light microscopes are equipped with a
graduated circular stage that can rotate through 360 degrees for quickly changing specimen
orientation with respect to the polarizer and analyzer transmission axes. In addition, one of
the eyepieces in a polarized light microscope should have a crosshair reticule to identify the
center and axes of the optical field. Advanced microscopes include a Bertrand lens for
conoscopic observation of interference patterns focused in the objective rear aperture, but
this accessory is not absolutely necessary for routine observations. Finally, polarized light
microscopes should have an accessory slot in the nosepiece or intermediate tube into which
a retardation plate or compensator can be inserted for quantitative analysis of birefringent
specimens.

Polarized Microscope Alignment Procedure

The first step in polarized light microscope alignment is to establish proper Köhler
illumination with both the polarizer and analyzer removed from the optical pathway. Place a
brightfield (stained) specimen on the stage and rotate the 10x objective into the observation
position. Focus the specimen with the objective and close the field diaphragm iris to its
smallest setting. Bring the partially closed field diaphragm into sharp focus in the same plane
as the specimen by adjusting the condenser height. Use the condenser centering screws to
translate the image of the field diaphragm iris opening to the center of the eyepiece
crosshairs, and then open the diaphragm until the leaves are just outside the viewfield.

Before inserting the polarizer and analyzer, the 360-degree circular rotating stage should be
centered to the 10x objective so specimens will remain in the exact center of the viewfield
when they are being rotated. Centering of the stage and objectives is also an important step
for quantitative analysis of birefringence and optical path differences. A majority of the
circular microscope stages pivot on three adjustable pins, one at the front of the stage for
correct positioning and the other two equally spaced at 120-degree angles near the rear of
the stage. The stage can be centered with minute adjustments to the rear pivot pins by
turning the centering screws. Start by locating a small circular feature on a relatively simple
brightfield specimen in an area devoid of other distracting structures. Next, relocate the
chosen feature to the center of the viewfield in the eyepiece crosshairs (see Figure 2(a)).
Rotate the stage through 180 degrees until the centered specimen feature is overlapped by
one of the vertical eyepiece crosshairs (Figure 2(b)). Using the pivot screws, relocate the
specimen feature to a distance approximately half way back to the crosshair center (Figure
2(c)). Finally, move the specimen feature back to the center of the crosshairs (Figure 2(a))
and repeat the alignment sequence. After a few cycles, the specimen feature should be
centered in the viewfield as the stage is rotated.

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After the stage has been centered with respect to the microscope optical axis with the 10x
objective installed in the optical train, the other objectives (4x, 20x, 40x, and 100x) should be
sequentially centered to the stage. Microscopes having centerable objectives contain a pair
of Allen (or a similar drive) setscrews in the nosepiece that translate the objective laterally
within its seat. Each objective can be centered using the procedure described above for the
circular stage by using the objective centering tools provided by the manufacturer. Accurate
centering of the stage and all objectives is paramount to quantitative analysis in polarized
light, and avoids the operator losing sight of specific features in the viewfield during stage
rotation. After completing the centering procedure, review the steps to ensure all of the
objectives on the microscope are accurately centered before continuing.

Establishing Köhler illumination and centering the microscope stage and objectives is easily
accomplished in brightfield mode (but can also be undertaken with a birefringent specimen in
polarized light mode, if necessary). To continue with alignment of the polarizing components,
install the fixed or rotating polarizing element in the condenser or a housing on the light
source exit port in the microscope base. Many polarized light microscopes are equipped with
a fixed polarizer that has a transmission vibration axis oriented East-West, or left-to-right
when standing in front of the microscope. Other microscopes have polarizers that can be
rotated through 180 or 360 degrees, and are often mounted into frames with graduated
increments ranging from 1 to 90 degrees. Ensure that the polarizer is correctly secured into
position and either permanently fixed into its holder or placed in the zero position. The
polarizer transmission vibration axis must be set to the East-West orientation in this step.

Insert the analyzer into the microscope nosepiece or intermediate tube. Virtually all research-
level polarized light microscopes have a rotating analyzer mounted in a rectangular frame
that enables rotation of the element through 180 or 360 degrees. Student microscopes often
have a fixed analyzer (as well as a fixed polarizer) to ease the burden of crossed polarizer
alignment. Rotate the analyzer while examining a birefringent specimen on the stage with
the 10x objective. When the transmission axes of the analyzer and polarizer are crossed at a
90-degree angle, maximum specimen birefringence (equivalent to maximum specimen
brightness) should be observed on a very dark background (a condition that is termed
maximum extinction). Under crossed polarized illumination, the vibration axis of the
analyzer should be oriented North-South or front-to-back while standing in front of the
microscope. A critical assessment for maximum extinction can only be accomplished while
observing the background intensity through the microscope eyepieces. Keep in mind that at
this point, maximum extinction only indicates that the polarizers are crossed, but does not
guarantee that their transmission axes are perfectly oriented East-West and North-South.
The latter aspect is addressed in the next step.

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Calibration of the polarizer and analyzer axis alignment can be accomplished by one of two
methods. Many manufacturers provide a polarized light microscope orientation plate with
their research-level microscopes to assist in alignment of the crossed polarizers. These
plates contain a perfectly cut rectangular thin birefringent crystal having an optical axis that is
parallel to the long edge of the crystal (see Figure 3). The crystal is mounted flat on a
microscope slide and designed specifically for use on circular polarized light microscope
stages. When the long axis crystal is oriented at a 45-degree angle to the polarizer and
analyzer transmission azimuths (Figure 3(a)), maximum birefringence is observed in
polarized light. Rotating the crystal towards the polarizer axis progressively reduces the level
of birefringence (Figure 3(b)) until it is eliminated when the long axis is exactly parallel to the
polarizer transmission azimuth (and the horizontal eyepiece crosshair; see Figure 3(c)).

