30 th october micrscope final

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MICROSCOPES

Author : Dr.P.R.SANJAYA M.D.S.,

Assistant Professor, Faculty of Dentistry

Department of Oral Maxillofacial Pathology & Microbiology

“COLLEGE OF DENTISTRY ”, UNIVERSITY OF HA’IL,

KINGDOM OF SAUDI ARABIA.

email – [email protected]

Co-author : Dr. RhutviVirani B.D.S.,

House Surgeon,

Mahuva, Bhavanagar, Gujarat, India

email- [email protected]

mailto:[email protected]






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Acknowledgement

I take this opportunity to express my profound gratitude and deepest

regards to The Mother & Sri Aurobindo for guiding me to take up

this topic & complete it on time.

Dr.P.R.Sanjaya

Immeasureable appreciation and deepest gratitude to my parents

and my sister who are all the reasons of my life.

I would like to express the deepest appreciation to Dr.P.R.Sanjaya

M.D.S, sincere, exemplary guidance and encouragement.

Dr.Rhutvi Virani



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CONTENT

Sr no. Topic Page. No.

1 Chapter I-History 6

2 Chapter II-Birth of the Light Microscope 8

3 Chapter III-Type of Microscopes 14

4 Chapter IV-Fundamentals of the Microscope 17

5 Chapter V-Components of the Microscope 28

6 Chapter VI-Care of the Microscope 70

7 Chapter VII-Micrometry 79

8 Chapter VIII-Alignment of Light Microscope for Bright Field 82

9 Chapter IX-Dark Field Optical Microscopy 90

10 Chapter X-Phase Contrast Microscopy 100

11 Chapter XI-Differential Interference Contrast Microscopy 110

12 Chapter XII-Polarised Light Microscopy 117

13 Chapter XIII-Flouresence Microscopy 134

14 Chapter XIV-The Confocal Microscope 157

15 Chapter XV-Electron Microscope 161

16 Chapter XVI-Stereomicroscope 188

17 Chapter XVII-Recent Advances 222

18 References 226



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List of Figure

Sr no. Figures Page no.

1 Robert Hook’s micoscope 10

2 Antony Van Leeuwenhoek 11

3 One of Leeuwenhoek’s microscope 12

4 Size, shape and motility of bacteria by Leeuwenhoek 12

5 Representation of a light ray showing wavelength

and amplitude

18

6 Diminished brightness as light gets further from the source 18

7 Various type of lenses 20

8 Phenomenon of retardation and refraction 22

9 Image formation-Real image , virtual image 23

10 Virtual image formation 24

11 Spherical and chromatic aberration 25

12 Components of microscope 28

13 Praboloid Condenser 30

14 Illumination of microscopic object with or without condenser 31

15 Swing out top lens condenser 35

16 Abbe condenser 36

17 Small microscope 39

18 Critical illumination 41

19 X-Y Translation mechanical stage 47

20 Universal stage 48

21 Body tube 51

22 Condenser centering and condenser adjusting screws 5323 Optical components 54

24 Effect of immersion oil on light rays in compound microscope 63

25 Dry and oil immersion objective lens 63

26 Simple eyepieces 66

27 Size of the real fields 67

28 Ocular micrometer disk 81

29 Inverted light microscope 89

30 Dark ground illumination 92

31 An excellent type of microscope lamp suitable both for ordinay

work and the dark illumination

93

32 DF condenser with lamp attached 93

33 Abbe condenser with dark field stop, Parabolid condenser,

Cardiod condenser

95



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34 Stops for dark field illumination 96

