1

Light Microscopy
Light or optical microscopy is the primary means for scientists and engineers to examine
microstructural
the microstructure of materials. The history of using a light microscope for
examination of mnaterials can be traced back to the 1880s. Since then, light microscopy has been
widely used by metallurgists to examine metallic materials. Light microscopy for metallurgists
became a special field named metallography. The basic techniques developed in metallography
polymers.
are not only used for examining metals, but also are used for examining ceramics and
microstructural examination
In this chapter, light microscopy is introduced as a basic tool for
of materials including metal, ceramics and polymers.

1.1 Optical Principles

1.1.1 Inage Formation
Reviewing the optical principles of microscopes should be the first step to understanding light
microscopy. The optical principles of microscopes include image formation, magnification and
resolution. Image formation can be illustrated by the behavior of a light path in a compound
light microscope as shown in Figure 1.1. A specimen (object) is placed at position A where it
is between one and two focal lengths from an objective lens. Light rays from the object firstly
converge at the objective lens and are then focused at position B to form a magnified inverted
lens)
image. The light rays from the image are further converged by the second lens(projector
toform a final magnified image of an object at C.
The light path shown in Figure 1.1 generates the real image at C on a screen or camera film,
which is not what we see with our eyes. Only areal image can be formed on a screen and
photographed. When we examine microstructure with our eyes, the light path in a microscope
goes through an eyepiece instead of projector lens to form a virtual image on the human eye
retina, as shown in Figure 1.2. The virtual image is inverted with respect to the object. The
virtual image is often adjusted to be located as the minimum distance of eye focus, which
is conventionally taken as 250mm from the eyepiece. A modern microscope is commonly
equipped with a device to switch between eyepiece and projector lens for either recording
images on photographic film or sending images to a computer screen.

Materials Characterization Yang Leng

© 2008 John Wiley & Sons (Asia) Pte Ltd
 Materials Characterization

Final image
Object

1st image

Figure 1.1 Principles of magnification in a microscope.

Advanced microscopes made since 1980 have more complicated optical arrangement
called 'infinity-corected' optics. The objective lens of these microscopes generates parallel
beams from a point on the object. A tube lens is added between the objective and eyepiece to
focus the parallel beams to form an image on a plane, which is further viewed and enlarged by
the eyepiece.
The magnification of a microscope can be calculated by linear optics, which tells us the
magnification ofa convergent lens M.

M= (1.1)

where f is the focal length of the lens and v is the distance between the image and lens. A
higher magnification lens has a shorter focal length as indicated by Equation 1.1. The total
Eyepiece
Objective
Object Eye Lens
Final Image

Primary Image
Retina

Virtual Image,

250mm

Figure 1.2 Schematic path of light in amicroscope with eyepiece. The virtual image is reviewed by a
human eye composed of eye lens and retina.
 Light Microscopy 3

magnification of acompound microscope as shown in Figure I.I should be the magnification

of the objective lens multiplied by that of the projector lens.
(U - fD(V2 - f2) (1.2)
M= M M) =

When an eyepiece is used, the total magnification should be the objective lens magnification
multiplied by eyepiece magnification.

1.1.2 Resolution
We naturally ask whether there is any limitation for magnification in light micrOscopes
because Equation 1.2 suggests there is no limitation. However, meaningful magni
fication of a light microscope is limited by its resolution. Resolution refers to the
minimum distance between two points at which they can be visibly distinguished as two
points. The resolution of a microscope is theoretically controlled by the diffraction of
light.
Light diffraction controlling the resolution of microscope can be illustrated with the images
of two self-luminous point [Link] the point object is magnified, its image is acentral
spot (the Airy disk) surrounded by a series of diffraction rings (Figure 1.3), not a single spot.
To distinguish between two such point objects separated by a short distance, the Airy disks
should not severely overlap each other. Thus, controlling the size of the Airy disk is the key
to controlling resolution. The size of the Airy disk (d) is related to wavelength of light (a)