At this point, the crystal birefringence is completely extinguished and should be

indistinguishable from the background. Note that the outline of the crystal is artificial in
Figure 3(c), and is included only to demonstrate that the long axis is parallel to the polarizer
azimuth. If the polarizer and analyzer are not positioned exactly in a East-West and North-
South orientation (respectively), then the long axis of the crystal will not be parallel to the
eyepiece crosshairs when the birefringence is completely extinguished. In this case, rotate
the long axis of the crystal until it is parallel to the horizontal microscope crosshair, and then
rotate the polarizer until minimum birefringence is observed in the eyepieces. Next, rotate
the analyzer until the crystal birefringence is completely extinguished.

If a Bertrand lens or focusing telescope is available with the polarized light microscope,
alignment of the polarizer and analyzer transmission azimuths can be performed using one
of these optical components. Without a specimen on the stage, cross the polarizers and
insert the eyepiece telescope into the observation tubes or swing the Bertrand lens into the
optical pathway. Focus the telescope or Bertrand lens on the objective rear aperture. A dark
polarization cross can be observed at maximum extinction (Figure 4(a)) with brighter regions
flanking the arms of the cross, which should be oriented vertically and horizontally. In most
cases, the horizontal and vertical components of the polarization cross are wider in the
center of the field and narrower at the periphery.

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The polarization cross geometry originates from the depolarizing effects of spherical lens
surfaces in the condenser and objective, and may vary in size and shape depending upon
the quality (strain reduction or elimination) of the lens systems. However, the presence of a
polarization cross is normal in most microscopes and can not generally be considered an
artifact. The cross should be centered in the viewfield (when superimposed on the eyepiece
crosshairs) and appear perfectly aligned in a North-South and East-West direction. If this is
not the case (as illustrated in Figure 4(c)), the polarizer and analyzer should be rotated in
combination until the proper orientation is achieved. As discussed above, perfect alignment
of the polarization cross (thus, the polarizers) with a transmission axis at zero degrees is
essential for performing quantitative measurements of birefringence azimuthal angles with or
without a retardation plate or compensator in the optical pathway. The polarization cross
reference calibration is also useful if a fixed polarizer or analyzer becomes misaligned in the
mount.
The final step in polarized light microscope alignment is to adjust the condenser aperture
diaphragm so the bright outer regions of the polarization cross that are visible at the edge of
the objective aperture are blocked. Figure 4(b) illustrates a properly adjusted condenser
diaphragm. This step dramatically improves the extinction factor of the microscope optical
system, and the field will appear maximally dark when viewed in orthoscopic mode (without a
Bertrand lens or focusing telescope). After the microscope has been aligned and the
polarization axes calibrated, it is ready for quantitative analysis of birefringent specimens

Polarization of Light

When light travels through a linear polarizing material, a selected vibration plane is passed
by the polarizer, while electric field vectors vibrating in all other orientations are blocked.
Linearly polarized light transmitted through a polarizer can be either passed or absorbed by
a second polarizer, depending upon the electric vector transmission azimuth orientation of
the second polarizing material. This tutorial explores the effect of rotating two polarizers on
an incident beam of white light.

The tutorial initializes with red, green, and blue sine waves (simulating white light)
originating from the left-hand side of the window and striking a linear polarizer (labeled
Polarizer 1). This polarizer allows only light waves that are vibrating parallel to the
transmission axis (oriented vertically) to pass. Polarizer 2 is initially positioned with its
transmission axis parallel to that of polarizer 1, and also passes linearly polarized light
transmitted through the first polarizer. To operate the tutorial, use the Angle 1 and Angle 2
sliders to adjust the orientation of the polarizers with respect to the incident white illumination

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(through a range of 360 degrees). When the slider bars are translated, the polarizers are
rotated, affecting the transmission of light through the virtual polarizing system.

In linear polarizers, incident light waves having electric vector vibrations that are parallel to
the crystal axis of the polarizer are absorbed. Many of the incident waves will have a vector
orientation that is oblique, but not perpendicular to the crystal axis, and will only be partially
absorbed. The degree of absorption for oblique light waves is dependent upon the vibration
angle at which they impact the polarizer. Those rays that have angles close to parallel with
respect to the crystal axis will be adsorbed to a much greater degree than those having
angles close to the perpendicular. The most common Polaroid filters (termed the H-series)
transmit only about 25 percent of the incident light beam, but the degree of polarization of
the transmitted rays exceeds 99 percent.

A number of applications, most notably polarized optical microscopy, rely on crossed

polarizers to examine birefringent or doubly refracting specimens. When two polarizers are
crossed, their transmission axes are oriented perpendicular to each other and light passing
through the first polarizer is completely extinguished, or absorbed, by the second polarizer,
which is typically termed an analyzer. The light-absorbing quality of a dichroic polarizing filter
determines exactly how much random light is extinguished when the polarizer is utilized in a
crossed pair, and is referred to as the extinction factor of the polarizer. Quantitatively, the
extinction factor is determined by the ratio of light that is passed by a pair of polarizers when
their transmission axes are oriented parallel versus the amount passed when they are
positioned perpendicular to each other. In general, extinction factors between 10,000 and
100,000 are required to produce jet-black backgrounds and maximum observable specimen
birefringence (and contrast) in polarized optical microscopy.