35 Spirochetes visualized by dark ground microscopy 99

36 Figure describing optical principle 102

37 Phase contrast microscope accessories 105

38 Phase contrast 106

39 Bright field ,Dark field, Phase contrast 108

40 a)Diffraction at slit b)Ray cross by diffraction at closely

adjacent slits c)Rays cross giving phase conditions for

amplitude differences d)wave peaks interfere in regular

pattern

111

41 Wallaston prism 118

42 Condenser 122

43 Polarizing filter 124

44 Birefringence in polarized light 127

45 Filters in fluorescence microscopy 144

46 Condenser in fluorescence microscopy 14647 Incident fluorescence illumination 148

48 Immuno fluorescence staining for diagnosis of oral blistering

disease

156

49 Electron microscope 166

50 Components of electron microscope 167

51 Transmission electron microscope 172

52 Flow chart illustrating the steps in the preparation of specimen

for diagnosis by electron microscope

173

53 Some examples of specimen gride apparatus for application of

plastic support films

175

54 Electron microscope of a bacteriophage without shadowing

and with shadowing

177

55 Effects of wavelength on resolution 178

56 Bayer pattern 199

57 Image analysis by computers 207



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Chapter- I

History

During that historic period known as the Renaissance, after the "dark

middle ages‖, there occurred the inventions of printing, gunpowder

and the mariner's compass, followed by the discovery of America.

Equally remarkable was the invention of the light microscope: an

instrument that enables the human eye, by means of a lens or

combinations of lenses, to observe enlarged images of tiny objects. It

made visible the fascinating details of worlds within worlds.

Long before, in the hazy unrecorded past, someone picked up

a piece of transparent crystal thicker in the middle than at the edges,

looked through it, and discovered that it made things look larger.

They were named lenses because they are shaped like the seeds of

a lentil. Someone also found that such a crystal would focus the sun's

rays and set fire to a piece of parchment or cloth. Magnifiers and

"burning glasses" or "magnifying glasses" are mentioned in the

writings of Seneca and Pliny the Elder, Roman philosophers during

the first century A. D., but apparently they were not used much until

the invention of spectacles, toward the end of the 13th century.

http://inventors.about.com/library/inventors/blprinting.htm

http://inventors.about.com/library/inventors/blrocketfirework.htm

http://inventors.about.com/library/inventors/blcompass.htm

http://inventors.about.com/library/inventors/bleyeglass.htm#Eyeglasses

http://inventors.about.com/library/inventors/bleyeglass.htm#Eyeglasses

http://inventors.about.com/library/inventors/blcompass.htm

http://inventors.about.com/library/inventors/blrocketfirework.htm

http://inventors.about.com/library/inventors/blprinting.htm



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The earliest simple microscope was merely a tube with a plate

for the object at one end, and at the other, a lens which gave a

magnification less than ten diameters -ten times the actual size.

These excited general wonder and were used to view fleas or tiny

creeping things and so were dubbed as ―flea glasses." 7



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Chapter- II

Birth of the Light Microscope

Birth of the Light Microscope

The first useful microscope was developed in the Netherlands

between 1590 and 1608. There is almost as much confusion about

the inventor as about the dates. Three different eyeglass makers

have been given credit for the invention. The possible inventors are

Hans Lippershey (who also developed the first real telescope), Hans

Janssen, and his son, Zacharias.

About 1590, two Dutch spectacle makers, Zaccharias Janssen

and Hans, while experimenting with several lenses in a tube,

discovered that nearby objects appeared greatly enlarged. That was

the forerunner of the compound microscope and of the telescope.

Galileo Galilei father of modern physics and astronomy, heard of

these early experiments, worked out the principles of lenses, and

made a much better instrument with a focusing device in 1610. 4

http://inventors.about.com/library/inventors/bltelescope.htm

http://en.wikipedia.org/wiki/Galileo_Galilei



http://inventors.about.com/library/inventors/bltelescope.htm



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Christian Huygens, another Dutchman, developed a simple 2-

lens ocular system in the late 1600's that was achromatically

corrected and therefore a huge step forward in microscope

development. 7

Robert Hooke made & used a compound microscope in the

1660‘s & described his fascinating explorations of the newly

discovered universe of the microscopic in his classic Micrographia,

published at the request of the royal society in London in 1665 which

contains beautiful drawings based on his microscopic observations.