-2.237 -1.22n .22n 2.23r

ndsino

Figure 1.3 Aself-luminous point object and the light intensity distribution along aline passing through
its center.
 Materials Characterization

Figure 1.4 Intensity distribution of two Aiy disks with adistance . I, indicates the maximum intensity
of each point and l, represents overlap intensity.

and angle of light coming into the lens. The resolution of a microscope (R) is defined as the
minimum distance between two Airy disks that can be distinguished (Figure I.4). Resolution
is a functionof microscope parameters as shown inthe following equation.
d 0.612
R= u sin a
(1.3)

where u is the refractive index of the medium between the object and objective lens and a
is the half-angle of the cone of light entering the objective lens (Figure 1.5 ). The
u sin a, is called the nunerical aperture (NA). product,
According to Equation 1.3, to achieve higher resolution we should use
length light and larger NA. The shortest wavelength of visible light is shorter wave
about 400 nm,

Aperture

Object
Optical Axis

Objective Lens

Figure 1.5 Thecone of light entering an

objective lens showing a the half angle.
 Light Microscopy

while the NA of the lens depends on a and the medium between the lens and object.
Two media between object and objcctive lens are commonly used: either air for which
=l,0r oilfor which L.5. Thus, the maximum value of NA is about 1.5. We estimate the
best resolutionof a light microscope from Equation I.3 as about 0.2 um.
Effective Magnification
Magnification is meaningful only in so far as the human eye can see the features resolved
by the microscope. Meaningful magnification is the magnification that is suficient to allow
the eyes to see the microscopic features resolved by the microscope. A microscope should
enlarge features to about 0.2 mm, the resolution level of the human eye. Thus, the effective
magnifcation of light microscope should approximately be Mef =0.2 +0.2 x 10° =1.0 x 10'.
Ahigher magnification than the effective magnification only makes the image bigger, may
make eyes more comfortable during observation, but does not provide more detail in an image.
Brightness and Contrast
Tomake amieroscale object in amaterial specimen visible, high magnification is not sufficient.
Amicroscope should also generate sufficient brightness and contrast of light from the object.
Brightness refers to the intensity of light. In a transmission light microscope the brightness is
related to the numerical aperture (NA) and magnification (M).

Brightness = M2
(NA (1.4)

In a reflected light microscope the brightness is more highly dependent on NA.

(NA)
Brightness = M2
(1.5)

These relationships indicate that the brightness decreases rapidly with increasing magnifica
tion, and controlling NA is not only important for resolution but also for brightness, particularly
in a reflected light microscope.
Contrast is defined as the relative change in light intensity (/) between an object and its
background.

Contrast
lobject - Ibackground (1.6)
Ibackground
Visibility requires that the contrast of an object exceeds a critical value called the contrast
threshold. The contrast threshold of an object is not constant for all images but varies with
image brightness. In bright light, the threshold can be as low as about 3% while in dim light
the threshold is greater than 200%.

1.1.3 Depth of Field

Depth of field is an important concept when photographing an image. It refers to the range of
position for an object in which image sharpness does not change. As illustrated in Figure 1.6,
an object image is only accurately in focus when the object lies in a plane within a certain
 Materials Characterization
6

A
Aperture

.-D
Focal Plane

Figure 1.6 Geometric relation among the depth of field (D), the half angle entering the objective lens
(a) and the size of Airy disk (d).

distance from the objective lens. The image is out of focus when the object lies either closer
to or farther from the lens. Since the diffraction effect limits the resolution R, it does not make
any difference to the sharpness of the image if the object is within the range of Dr shown in
Figure 1.6. Thus, the depth of field can be calculated.

d 2R 1.222
Df= tan a tan a (1.7)
L sin tan a

Equation 1.7 indicates that a large depth of field and high resolution cannot be obtained
simultaneously; thus, a larger D, means a larger Rand worse resolution. We may reduce angle
ato obtain a better depth of field only at the expense of resolution. For a light
a is around 45° and the depth of field is about the same as its microscope,
resolution.
We should not confuse depth offield with depth of focus. Depth of focus refers to the
range
of image plane positions at which the image can be viewed without appearing out of focus for
a fixed position of the object. In other words, itis the range of screen
positions in which and
images can be projected in focus. The depth of focus is M² times larger than the depth of field.