The amount of light passing through a crossed pair of high-quality polarizers is determined
by the orientation of the analyzer with respect to the polarizer. When the polarizers are
oriented perpendicular to each other, they display a maximum level of extinction. However, at
other angles, varying degrees of extinction are obtained, as illustrated by the vector
diagrams presented in Figure 1. The analyzer is utilized to control the amount of light
passing through the crossed pair, and can be rotated in the light path to enable various
amplitudes of polarized light to pass through. In Figure 1(a), the polarizer and analyzer have

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parallel transmission axes and the electric vectors of light passing through the polarizer and
analyzer are of equal magnitude and parallel to each other.

Rotating the analyzer transmission axis by 30-degrees with respect to that of the polarizer
reduces the amplitude of a light wave passing through the pair, as illustrated in Figure 1(b).
In this case, the polarized light transmitted through the polarizer can be resolved into
horizontal and vertical components by vector mathematics to determine the amplitude of
polarized light that is able to pass through the analyzer. The amplitude of the ray transmitted
through the analyzer is equal to the vertical vector component (illustrated as the yellow arrow
in Figure 1(b)).

Continued rotation of the analyzer transmission axis, to a 60-degree angle with respect to
the transmission axis of the polarizer, further reduces the magnitude of the vector component
that is transmitted through the analyzer (Figure 1(c)). When the analyzer and polarizer are
completely crossed (90-degree angle), the vertical component becomes negligible (figure
1(d)) and the polarizers have achieved their maximum extinction value.

The amount of light passing through a pair of polarizers can be quantitatively described by
applying Malus' cosine-squared law, as a function of the angles between the polarizer
transmission axes, utilizing the equation:

I = I(o) • cos2θ

where I is the intensity of light passing through the analyzer (and the total amount of light
passed through the pair of crossed polarizers), I(o) is the intensity of light that is incident
upon the polarizer, and θ is the angle between the transmission axes of the polarizer and
analyzer. By examining the equation, it can be determined that when the two polarizers are
crossed (θ = 90 degrees), the intensity is zero. In this case, light passed by the polarizer is
completely extinguished by the analyzer. When the polarizers are partially crossed at 30 and
60 degrees, the light transmitted by the analyzer is reduced by 25 percent and 75 percent,
respectively

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 Introduction to Fluorescence Microscopy

The absorption and subsequent re-radiation of light by organic and inorganic specimens is
typically the result of well-established physical phenomena described as being either
fluorescence or phosphorescence. The emission of light through the fluorescence process
is nearly simultaneous with the absorption of the excitation light due to a relatively short time
delay between photon absorption and emission, ranging usually less than a microsecond in
duration. When emission persists longer after the excitation light has been extinguished, the
phenomenon is referred to as phosphorescence.

British scientist Sir George G. Stokes first described fluorescence in 1852 and was
responsible for coining the term when he observed that the mineral fluorspar emitted red
light when it was illuminated by ultraviolet excitation. Stokes noted that fluorescence
emission always occurred at a longer wavelength than that of the excitation light. Early
investigations in the 19th century showed that many specimens (including minerals, crystals,
resins, crude drugs, butter, chlorophyll, vitamins, and inorganic compounds) fluoresce when
irradiated with ultraviolet light. However, it was not until the 1930s that the use of
fluorochromes was initiated in biological investigations to stain tissue components, bacteria,
and other pathogens. Several of these stains were highly specific and stimulated the
development of the fluorescence microscope.

The technique of fluorescence microscopy has become an essential tool in biology and the
biomedical sciences, as well as in materials science due to attributes that are not readily
available in other contrast modes with traditional optical microscopy. The application of an
array of fluorochromes has made it possible to identify cells and sub-microscopic cellular
components with a high degree of specificity amid non-fluorescing material. In fact, the
fluorescence microscope is capable of revealing the presence of a single molecule. Through
the use of multiple fluorescence labeling, different probes can simultaneously identify several

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target molecules simultaneously. Although the fluorescence microscope cannot provide
spatial resolution below the diffraction limit of specific specimen features, the detection of
fluorescing molecules below such limits is readily achieved.

A variety of specimens exhibit autofluorescence (without the application of fluorochromes)

when they are irradiated, a phenomenon that has been thoroughly exploited in the fields of
botany, petrology, and the semiconductor industry. In contrast, the study of animal tissues
and pathogens is often complicated with either extremely faint or bright, nonspecific
autofluorescence. Of far greater value for the latter studies are added fluorochromes (also
termed fluorophores), which are excited by specific wavelengths of irradiating light and emit
light of defined and useful intensity. Fluorochromes are stains that attach themselves to
visible or sub-visible structures, are often highly specific in their attachment targeting, and
have a significant quantum yield (the ratio of photon absorption to emission). The
widespread growth in the utilization of fluorescence microscopy is closely linked to the
development of new synthetic and naturally occurring fluorophores with known intensity
profiles of excitation and emission, along with well-understood biological targets.

Fundamentals of Excitation and Emission

The basic function of a fluorescence microscope is to irradiate the specimen with a desired
and specific band of wavelengths, and then to separate the much weaker emitted
fluorescence from the excitation light. In a properly configured microscope, only the emission
light should reach the eye or detector so that the resulting fluorescent structures are
superimposed with high contrast against a very dark (or black) background. The limits of
detection are generally governed by the darkness of the background, and the excitation light
is typically several hundred thousand to a million times brighter than the emitted
fluorescence.

