FACULTY OF ENGINEERING

TECHNOLOGY
MATERIAL ENGINEERING TECHNOLOGY
BNQ30503
INDIVIDUAL ASSIGNMENT
TITLE: OPTICAL MICROSCOPIC EXAMINATION
AND ANALYSIS

NAME
MATRIC
NUMBER
LECTURERS
NAME
DATE OF
SUBMISSION

: KHAIRUL ANWAR BIN ROSLI

: AN120228
: ENGR. DR. NASRUL FIKRY
BIN CHE PA
: 5TH MAY 2015

INDEXES

CONTENTS
1.0 Objective
2.0 Literature Review

PAGE
1

2.1 Background and history of optical microscopic.

2.2 Fundamental of image formation of optical

microscopy examination.
2.3 Specimen illumination
2.4 Image magnification
3.0 Discussion

5
5

3.1 Techniques of Optical Microscopy

3.2 Samples preparation in Optical Microscopy

analysis
3.3 Examples and Product Safety of Optical
Microscopy analysis
4.0 Conclusion
References

10
13
14

1.0 Objective of the Study.

2.0 The aims of this study are:
i.

To discuss the basic principle and fundamental of optical microscopy

ii.
iii.
iv.

techniques.
To introduce the available techniques in optical microscopic.
To study the uses and application of optical microscopy.
To relate the importance of optical microscopy analysis on product safety.

3.0
4.0 Literature Review.
5.0 2.1 Background and history of optical microscopic.
6.0 The history of optical microscopy began a long time ago during 15 th century.
According to Vignati (2005) the history begins with the invention of simple
microscope which it is credited to Zacharias Jansen, in Middleburg, Holland
around the year of 1595. The microscope was a single convex lens through which
specimen can be focused and magnifies on the observers eye. The development
continues in the 1600s whereas Anton von Leeunwenhoek successfully develops
compound microscope which was able to observe larger bacteria. The compound
microscope consists of an objectives lens placed near to the specimen and an
eyepiece close to observers eyes. This development enables a two staged
magnification. However, compound microscope with multiple lenses encounter
more spherical and chromatic aberration compared to the simple microscope. To
counter the problems, more research and development on microscope are done.
In 19th century chromatically corrected microscope are invented which is built
with different coloured dispersion lenses. By the end of 19th century, there were
many competitions among microscope manufacturers to develop mechanically
and optically high quality of compound microscope. Modern microscope in the
1990s undergoing advancement such as in glass formulation and lens technology
allowing

an

excellent

optical

aberration

correction.

Furthermore,

the

advancement also enables the resulting image to be detected directly by the eyes
or by various types of light detecting devices such as photographic plate, chargecouple device (CCD) cameras, photodiodes and other optical sensors.
7.0

8.0

9.0 Figure 2.1: Simple and compound microscope in the early development. (a)
Anton von Leeunwenhoek's simple microscopes; (b) Hooke compound
microscope
10.0

11.0

12.0

Figure 2.2: Past and present microscopes. (a) Powell and Lealand No 1
microscope 1850; (b) Olympus Provis AX 70

13.0

14.0

2.2

Fundamental of image formation of optical microscopy

examination.
15.0

In the optical microscope, when light from the lamp passes through the

condenser and pass through the specimen, some of the light passes around and
through the specimen then produce magnified images the specimen on a detector
such as human eyes or light detecting devices such as camera (Davidson M. W.
and Abramowitz M., 2002). In human eyes, there are 2 types of receptors which
3

are cone cells and rod cells. Cone cells are used for detecting colours while rod
cells are used to distinguishing levels of intensity. These cells are located on the
retina, iris, and cornea while the crystalline lens is the device for disclosing the
light and focusing it on the retina.
16.0

Optical camera on the other hands, the image was formed on the

films. Digital cameras comprises of a dense matrix of photodiodes that accumulate

electric charges as light imposes on them. The magnified image must meet these
requirements to be seen clearly by the detector:
i.

It must be spread on the detector at a sufficient angle, unless the light falls on

ii.

non-adjacent receptor.
There must be adequate contrast between adjacent details and background to
condense the image visible.
17.0

Conventional optical microscopy containing one or more lenses that

produce enlarged image of an object placed in the focal of the lens. There are two
possible configurations for optical microscopy:
i.

Transmission (Figure 2.3 (a) )

a. The beam of light passes through the sample.
b. The specimen is placed between the objective and the condenser lens.
The illuminating light formed by the condenser lens falls in the
specimen and collected by the objective.
c. Example of transmission microscopy is polarizing or petrographic

ii.

microscope.
d. Sample types are usually fine powder or thin transparent slices.
Reflection (Figure 2.3 (b))
a. The beam of light reflected off the sample surface.
b. The objective is used as matching well-corrected condenser and as
image forming lens which the light passes the sample twice.
c. Example of reflection microscopy is metallurgy microscope.
d. The microscopes usually use to analyse the surface of the material
especially the opaque material.