1.1.4 Aberrations
The aforementioned calculations of resolution and depth of field are based on the
assumptions
that all components of the microscope are perfect, and that light rays from any point on an
object focus on a correspondingly unique point in the image. Unfortunately, this is almost
impossible due to image distortions by the lens called lens aberrations. Some aberrations
affect the whole field of the image (chromatic and spherical aberrations), while others affect
only off-axis points of the image (astigmatism and curvature of field). The true resolution and
depth of field can be severely diminished by lens aberrations. Thus, it is important for us to
have a basic knowledge of aberrations in optical lenses.
Chromatic aberration is caused by the variation in the refractive index of the lens in the
range of light wavelengths (light dispersion). The refractive index of lens glass is greater for
 Light Microscopy 7

Blue
Red
Optical Axis Red
Blue

Figure 1.7 Paths of rays in white light illustrating chromaticaberration.

shorter wavelengths (for example, blue) than for longer wavelengths (for example, red). Thus,
the degree of light deflection by a lens depends on the wavelength of light. Because a range of
wavelengths is present in ordinary light (white light), light cannot be focused at a single point.
This phenomenon is illustrated in Figure 1.7.
Spherical aberration iscaused by the spherical curvature of a lens. Light rays from apoint
on the object on the optical axis enter a lens at different angles and cannot be focused ata single
point, as shown in Figure 1..The portionof the lens farthest from the optical axis brings the
rays to a focus nearer the lens than does the central portion of the lens.
Astigmatism results when the rays passing through vertical diameters of the lens are not
focused on the same image plane as rays passing through horizontal diameters, as shown in
Figure 1.9. In this case, the image of a point becomes an elliptical streak at either side of the
best focal plane. Astigmatism can be severe in a lens with asymmetric curvature.
Curvature of feld is an off-axis aberration. It occurs because the focal plane of an image
is not flat but has a concave spherical surface, as shown in Figure 1.10. This aberration is
especially troublesome with a high magnification lens with a short focal length. It may cause
unsatisfactory photography.

Optical
Axis

Figure 1.8 Spherical aberration.

 Materials Characterization
8

Optical Axis

aberration.
Figure 1.9 Astigmatism is an off-axis

Figure 1.10 Curvature of field is an off-axis aberration.

There are a number of ways to compensate for and/or reduce lens aberrations. For example,
combining lenses with different shapes and refractive indices corrects chromatic and spherical
aberrations. Selecting single wavelength illumination by the use of flters helps eliminate
chromatic aberrations. We expect that the extent to which lens aberrations have been corrected
is reflected in the cost of the lens. It is a reason that we see huge price variation in microscopes.

1,2 Instrumentation
Alight microscope includes the following main components:
" Illumination system;
" Objective lens;
" Eyepiece;
" Photomicrographic system; and
Specimen stage.

A light microscope for examining material microstructure can use either transmitted or
reflected light for illumination. Reflected light microscopes are the most commonly used for
metallography, while transmitted light microscopes are typically used to examine transparent
Light Microscopy 25

material (such as platinun) as the cathode in an electochemical cell filled with electrolyte.
The clectrochemical reaction on the anode produces selectiveetching onthespecimen surface.
Since clectrochemicaletching is a chemical reaction, hesides choosing a suitable etchant and
clectrolyte, temperature and time are thc key parameters to avoiding under-etching and over
ctching of specimens.
We may also use the method of tintetching to produce color contrast in microstructure. Tint
etchants, usually acidic, areable to deposit a thin (40 500 nm) film such as an oxide or sulfide
on specimen surfaces. Tint etching require avery high-quality polished surface for best results.
Tint etching can also be done by heat tinting, a process by which a specimen is heated to a
relatively low temperature in air. As it warms, the polished surface is oxidized. The oxidation
rate varies with the phase and chemical composition of the specimen. Thus, differences in the
thickness of oxidation films on surfaces generate variations incolor. Interferernce colors are
obtainedonce the film reaches a certain thickness. Effectivencss of heat tinting depends on the
material of specimens: it is effective for alloy secls and other non-ferrous metals and carbides,
but not for carbon or low alloy steels.