Illustrated in Figure 1 is a cutaway diagram of a modern epi-fluorescence microscope

equipped for both transmitted and reflected fluorescence microscopy. The vertical illuminator
in the center of the diagram has the light source positioned at one end (labeled the episcopic
lamphouse) and the filter cube turret at the other. The design consists of a basic reflected
light microscope in which the wavelength of the reflected light is longer than that of the
excitation. Johan S. Ploem is credited with the development of the vertical illuminator for
reflected light fluorescence microscopy. In a fluorescence vertical illuminator, light of a
specific wavelength (or defined band of wavelengths), often in the ultraviolet, blue or green
regions of the visible spectrum, is produced by passing multispectral light from an arc-
discharge lamp or other source through a wavelength selective excitation filter.
Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also
termed a dichroic) mirror or beamsplitter, through the microscope objective to bath the
specimen with intense light. If the specimen fluoresces, the emission light gathered by the
objective passes back through the dichromatic mirror and is subsequently filtered by a
barrier (or emission) filter, which blocks the unwanted excitation wavelengths. It is important
to note that fluorescence is the only mode in optical microscopy where the specimen,
subsequent to excitation, produces its own light. The emitted light re-radiates spherically in
all directions, regardless of the excitation light source direction.

Epi-fluorescence illumination is the overwhelming choice of techniques in modern

microscopy, and the reflected light vertical illuminator is interposed between the observation
viewing tubes and the nosepiece housing the objectives. The illuminator is designed to direct
light onto the specimen by first passing the excitation light through the microscope objective
(which in this configuration, acts as a condenser) on the way toward the specimen, and then

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using that same objective to capture the emitted fluorescence. This type of illuminator has
several advantages. The fluorescence microscope objective serves first as a well-corrected
condenser and secondly as the image-forming light gatherer. Being a single component, the
objective/condenser is always in perfect alignment. A majority of the excitation light reaching
the specimen passes through without interaction and travels away from the objective, and
the illuminated area is restricted to that which is observed through the eyepieces (in most
cases). Unlike the situation in some contrast enhancing techniques, the full numerical
aperture of the objective is available when the microscope is properly configured for Köhler
illumination. Finally, it is possible to combine with or alternate between reflected light
fluorescence and transmitted light observation and the capture of digital images.

As presented in Figure 1, the reflected light vertical illuminator comprises an arc-discharge

lamphouse at the rear end (usually a mercury or xenon burner). Excitation light travels along
the illuminator perpendicular to the optical axis of the microscope, passes through collector
lenses and a variable, centerable aperture diaphragm, and then through a variable,
centerable field diaphragm (see Figure 1). The light then impinges upon the excitation filter
where selection of the desired band and blockage of unwanted wavelength occurs. The
selected wavelengths, after passing through the excitation filter, reach the dichromatic
beamsplitting mirror, which is a specialized interference filter that efficiently reflects shorter
wavelength light and efficiently passes longer wavelength light. The dichromatic beamsplitter
is tilted at a 45-degree angle with respect to the incoming excitation light and reflects this
illumination at a 90-degree angle directly through the objective optical system and onto the
specimen. Fluorescence emission produced by the illuminated specimen is gathered by the
objective, now serving in its usual image-forming function. Because the emitted light consists
of longer wavelengths than the excitation illumination, it is able to pass through the
dichromatic mirror and upward to the observation tubes or electronic detector.

Most of the scattered excitation light reaching the dichromatic mirror is reflected back toward
the light source, although a minute quantity often passes through and is absorbed by the
internal coating of the mirror block. Before the emitted fluorescence can reach the eyepiece
or detector, it must first pass through the barrier or suppression filter. This filter blocks
(suppresses) any residual excitation light and passes the desired longer emission
wavelengths. In most reflected light illuminators, the excitation filter, dichromatic mirror, and
barrier filter are incorporated into an optical block (often referred to as a cube), as illustrated
in Figure 2. Modern fluorescence microscopes are capable of accommodating between four
and six fluorescence cubes (usually on a revolving turret or through a slider mechanism; see
Figure 1) and permit the user to easily attach replacement aftermarket excitation and barrier
filters, as well as dichromatic mirrors.

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The vertical illuminator design should enable the user to adjust the microscope for Köhler
illumination, providing a bright and even illumination aperture across the entire field of view.
The corrected condensing lenses of the optical system should ensure that the image of the
centerable aperture diaphragm is conjugate with the rear aperture of the focused objective.
In modern illuminators, the image of the pre-focused, centerable field diaphragm is conjugate
to the focused specimen and the plane of the fixed eyepiece diaphragm.

The illuminator lamphouse usually incorporates an infrared light suppression filter. The
lamphouse itself should not leak harmful ultraviolet wavelengths and, preferably, should
incorporate a switch to automatically shut down the lamp if the housing is inadvertently
opened during operation. The lamphouse should be sturdy enough to withstand a possible
burner (arc-discharge lamp) explosion during operation. In modern lamphouses, the lamp
socket is equipped with adjustment knobs to permit centering the arc lamp image within the
rear aperture of the objective (in Köhler illumination, these planes are conjugate).
Somewhere in the light path, usually closer to the lamphouse and preceding the excitation
filter, it is desirable to have a shutter in order to completely block excitation light when the
specimen is not being viewed or imaged with the detector. In addition, provisions for neutral
density filters should be made available (either on a wheel, turret, or slider) in order to enable
the user to reduce the intensity of excitation illumination.