18.0

19.0

20.0

Figure 2.3: Type of microscopy light configuration. Khler illumination for

transmitted and reflected microscopy (a) Brightfield transmitted light; (b)
Brightfield reflected light.
5

21.0

2.3 Specimen illumination

22.0

Illumination is the important variable in achieving high quality images and it

is use for viewing thick specimen. The illumination should be adequately bright,
glare free and evenly dispersed in the field of view. Most of microscope's
manufacturers have adjusted the arrangement of optical elements in the Khler
illumination configuration to optimise the illumination (Vignati E., 2005). This
arrangement establishes two sets of conjugates planes. The ones in the path of the
illuminating rays are:

23.0

The lamp filament.

The condenser aperture diaphragm.
The back focal plane of the objective.
The eye-point of the eyepiece.
The other set of conjugates planes are related to image formation light path

and consist of:

24.0

The field diaphragm.

The focused specimen.
The intermediate image plane.
The eye's retina or the film plate of the camera.

25.0

2.4 Image magnification

26.0

Magnification of microscope is dependent on the objective, the tube lens, the

eyepieces and additional lenses. Consider an object of size lobj at a distance a

from a lens of focal length f. The object's image, of size limg, is formed at a
distance b according to the lens law (Vignati E., 2005):

27.0
28.0

1 1 1
+ =
a b f

The lateral magnification M is the ratio of the linear size of the image to the

linear size of the object:

29.0

M=

limg b
=
l obj a

30.0

3.0

Discussion

31.0

In material engineering, the optical microscopy is used to analyse the

microstructure, optical illumination system of the elements. For materials that are
opaque to visible light such as all metals, some ceramics and polymers, the
surface is the subject to be observed and the reflective light microscope must be
use. Different types of metals or compounds required the light microscope to be
set in different mode. Thus, optical microscopy analysis introduces techniques for
analysing different types of materials.
32.0

3.1

Techniques of Optical Microscopy

33.0

3.1.1

Darkfield Microscopy.

34.0

In Darkfield microscopy, the light illumination involves blocking out of the

central light pass through or around the specimen and permitting the only oblique
rays to the specimen to be illuminated. This is the most simple and popular for
imaging unstained specimens, which will appear as bright illumination on dark
background. The right rays came from darkfield condenser strike the specimen
from every azimuth, diffracted and reflected into the objectives lens.
35.0

3.1.2

Phase Contrast Microscopy

36.0

Phase Contrast microscopy is primarily used to detect phase specimens.

Phase specimens is the unstained object which do not absorb light and slightly
alter the light diffracted by the specimen, usually retarding light to wavelength
but remains in amplitude. Phase Contrast involves the separation of the direct
zeroth order light from the diffracted light at the rear focal plane of the objective.
This is done by placing a ring annulus is in direct position under the lower lens of
the condenser at the front focal plane of the condenser, conjugate to the objective
rear focal plane.
37.0

3.1.3: Polarized Light

38.0

A polarizer place under the sub-stage condenser is focused on the polarized

light exiting the polarizer. The plane polarized in a vibration direction with
respect to the optic axis of the microscope. The polarized light waves then pass
through the specimen and objective before reaching a second polarizer or
7

analyser that is oriented to pass a polarized vibration direction perpendicular to

that of the sub-stage polarizer. Therefore, the analyser passes only those
components of the light waves that are parallel to the polarization direction of the
analyser. The retardation of one ray with respect to another is caused the
difference in speed between the ordinary and extraordinary rays refracted by the
anisotropic crystal.
39.0

3.1.4

Fluorescence Microscope

40.0

Fluorescence microscopy is an excellent technique to study material that can

fluoresce, either naturally which is auto-fluorescence or when treated with

chemicals capable of fluorescing. The basic fluorescence microscope is to allow
light to irradiate the specimen and then separate the much weaker re-radiating
fluorescent light from the brighter excitation light, resulting fluorescing areas
shine against a dark background with sufficient contrast to allow detection. For
example, ultraviolet (UV) light of a specific wavelength or set of wavelengths is
produced by passing light from a UV-emitting source through the exciter filter.
The filtered UV light illuminates the specimen, which emits fluorescent light of
longer wavelengths while illuminated with ultraviolet light. Visible light emitted
from the specimen is then filtered through a barrier filter that does not allow
reflected UV light to pass.
41.0
42.0

Table 3.1: The schematic diagram of optical microscopy techniques and

description (Davidson M.W and Abramowitz M., 2002)

43.0

Techniques of Optical Microscopy

44.0

Description

45.0

46.0

Schematic configuration for

darkfield

microscopy.