1.4 Imaging Modes

The differences in properties of the light waves reflected from microscopic objects enable us
to observe these objects by light microscopy. The light wave changes in either amplitude or
phase when it interacts with an object as illustrated in Figure 1.27. The eye can only appreciate
amplitude and wavelength differences in light waves, not their phase difference. The most
commonly used examination modes, bright-field and dark-field imaging, are based on contrast
due to ifferences in wave amplitudes. The wave phase differences have to be converted to
amplitude differences through special optical arrangements such as in the examination modes
of phase contrast, polarized light and Nomarski contrast. This scction introduces commonly
used modes of light microscopy for materials characterization.

1.4.1 Bright-Field and Dark-Field Imaging

Bright-field imaging is the predominant mode for examining microstructure. Dark-field imag
ing is also widely used to obtain an image with higher contrast than in bright-field. Figure 1.28

Reference
a) wave

Reduced
Amplitude
b) Object amplitude

Phase Retarded
c) object phase

Figure 1.27 (a) Reference wave; (b) amplitude difference; and (c) phase difference generated by
objects. (Reproduced with permission from D.B. Murphy, Fundamnentals of Light Microscopy and
Electronic Imaging,Wiley-Liss, O2001 John Wiley &Sons Inc.)
 Materials Characterization
26

OBJECTIVE

b
-OBJECT

-CONDENSER
-APERTURE

ANNULAR STOP-IRIS CENTRAL STOP

Figure 1.28 (a) Bright-field illumination; and (b) dark-field illumination in transmitted mode. Shaded
areas indicate where the light is blocked.

illustrates the difference in optical arrangement between these modes in transmitted illumina
tion. In bright-field imaging, the specimen is evenly illuminated by a light source. Dark-field
imaging requires that the specimen is illuminated by oblique light rays. There is a central
stop in the light path to block the central portion of light rays from illuminating the specimen
directly. Thus, the angle of the light rays illuminating the specimen is so large that light from
the specimen cannot enter the objective lens unless it are scattered by microscopic objects.
The dark fieldin reflected illumination is also realized using a central stop in the light path
(Figure 1.29), similar to that of transmitted illumination. The light rays in a ring shape will be
further reflected in order to illuminate a specimen surface with an oblique angle. Figure 1.30
shows the comparison between bright- and dark-field images of an identical field in a high
carbon steel specimen under areflected light microscope. Microscopic features such as grain
boundaries and second phase particles appear self-luminous in the dark field image as shown
inFigure 1.30.

1.4.2 Phase Contrast Microscopy

inherent
Phase contrast is a useful technique for specimens such as polymers that have little
diffraction
contrast in the bright-field mode. In the technique, a phase change due to lightinterference
based on
by an object is converted to an amplitude change. This conversion is
 Light Microscopy
27

Central stop

Figure 1.29 Dark-field illumination in a reflected light microscope.

Figure 1.30 Comparison between: (a) bright-field: and (b) dark-field images of AISI 1080 high
carbon steel. In addition to grain boundaries and oxide particles, annealing twins are revealed in
the dark-field image. (Reproduced with permission of ASM International®. All Rights Reserved.
[Link]. G.F. Vander Voort, Metallography Principles and Practice, McGraw-Hill.
New York. O 1984 ASM International®.)
 Materials Characteri
28

phenomenon of light waves as illustrated in Figure 1.31. Constructive interference OCCurS