Stokes' Shift

Vibrational energy is lost when electrons relax from the excited state back to the ground
state. As a result of the energy loss, the emission spectrum of an excited fluorophore is
usually shifted to longer wavelengths when compared to the absorption or excitation
spectrum (note that wavelength varies inversely to radiation energy). This well-documented
phenomenon is known as Stokes' Law or Stokes' shift. As Stokes' shift values increase, it
becomes easier to separate excitation from emission light through the use of fluorescence
filter combinations.

The fluorophore emission (or absorption) intensity peak is usually lower in wavelength and
magnitude than that exhibited by the excitation peak, and the emission spectral profile
(curve) is often a mirror image (or nearly so) of the excitation curve, but shifted to longer
wavelengths, as illustrated in Figure 3 for Alexa Fluor 555, a useful probe that absorbs light
in the yellow-green region and produces yellow-orange emission. In order to achieve
maximum fluorescence intensity, a fluorophore (often termed a dye) is usually excited at
wavelengths near or at the peak of the excitation curve, and the widest possible range of
emission wavelengths that include the emission peak are selected for detection. The
selection of excitation and emission wavelengths is typically based on interference filters
(Figure 2). In addition, the spectral response of a microscope optical system will also depend
on such factors as glass transmission efficiency (due to anti-reflection coatings), the number
of lens and mirror elements, and the responsivity of the detector system.

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The effective separation and detection of excitation and emission wavelengths is achieved in
fluorescence microscopy through the proper selection of filters to block or pass specific
wavelength bands in the ultraviolet, visible, and near-infrared spectral regions. Fluorescence
vertical illuminators are designed with the purpose of controlling the excitation light through
the application of readily interchangeable filter (neutral density and interference excitation
balancers) insertions into the light path on the way toward the specimen, and again in the
path between the specimen and the observation tubes or camera detector system. Perhaps
the most important criteria, in view of relatively low fluorescence emission intensities (see
discussion above), is that the light source utilized for excitation be of sufficient brightness so
that the weak emission light can be maximized, and that the fluorochromes possess
adequate absorption properties and emission quantum yields.

The efficiency with which a particular fluorophore absorbs a photon of the excitation light is a
function of the molecular cross-section, and the likelihood of absorption is known as the
extinction coefficient. Larger extinction coefficients indicate that the absorption of a photon
(or quantum) in a given wavelength region is more likely. The quantum yield denotes the
ratio of the number of quanta emitted compared to those absorbed (and is usually a value
between 0.1 and 1.0). Quantum yield values below 1 are the result of the loss of energy
through nonradiative pathways, such as heat or a photochemical reaction, rather than the re-
radiative pathway of fluorescence. Extinction coefficient, quantum yield, mean luminous
intensity of the light source, and fluorescence lifetime are all important factors that contribute
to the intensity and utility of fluorescence emission.

Fading, Quenching, and Photobleaching

A wide spectrum of conditions often come into play that ultimately affect the re-radiation of
fluorescence emission and thus reduce the intensity. The general term for a reduction of
fluorescence emission intensity is fading, a catch-all category that is usually further
subdivided into quenching and photobleaching phenomena for more precise descriptions.
Photobleaching is the irreversible decomposition of the fluorescent molecules in the excited
state because of their interaction with molecular oxygen before emission. The occurrence of
photobleaching is exploited in a technique known as fluorescence recovery after
photobleaching (FRAP), a very useful mechanism for investigating the diffusion and motion
of biological macromolecules. The method is based upon photobleaching a sharply defined
region of the specimen by an intense burst of laser light, accompanied by the subsequent

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observation of the rates and pattern of fluorescence recovery in the photobleached area. A
related technique, known as fluorescence loss in photobleaching (FLIP), is employed to
monitor the decrease of fluorescence in a defined region lying adjacent to a photobleached
area. Similar to FRAP, the latter technique is useful in the investigation of molecular mobility
and dynamics in living cells.

Presented in Figure 4 is a typical example of photobleaching (fading) observed in a series of

digital images captured at different time points for a multiply-stained culture of Indian Muntjac
deer epidermis fibroblast cells. The nuclei were stained with a bis-benzimidazole derivative
(Hoechst 33258; blue fluorescence), while the mitochondria and actin cytoskeleton were
stained with MitoTracker Red CMXRos (red fluorescence) and a phalloidin derivative
conjugated to Alexa Fluor 488 (green fluorescence), respectively. Time points were taken in
two-minute intervals using a fluorescence filter combination with bandwidths tuned to excite
the three fluorophores simultaneously while also recording the combined emission signals.
Note that all three fluorophores have a relatively high intensity in Figure 4(a), but the
Hoechst fluorophore (blue) intensity starts to drop rapidly at two minutes and is almost
completely gone at 6-8 minutes. The mitochondrial and actin stains are more resistant to
photobleaching, but the intensity of both drops significantly over the course of the timed
sequence (10 minutes).

The excited state relaxation process of quenching results in reduced fluorescence intensity
through a variety of mechanisms involving non-radiative energy loss and frequently occurs
as a result of oxidizing agents or the presence of salts or heavy metals or halogen
compounds. In some cases, quenching results from the transfer of energy to another
molecule (termed the acceptor), which resides physically close to the excited fluorophore
(the donor), a phenomenon known as fluorescence resonance energy transfer (FRET). This
particular mechanism has become the basis for a useful technique involving the study of
molecular interactions and associations at distances far below the lateral resolution of the
optical microscope.