The

central opaque light stop under

the condenser to eliminate zeroth
order

illumination.

The

condenser produces a hollow

cone

of

reflected,
diffracted

illumination.

The

refracted,

and

light

from

the

specimen enters the objective

front lens.

47.0

48.0

Schematic configuration for

phase contrast microscopy. Light

passing through the phase ring is
first

concentrated

onto

the

specimen by the condenser. The

light enters the objective before
interference at the rear focal
plane of the objective.

49.0

50.0

Schematic

microscope

configuration

for

observing

specimens

under

crossed

polarized

illumination.

White

light

passing

through

the

polarizer is plane polarized and

concentrated onto the specimen
by the condenser. Light rays
emerging from the specimen
interfere

when

they

recombined

in

the

subtracting

some

are

analyser,
of

the

wavelengths of white light, thus

producing a myriad of tones and
colours

51.0

52.0

Schematic

reflected

diagram

light

of

fluorescence

microscopy. Light emitted from

UV concentrated by the collector
lens

before

passing

through

aperture and field diaphragms

which
through

are

reflected

the

objective

down
to

illuminate the specimen. Longer

wavelength emitted through the
objective and dichroic mirror
before filtered by the emission
filter.

53.0

3.2

Sample preparation in Optical Microscopy analysis

10

54.0

55.0

Figure 3.1: Basic general steps for specimen preparation microscopy

(Adapted from Mukhopadhay S. M., 2003)

56.0

57.0 Table 3.2: Description of steps in sample preparation for optical microscopy
techniques
58.0

St 59.0

eps
60.0 C
leani

61.0

ng
64.0 G
rindin
g
68.0

olishi
ng

Cutting process is carried out in order to get the desired size and

dimension of the sample so that it can be easily molded and handle.

ng
62.0 M 63.0
ouldi

Description and function of the steps

Molding process is carried out to make the sample easier to hold

throughout the polishing procedure.

65.0

Rough grinding: To remove rough scale and gross imperfections

on the surface of sample.

67.0 Fine grinding: To improve the specimen's surface so it is shines
and reflects light slightly.
69.0 Rough polishing: To remove the imperfections that grinding has
left.
71.0 Fine polishing: To remove scratches and leave a mirror like
surface.
11

72.0

Et 73.0

ching

Various etchants are used to selectively attack the surfaces of

metals to reveal grain boundaries, phase, precipitates, inclusion and

composition.
Examples and Product Safety of Optical Microscopy analysis

74.0

3.3

75.0

Optical microscopic are the methods that are extensively applied in material

characterization. This is due to the capability of optical microscopy to allow the

observation of the internal structure of the material. Microstructure features of
the materials such as grains and grain sizes, pores and pore sizes, precipitates,
inclusions, alteration products, textures, shapes and morphology of crystals or
aggregates, cracking and interfacial reactions, twin boundaries can be recognized
and measured.
76.0

For the determination and recognition of minerals and mineral phases,

a polarized optical microscope is usually used and considered to be the basic tool.
Two main types of polarizing optical microscopes that are used for the
characterization includes transmitted light and reflected light. A transmitted light
polarizing microscope is used for the observation of transparent minerals while a
reflected or incident light microscope is used for the observation of metallic and
opaque minerals.
77.0

Optical microscopy has the advantages of direct imaging and

straightforward information. Certain features are better visualized at low

magnification, for example directional deformation and twin boundaries, while
porosity and phase fractions can be better quantified. The disadvantage of this
method is the low resolution. Due to the light diffraction limit, a common optical
microscope can distinguish objects that range with size from 1 to100m, at a
magnification of about 1500X. Another drawback can be the sample preparation
since the thin sections are required from all samples studied under the polarizing
microscope.
78.0
79.0

3.3.1 Detection of deterioration

80.0

Most building material is porous and susceptible to deterioration due to the

long and slow effect of the environment conditions on the materials. Microscopic
12

techniques can assist on the study of the whole spectrum of deterioration and find
the major mechanism starting from the macroscopic image and even deeper up to
the crystal unit means that a researcher can see all the path of the procedure.
81.0

The cause, the type and the complexity of deterioration is possible to

be determined by microstructure investigation. Determining the cause of

deterioration is important to understand errors on design or materials use. The
information is important to improve the property of the material or the type of
material use and also it is essential in order to organize the appropriate restoration
procedure. For the heterogeneous materials such as concrete and mortars with
features ranging from nano to centimetres, microscopy can give information
concerning the size, the distribution and the topology in detail.
82.0

83.0

Figure 3.2: Mortar from the medieval walls in Rhodes (a) under Stereoscope
(x8) where the pore within crystallized salts is observed; (b) under Polarized
microscope (x150) the presence of angular crystal are analysed.