a phase difference betwee
when combining two same-wavelength waves that do not have combining two waves witk
them. However, completely destructive interference occurS when
phase difference of a half wavelength (). arTangement in a
Figure 1.32 illustrates generation of phase contrast by a special optical
inter
transmitted light microscope. This optical arrangement creates completely destructive
ference when light is diffracted by an object in the specimen. A condenser annulus, an opaque
lens.
black plate with a transparent ring, is placed in the front focal plane of the condenser
Thus, the specimen is illuminated by light beams emanating from a ring. The light beam
that passes through a specimen without diffraction by an object (the straight-through light
beam) will pass the ring of a phase plate placed at the back focal plane of the objective lens.
"The phase plate is plate of glass with an etched ring of reduced thickness. The ring with

Wave 1 Scattering
event
Wave 1
-

Amplitude

2A

Wave 2 Wave 2'

Position
(a)
P Wave 3 Scattering
event
Wave 3

Amplitude
A
Wave 4
P Wave 4'
Position
(a)

Figure 1.31 llustration interference between waves: (a) constructive interference; and (b) completely
destructive interference. (Reproduced with permission from WJ. Callister Jr, Materials Science and
Engineering: An Introduction, 7th ed., John Wiley & Sons Inc., New York. O 2006 John Wiley & Sons
Inc.)
Light Microscopy 29

Image plane

Diffracted
light
Phase plate

Non diffracted
light
Objective

Condenser

Condenser
annulus

microscopy. Shading marks the paths of diffracted

Figure 1.32 Optical arrangement of phase contrast
Microscopyand Electronic
light. (Reproduced with permission from D.B. Murphy, Fundamentals ofLight
Imaging, Wiley-Liss. © 2001 John Wiley & Sons Inc.)

straight-through beam to be
reduced thickness in the phase plate enables the waves of the
through
advanced by .4 The light beam diffracted by the object in the specimen cannot passdiffracted
plate. If the
the ring of the phase plate but only through the other areas of the phase difference in phase is
beam is delayed by while passing through the object, a total ()
generated.
plane, com
When the straight-through beam and diffracted beam recombine at the image
pletely destructive interference occurs. Thus, we expect a dark image of the object in phase
produces variations in
contrast microscopy. Variation in phase retardation across the specimen
contrast images
contrast. Figure 1.33 shows image differences between bright field and phase
of composites in transmission light microscopy. In reflected light microscopy, phase contrast
can also be created with a condenser annulus and phase plate similar to those in transmitted
light microscopy.
 Materials Characterization
38

(a)

Figure 1.44 Micrographs of asphalt-polyolefin elastomer (POE) blend obtained with transmitted light
microscopy: (a) bright-field image that cannot reveal the two phases in the blend; and (b) a fluorescence
labeled image that reveals two-phase morphology. (Reproduced with permission of Jingshen Wu.)

fluorescence microscopy with epi-illumination. Ahigh pressure mercury or xenon light can be
used for generating high intensity, short wavelength light. The light source should be ultravio
let, violet or blue,depending on the types of fluorchromes used in the specimen. A fluorescence
filter set, arranged in a cube as shown in Figure 1.43, includes an exciter filter, dichroic mir
ror and a barrier filter. The exciter filter selectively transmits a band of short wavelengths
for exciting a specific fluorchrome, while blocking other wavelengths. The dichroic mirror
reflects short wavelength light to objective lens and specimen, and also transmits returning
fluorescent light toward the barrier filter. The barrier filter transmits excited fluorescent light
only, by blocking other short wavelength light. Figure 1.44 shows an example of fluorescence
microscopy used for examining apolymer blend. Fluorescence labeling reveals the dispersed
polymer particles in an asphalt matrix, which cannot be seen in the bright-field image.