Fluorescence Light Sources

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An unfortunate consequence of low emission levels in most fluorescence microscopy
applications is that the number of photons that reach the eye or camera detector is also very
low. In most cases, the collection efficiency of optical microscopes is less than 30 percent
and the concentration of many fluorophores in the optical path ranges in the micromolar or
nanomolar regions. In order to generate sufficient excitation light intensity to produce
detectable emission, powerful compact light sources, such as high-energy short arc-
discharge lamps, are necessary. The most common lamps are mercury burners, ranging in
wattage from 50 to 200 Watts, and the xenon burners that range from 75 to 150 Watts (see
Figure 5). These light sources are usually powered by an external direct current supply,
furnishing enough start-up power to ignite the burner through ionization of the gaseous vapor
and to keep it burning with a minimum of flicker.

The microscope arc-discharge lamp external power supply is usually equipped with a timer
to track the number of hours the burner has been in operation. Arc lamps lose efficiency and
are more likely to shatter if used beyond their rated lifetime (200-300 hours). The mercury
burners do not provide even intensity across the spectrum from ultraviolet to infrared, and
much of the intensity of the lamp is expended in the near ultraviolet. Prominent peaks of
intensity occur at 313, 334, 365, 406, 435, 546, and 578 nanometers. At other wavelengths
in the visible light region, the intensity is steady although not nearly so bright (but still
useable in most applications). In considering illumination efficiency, mere lamp wattage is not
the prime consideration. Instead, the critical parameter is the mean luminance must be
considered, taking into account the source brightness, arc geometry, and the angular spread
of emission.

In the past few years, optical microscopy has experienced an increase in the application of
laser light sources, particularly the argon-ion and argon-krypton (ion) lasers. These lasers
have the virtues of small source size, low divergence, near-monochromicity, and high mean
luminance. They have become essential in scanning confocal microscopy, a technique that
has proven to be a powerful tool in rendering very sharp fluorescence images through
rejection of non-focused light removed from the specimen focal plane. Confocal microscopes
accomplish this task through point or line scanning with coincident imaging through a
conjugate aperture. Optical sections of the specimens can be stored in a host computer and
reconstructed into the final image, which is then displayed on the monitor.

Filter Terminology

The common terminology applied to fluorescence microscopy filter combinations has

become confusing as a result of the various initials and codes utilized by different
manufacturers to identify their filters. Basically, there are three major categories of filters:
excitation (often referred to as exciters), barrier (emission), and dichromatic beamsplitters
(or dichroic mirrors). Fluorescence filters were formerly almost exclusively constructed from
dyed glass or gelatin sandwiched between two glass plates. However, the current trend is to
manufacture high-resolution filters with interference optics for excitation filters to pass or

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reject wavelengths of light with a great specificity and high transmission. Dichromatic
beamsplitters are specialized interference filters designed to reflect or pass light of specific
wavelengths when placed into the light path at a 45-degree angle (see Figures 1 and 2).
Barrier filters are fabricated with both colored glass or interference coatings (or a
combination of the two).

Abbreviations employed by manufacturers to identify the properties of their excitation filters

include: UG (ultraviolet glass) and BG (blue glass). Shortpass filters often are denoted as KP
(K is an abbreviation for kurz, which means "short" in German) or simply as SP. Several
manufacturers now label their interference filters with the designation IF. Narrow band
excitation interference filters are especially helpful if the Stokes' shift is small.

Acronyms or abbreviations for barrier filters include: LP or L for longpass filters, Y or GG for
yellow or gelb (German) glass, R or RG for red glass, OG or O for orange glass, K for kante,
a German term for edge (filter), and BA for barrier filter. When the filter type is also
associated with a number, such as BA515, that designation refers to the wavelength (in
nanometers) at 50-percent of its maximum transmission.

Dichromatic beamsplitters also are described by numerous abbreviations including CBS for a
chromatic beam splitter, DM for dichroic mirror, TK for "teiler kante", German for edge
splitter, FT for "farb teiler" (German for color splitter), and RKP for reflection short pass. All of
these terms should be considered interchangeable, and modern dichromatic beamsplitters
are always manufactured with interference coatings on optical glass (as opposed to organic
or metallic dyes). The interference thin films are designed to produce high reflectivity for
shorter wavelengths and high transmission for longer wavelengths. Dichromatic
beamsplitters are oriented at a 45-degree angle to the path of the excitation light entering the
optical block through the reflected light fluorescence illuminator. Their primary function is to
re-direct the selected excitation (shorter) wavelengths through the objective and onto the
specimen. These specialized filters also have the additional functions of passing longer
wavelength fluorescence emission to the barrier filter, and reflecting any scattered excitation
light back in the direction of the lamphouse.

Presented in Figure 6 are the transmission profiles for a typical fluorescence filter
combination used in modern microscopes. The excitation filter spectrum (red curve) exhibits
a high level of transmission (approximately 75 percent) between 450 and 490 nanometers
with a center wavelength (CWL) of 470 nanometers. The dichromatic mirror (yellow curve)

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reflects wavelengths in the region of the excitation spectrum, while passing higher and lower
wavelengths with relatively high efficiency. Note that zero percent transmission on the
dichromatic mirror curve corresponds to 100 percent reflection. The pronounced dip in the
transmission profile between 450 and 500 nanometers, which represents a peak in
reflectance, serves to reflect the band of wavelengths passing from the excitation filter at a
90-degree angle and onto the specimen. The final component in the optical train, an
emission or barrier filter (white curve), transmits wavelengths in the green visible light region,
in the range between 520 and 560 nanometers. Boundaries between transmitted and
reflected wavelength bands of the various superimposed spectra are designed to be as
steep as possible to assure nearly complete separation of the reflected and transmitted
wavelengths. A pattern of sinusoidally rising and falling spikes appearing in the dichromatic
mirror spectrum is a common effect of the thin-film deposition process known as ringing.
The performance of this filter combination is remarkable and is a clear demonstration of the
rapid advances being achieved in thin film interference filter technology.