84.0
85.0

3.3.2

Characterization of porosity and crack pattern

13

86.0

Porosity is a basic characteristic of materials that influence most of the

material properties such as mechanical strength, deformation, resistance to

weathering and insulting properties. Optical microscopy is among the widely
applied techniques for measurement of porosity. The advantage of optical
microscopic techniques is the exact size and position of the pore can be identified
which the pores and cracks occurring inside the matrix or in the transition zones
can be distinguished. Cracks in the microstructure of a material might be preexist or are formed during the service life of the materials. Their structure
recorded by microscopy of different magnification capacity, allows to
determination of the main mechanism responsible for the crack. For example,
cracks in the cement matrix of concrete may be related to sulfate attack and
ettringite or thaumasite can be easily identified from the characteristic crystals.
Cracks in lime based mortars in the other hands are mainly localized in the
transition region around aggregates and they are usually filled with recrystallized calcite which contributes to the increase in density and strength of
mortars.

87.0
88.0

Figure 3.3: Re-crystallization of calcite in the contact zone of binderaggregate filling the crack

89.0

3.3.3

Detection of interfaces

90.0

In composite materials, the existence of many interface are considered as the

weak phases of the material and requires special treatment to reinforce the
14

material. Good compaction, proper additives often use to avoid weak zones and
enhance the material strength and other properties. The microscopic analysis of
the interfaces provides information about the techniques used for building with
these materials. For examples, high quality old mortars taken from monuments,
the interfaces between brick-mortars or bedding mortar-renders or even mortarmortar are quite compact (Figure 3.4). In many cases the microscopic
examination shows the chemical reaction products between the bonding
materials. In the case of a roller compacted concrete pavement the cold joint due
to premature setting and inadequate compaction of concrete implies for a low
performance pavement.
91.0

92.0

Figure 3.4: Interface zones under polarized microscope (x150) (a) brickmortar; (b) mortar-render; (c) two mortar layers.

93.0

94.0

4.0

Conclusion

95.0

Optical microscopy applying the light from the sources that passes through

the condenser and specimen either will be reflected or transmitted to produce

magnified images of the specimen on the detector such as human eyes or devices
such as camera. The main components of optical microscopy are the objective
15

lens, eyepiece, condenser, detection devices and light sources. There are many
available techniques in optical microscopy such as Darkfield microscope, Phase
Contrast microscopy, Polarized Light microscopy, Reflected Light Fluorescence
microscopy and many others. The techniques are different depends on the as the
light ray path, the position of the samples, sample types and characteristics and
others. The optical microscopy is used in materials analysis. For examples,
optical microscope is used in the investigation and analysis of the behaviour of
materials such as brick, mortar in the building and construction field. Most
common application of optical microscope is in the detection of deterioration in
materials, characteristics of porosity and crack pattern on the materials, detection
of interface and others. This application is very crucial in building and
construction field, as the materials need to be first analysed and determine the
strength, compatibility of the material to be use, durability or to be checked any
defects so that the quality and the function of the materials can be fully utilized.
96.0
97.0

References

1. Davidson, M. W. and Abramowitz, M. 2002. Optical Microscopy.

Encyclopedia of Imaging Science and Technology. .
2. Lee T. et al., 2011. Optical Microscopy Of Soft Matter System
3. Mukhopadhyay, S. M., 2003. Sample Preparation for Microscopic and
Spectroscopic Characterization Of Solid Surface And Films. Sample
Preparation Techniques in Analytical Chemistry. John Wiley & Sons, Inc. (pp.
377412).
4. Patzelt W.J, 1985. Polarized Light Microscopy: Principles, Instruments,
Application 3rd Edition. Ernst Leitz Wetzlar GmbH, Germany.
5. Stefanidou M. et al., 2014. Applying Microscopic Techniques for the
Investigation of the Behaviour of Building Materials. Microscopy: Advances
In Scientific Research and Education, FORMATEX 2014, p:1065-1070
6. Venkannah S., 2004. Metallurgy Laboratory, Material Science. University of
Mauritius.
7. Vignati E. 2005. Optical Methods for Investigation of Application-Oriented
Complex Fluids. Ph.D Thesis, Radiation Science & Technology, Polytechnic
University of Milan.
98.0
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99.0

17