1.5 Confocal Microscopy

Confocal microscopy is a related new technique that provides three-dimensional (3D) optical
resolution. Image formation in a confocal microscope is significantly different from a con
ventional light microscope. Compared with a conventional compound microscope, a modern
confocal microscope has two distinctive features in its structure: a laser light source and a
Light Microscopy 39

scanning device. Thus,the confocalmieroscope is often referred to as the confocal laser scan
ning micOscope (CLSM). The laser light provides ahigh-intensity beam to generate image
signals from individual microscopic spots in the specimen. The scanning device moves the
beam ina rectangular area of specimen to construct a 3D image on a computer.

1.5.1 Working Principles

The optical principles of confocal microscopy can be understood by examining the CLSM
beam is
optical path that has reflected illumination as illustrated in Figure 1.45. The laser which
lens,
focused as an intense spot on acertain focal plane of the specimen by a condenser
aperture is placed
is also serves as an objective lens to collect the reflected beam. A pinhole
from the focal plane in
at a confocal plane in front of the light detector. The reflected beam
aperture blocks the
aspecimnen becomes a focused point at the confocal plane. The pinhole the light signals
reflected light from the out-of-focal plane from entering the detector. Only

detector

Pinhole aperture

Dichroic
mirror

Laser
point source

Objective lens

Focal plane
Specimen

Figure 1.45 Optical path in the confocal microscope. (Reproduced with permission from D.B. Murphy.
Fundamentals of Light Microscopy and Electronic Imaging, Wiley-Liss. O 2001 John Wiley &Sons Inc)
 Materials Characterization
40

photo
detector
photo confocal
detector
pinholes
confocal
pinholes

dichroic laser
mirror
dichroic
mirror
Scan
mirror
SCan
miror

on-axis
on-axis
conjugate off-axis
planes

entrance
pupil plane

Figure 1.46 Two scanning methods in the confocal microscope: (a) specimen scanning: and (b)
laser scanning. (Reproduced with permission from M. Müller, Introduction to Confocal Fluorescence
Microscopy, 2nd ed., SPIE Press, Bellingham, Washington. © 2006 Michiel Müller)

from the focal point in the specimen are recorded each time. Since the pinhole aperture can
block a large amount of reflected light, high-intensity laser illumination is necessary to ensure
that sufficient signals are received by the detector. The detector is commonly a photomultiplier
tube (PMT) that converts light signals to electric signals for image processing in a computer.
To acquire an image of the focal plane, the plane has to be scanned in its two lateral directions
(y directions).To acquire a 3D image of a specimen, the plane images at different vertical
positions should also be recorded. A scanning device moves the focal laser spot in the x-y
directions on the plane in a regular pattern called a raster. After finishing one scanning plane,
the focal spot is moved in the vertical direction to scan a next parallel plane.
Figure 1,46 illustrates two different methods of scanning in CLSM: specimen and laser
scanning. Specimen scanning was used in early confocal microscopes. The specimen moves
with respect to the focal spot and the optical arrangement is kept stationary as shown in Figure
1.46a. The beam is always locatedat the optical axis in the microscope so that optical aberration
is minimized. The main drawback of this method is the low scanning speed. Laser scanning
is realized by two scanning mirrors rotating along mutually perpendicular axes as shown in
Figure 1.46b. The scan mirror can move the focal spot in the specimen by sensitively changing
the reflecting angle of miror. Changing the vertical position of the spot is still achieved by
moving the specimen in the laser scanning method.
The resolution of theconfocal microscope is mainly deternined by the size of focal spot of
the laser beam. High spatial resolution about 0.2 um can be achieved. A3D image of specimen
20 um
 Materials Characterization
42

Figure 1.48 Confocal micrographs of polyurethane foam with labeled particulates: (a)
highlights of
particulates; and (b) locations of particulates on the foam surfaces. The scale bar is Imm. (Reproduced
with permission from A.R. Clarke and C.N. Eberhardt,
Woodhead Publishing Ltd, Cambridge UK. O 2002 Woodhead MicroscopyTechniques for Materials Science,
Publishing Ltd.)
shows an example of how a 3D image is formed with
biological specimen, a Spathiphyllum pollen grain, wasconfocal fluorescence microscopy. A
fluorescently
orange. In total, 80sections of the pollen grain were imaged for 3D labeled with acridine
tical distance between sections is 1.1 um. Figure 1.47 reconstruction. The ver
reconstructed 3D image. shows 11 of 80 plane images and the
Although its major applications are in biology, confocal microscopy can also
examining the surface topography and internal structure of be used for
ure 1.48 shows an example of using confocal semi-transparent materials. Fig
fluorescent
penetration through an air filter made of polymer foam. microscopy to examine particulate
labeled and their locations in the polymer foam are revealedThe particulates were fluorescently
against the polymer foam surfaces.