The filter nomenclature employed by Nikon derives from a mixture of terms dating back to
the early 1990s. At that time, all of the Nikon complementary filter combinations were
produced using the hard coat sputter technique, but many of the currently available filters
take advantage of newer softer coating methods. Although soft coats are more susceptible to
humidity and heat degradation, and must be handled more carefully than hard coat filters,
they exhibit higher blocking value optical densities and provide greater ease of fine-tuning
specific wavelength bands. Understanding the Nikon filter combination code nomenclature
provides a mechanism to quickly determine whether a particular set will perform adequately
for a specific fluorophore.

The first letter in the Nikon proprietary alphanumeric filter designation code indicates the
wavelength excitation spectral region (for example, UV, V, B, and G, which are simple
abbreviations for ultraviolet, violet, blue, and green, respectively). The number following the
excitation code relates to the excitation filter passband width: 1 for narrow band excitation, 2
for medium and wide band excitation, and 3 for very wide band excitation. Finally, one or
more letters following the excitation bandpass size number identifies the barrier filter
characteristics. The code letter A indicates a standard longpass barrier filter with the lowest
cut-on wavelength, while B designates a higher cut-on wavelength value for a longpass
emission filter. Bandpass emission filters are identified with the letter E (referring to the term
"enhanced") to indicate their superior performance with regard to eliminating crossover. The
E/C filters are soft coat interference combinations designed for best performance with
specific probes, such as DAPI, FITC, TRITC, and Texas Red.

The Fluorescence Light Budget

An estimation of the light fluxes in a typical fluorescence microscope is useful to outline

constraints that will be encountered in producing digital images or during the visual
observation of specimens. The excitation source is assumed, for this exercise, to be a
standard 75-Watt xenon arc-discharge lamp having a mean luminous flux density of
approximately 400 candelas per square millimeter (for other sources, see Table 1). When the
lamp output is collected and directed through a 490-nanometer interference filter (having a
10-nanometer bandwidth and 75 percent transmission), about 2 milliWatts of light will pass
through. After reflection by a 90-percent efficient dichromatic mirror, a light flux of 1.8
milliWatts enters the rear aperture of the microscope objective as the excitation beam.

With a 100x objective having a numerical aperture of 1.4, the area of the specimen
illuminated will be 12 x 10•E(-6) square centimeters, assuming a circular field of view about

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40 micrometers in diameter. The light flux on the specimen is then about 150 Watts per
square centimeter, which corresponds to a flux density of 3.6 x 10•E(20) photons per square
centimeter. Thus, the specimen illumination intensity is about 1000 times higher than that
incident on the Earth's surface on a sunny day.

The fluorescence emission that results from the light flux discussed above depends on the
absorption and emission characteristics of the fluorophore, its concentration in the specimen,
and the optical path length of the specimen. In mathematical terms, the fluorescence
produced (F) is given by the equation:

F=σ •Q•I

where σ is the molecular absorption cross-section, Q is the quantum yield, and I is the
incident light flux (as calculated above). Assuming that fluorescein is the fluorophore, the
absorption cross-section (σ) is 3 x 10•E(-16) square centimeters per molecule, Q equals
0.99, resulting in a value for F of 100,000 photons per second per molecule. If the dye
concentration is 1 micromole per liter and is uniformly distributed in a 40-micrometer
diameter disk with a thickness of 10 micrometers (volume equal to 12 picoliters), there are
approximately 1.2 x 10•E(-17) moles of dye or 7.2 million molecules in the optical path. If all
of the molecules were excited simultaneously, the fluorescence emission rate would be 7.2 x
10•E(11) photons per second (given the product of F and the number of dye molecules). The
question of interest is how many of the emitted photons would be detected and for how long
could this emission rate continue?

Luminous Density of Selected Light Sources

Arc Size
Current Luminous Flux Mean Luminous
Lamp (H x W)
(Amperes) (Lumens) Density (cd/mm2)
(Millimeters)
Mercury Arc
5 2200 1700 0.25 x 0.25
(100 Watt)
Xenon Arc
5.4 850 400 0.25 x 0.50
(75 Watt)
Xenon Arc
30 9000 3500 0.30 x 0.30
(500 Watt)
Tungsten
8 2800 45 4.2 x 2.3
Halogen

Table 1

The efficiency of detection is a function of the optical collection efficiency and the detector
quantum efficiency. A 1.4-numerical aperture objective with 100-percent transmission (an
unrealistic condition) has a maximum collection efficiency, limited by the acceptance angle of
about 30 percent. The transmission efficiency of the dichromatic mirror is 85 percent and that
of the barrier filter is 80 percent. The overall collection efficiency is then about 20 percent or
140 billion photons per second. If the detector is a conventional charge-coupled device
(CCD), the quantum efficiency is about 50 percent for the green fluorescein emission (at 525
nanometers), so the detected signal would be 70 billion photons per second or about 10
percent of the emitted fluorescence. Even with a perfect detector (100 percent quantum
efficiency), only about 20 percent of the fluorescence emission photons can be detected.

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The duration of fluorescence emission depends upon the rate of fluorophore destruction as a
result of photobleaching. For fluorescein in an oxygenated saline solution, measurements
indicate that each molecule can only emit about 36,000 photons before being destroyed. In a
deoxygenated environment, the rate of photodestruction diminishes about tenfold, so
360,000 photons are produced per fluorescein molecule. The entire dye pool, in this example
(7.2 million molecules), would then be capable of producing a minimum of 2.6 x 10•E(11) and
a maximum of 2.6 x 10•E(12) photons. Assuming the emission rate of 100,000 photons per
second per molecule calculated above, fluorescence could continue for only 0.3 to 3
seconds before photodestruction. In the case where 10 percent of the photon flux is
detected, a signal of 7.2 x 10•E(10) electrons per second would be obtained.

Following the argument of this example, if the detector is a 1000 x 1000 pixel CCD camera,
this signal would be distributed over a million sensors, with approximately 72,000 electrons
per sensor. For a scientific-grade CCD with 9-micrometer square sensors, the full well
storage capacity is about 80,000 electrons and the read-out noise is less than 10 electrons.
The signal-to-noise ratio would then be largely determined by photon statistical noise equal
to the square root of the signal, approximately 268. In almost all cases, this high signal level
could only continue for a very brief period of time before photodestruction occurs. The
compromise utilized by most microscopists to prolong the observation period is a reduction
in the incident light flux intensity so that only a fraction of the fluorophore molecules in the
dye pool are excited and subjected to photodestruction. Thus, the signal-to-noise ratio rarely
equals the theoretical maximum and typically ranges between 10 and 20 in fluorescence
microscopy.

Detecting Single Molecules

Under ideal conditions, it is often possible to detect the fluorescence emission from a single
molecule, provided that the optical background and detector noise are sufficiently low. As
discussed above, a single fluorescein molecule could emit as many as 300,000 photons
before it is destroyed by photobleaching. Assuming a 20-percent collection and detection
efficiency, about 60,000 photons would be detected. Using avalanche photodiode or electron
multiplying CCD detectors for these experiments, investigators have been able to monitor
the behavior of single molecules for many seconds and even minutes. The major problem is
adequate suppression of the optical background noise. Because many of the materials
utilized in construction of microscope lenses and filters display some level of
autofluorescence, efforts were initially directed toward the manufacture of very low
fluorescence components. However, it soon became evident that fluorescence microscopy
techniques utilizing total internal reflection (TIR) provided the desired combination of low
background and high excitation light flux.

Total internal reflection fluorescence microscopy (TIRFM) takes advantage of the evanescent
wave that is developed when light is totally internally reflected at the interface between two
media having dissimilar refractive indices. The principle employing an external light source is
illustrated in Figure 7(a). In this technique, a beam of light (usually an expanded laser beam)
is directed through a prism of high refractive index, such as glass or sapphire, which abuts a
lower refractive index medium of glass or aqueous solution. If the light is directed into the
prism at higher than the critical angle, the beam will be totally internally reflected at the
interface. The reflection phenomenon develops an evanescent wave at the interface by the
generation of an electromagnetic field that permeates about 200 nanometers or less into the
lower refractive index space. The light intensity in the evanescent wave is sufficiently high to
excite the fluorophores within it, but because of its shallow depth, the volume excited is very
small. The result is an extremely low-level background because so little of the specimen is

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exposed to the excitation light (only that portion within a 200-nanometer distance of the
interface).

Total internal reflection fluorescence microscopy can also be conducted through a

modification of the epi-illumination approached utilized in widefield techniques (as illustrated
in Figure 7(b)). This method requires a very high numerical aperture objective (at least 1.4,
but preferably 1.45 to 1.6) and partial illumination of the microscope field from one side by a
small sport or more uniform illumination by a thin annulus. High refractive index lens
immersion medium and microscope cover glass are required to achieve the illumination
angle resulting in total internal reflection. As presented in Figure 7(b), light rays exiting the
objective front lens element at an angle less than the critical angle (denoted as A(1)) in
figure 7(b)) are transmitted away from the microscope. When the angle is increased to or
beyond the critical angle (indicated a angle A(2) in Figure 7(b)), total internal reflection
results.

Other popular advanced fluorescence techniques, such as fluorescence resonance energy

transfer (FRET) and fluorescence recovery after photobleaching (FRAP), as well as
spectroscopy, are often combined with total internal reflection to achieve additional
information. The result is a very powerful tool for the study of individual fluorophores and
fluorescently labeled molecules. The advantages resulting from the study of the properties of
single molecules are only beginning to be appreciated. Thus, the current range of optical
microscopy now extends from the single molecule to the entire animal.

Conclusions

The modern fluorescence microscope combines the power of high performance optical
components with computerized control of the instrument and digital image acquisition to
achieve a level of sophistication that far exceeds that of simple observation by the human
eye. Microscopy now depends heavily on electronic imaging to rapidly acquire information at
low light levels or at visually undetectable wavelengths. These technical improvements are
not mere window dressing, but are essential components of the light microscope as a
system.

The era when optical microscopy was purely a descriptive instrument or an intellectual toy is
past. At present, optical image formation is only the first step toward data analysis. The
microscope accomplishes this first step in conjunction with electronic detectors, image
processors, and display devices that can be viewed as extensions of the imaging system.

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Computerized control of focus, stage position, optical components, shutters, filters, and
detectors is in widespread use and enables experimental manipulations that were not
humanly possible with mechanical microscopes. The increasing application of electro-optics
in fluorescence microscopy has led to the development of optical tweezers capable of
manipulating sub-cellular structures or particles, the imaging of single molecules, and a wide
range of sophisticated spectroscopic applications

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