Figure 149 3D confocal fluorescent micrograph of silica particles in

low density polyethylene.
(Reproduced with permission from A.R. Clarke and C.N. Eberhardt, Microscopy
Materials Science, Woodhead Publishing Ltd, Cambridge UK. O 2002 Woodhead Techniques for
Publishing Ltd.)
Light Microscopy
43

Figure .49 shoWs an example of using

features in a specimen. The specimen isconfocal fluorcscent nicroscopy to reveal microscopie
lowdensity polyethylene
cently labeled slica particles. The (LDPE)containing nuoes
clearly revealed by 3D confocal particle size and distribution in the polymer matrix can e
in microscopy.
dimension light microscopy for materials Thus, confocal microscoPY provides us a new
materials science are not as broad as in characterization, even though its applicatos
biology.
References
| Bradbury, S.S. and
|21 Rawlins, D.J. (1992)Bracegirdle, B. (1998) Intoduction to Light
Light Microscopy, BIOS scientific Microscopy, BIOS Scicntific Publshers.
(3] Murphy, D.B. (2001) Fundamentals of Light Publishers.
Microscopy and Electronic Imaging, Wiley-Liss.
[4] Vander Voort, G.F. (1984) Metallography Principles and Practice. McGraw-Hill Book Co., New York.
[5] Callister Jr., W.J. (2006) Materials Science and
New York.
Enoineerine: An Introduction, 7th cd., John Wiley & Sons,

[6] Clarke, A.R. and Eberhardt, C.N. (2002) Microscopy Techniaues for Materials Science, Woodhead Publishing
Ltd, Cambridge UK.
17]Hemsley, D.A. (1989) Applied Polymer Light Microscopy, Elsevier Applied Science, London.
[8] Swayer, L.C. and Grubb, D.T. (1996) Polymer Microscopy, Chapman & Hall, London.
(9] American Society for Metals (1986) Metals Handbook. Volume 10. 9th edition, American Society for Metals,
Metals Park.
[10] Scheirs,J. (2000) Compositional and Failure Analysis of Polymers:APractical Approuch, John Wiley &Sons,
Ltd, Chichester.
|11] Müller, M.(2006) Introduction to Confocal Fluorescence Microscopy, 2nd edition, The Intemational Society for
OpticalEngineering, Bellingham, Washington.

Questions
1.1 Three rules govern light path for asimple lens:
1. A light ray passing through the center of alens is notdeviated;
2. A Light ray parallel with optic axis will pass through the rear focal point; and
3. A ray passing through the front focal point will be refracted in a
direction parallel
to the axís. Sketch the light paths from object to image in a single lens system in
following situations.
distance of
(a) a <f (b) a=f (c) 2f> a>f; (d) a=2f; and (e) a> 2f, where a is the
obiect from lens and fis the focal length of lens. By this exercise, you may understand
that 2f> a>fis necessary to obtain a real magnified image. magni
12 The working distance between the specimen and objective lens is determined by
fcations of the lens. Estimate the difference in the working distance for objective lens
50x.
with power of 5 x, 20xand a light microscope,
13 Calculate the resolution and the depth of fieldof the objective lenses oftreated as 1. Assume
listed below. The refractive index of vacuum is 1, and that of air can be
blue light is used in the microscope.
Magnification/NA
5x/0.13
10x/0.25
20x/0.40
50x/0.70
100×/0.90