Robert Hooke re-confirmed Antony van Leeuwenhoek's discoveries

of the existence of tiny living organisms in a drop of water. The first to

record small living organism from plaque in his mouth.

http://en.wikipedia.org/wiki/Christiaan_Huygens

http://en.wikipedia.org/wiki/Christiaan_Huygens



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Robert Hooke‘s microscope, published in 1665

Although Hooke‘s highest magnifications were possibly enough to

reveal bacteria, he apparently made no observations of them,

probably because he studied mainly opaque objects in the dry state

by reflected light, conditions that are not optimal for observation of

microorganisms. 4

Antonj van Leeuwenhoek (1632-1723) is the father of microscopy. A

contemporary of Hooke, & the man mainly responsible for revealing

the hitherto unknown & unseen world of micro-organisms, did not use

a compound microscope. He started as an apprentice in a dry goods

store where magnifying glasses were used to count the threads in



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cloth. He was not a trained scientist but was self-educated. He taught

himself new methods for grinding and polishing tiny lenses of great

curvature which gave magnifications up to 270 diameters, the finest

known at that time. These led to the building of his microscopes and

the biological discoveries for which he is famous. He was the first to

see and describe bacteria, yeast plants, the teeming life in a drop of

water, and the circulation of blood corpuscles in capillaries. During a

long life he used his lenses to make pioneer studies on an

extraordinary variety of things, both living and non living, and reported

his findings in over a hundred letters to the Royal Society of England

and the French Academy.

Unlike Hooke, Leeuwenhoek made many of his observations by light

transmitted through the object & that the microorganisms were

Antony Van Leeuwenhoek. Afanciful delineation based on a

famous portarait. The picture

shows accurately the size andshape of the first microscopes,

the manner in which they were

used , and the simple lab

apparatus of the “ Father of

bacteriology”



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suspended in various fluids, not immobilized or otherwise altered by

drying.4

Later, few major improvements were made until the

middle of the 19th century. Then several European countries began

to manufacture fine optical equipment but none finer than the

One of Leeuwenhoek’s microscopes: front, back and side views. The tiny spherical orhemispherical lens is held in the slightly raised structure in the upper part of the metal

plate. The object to be examined was mounted at the tip of the sharp pointed mounting pin. Focusing was accomplished by means of the three thumb screws to which mounting

pin is attached. These are approximately actual size.

In letters to the Royal Society, Leeuwenhoek

described the sizes, shapes and even the

motility of bacteria. These are his draings of

bacteria from the human mouth.A. A motile

Bacillus.B to D. Selenomonas sputigena.E.Micrococci. F.Leptothrix buccans. G.

Probably Spirochaeta buccalis. ( From Dobell:

Anton van Leeuwenhoek and His “ Little

Animals.” Harcourt, Brace and Co, 1932.



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marvelous instruments built by the American, Charles A. Spencer,

and the industry he founded. Present day instruments, changed but

little, give magnifications up to 1250 diameters with ordinary light and

up to 5000 with blue light. 7



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Chapter- III

Types of

Microscopes

Microscopes can largely be separated into two classes, optical

theory microscopes and scanning probe microscopes.

I. Optical theory microscopes (OTM) are microscopes which

function through the optical theory of lenses in order to magnify the

image generated by the passage of a wave through the sample. The

waves used are either electromagnetic in optical microscopes or

electron beams in electron microscopes. The types are the

Compound Light, Stereo, and the electron microscope.

II. In scanning probe microscopy (SPM), a physical probe is

used either in close contact to the sample or nearly touching it. By

http://en.wikipedia.org/wiki/Optical


http://en.wikipedia.org/wiki/Wave

http://en.wikipedia.org/wiki/Electromagnetic_waves

http://en.wikipedia.org/wiki/Optical_microscope

http://en.wikipedia.org/wiki/Electron

http://en.wikipedia.org/wiki/Electron_microscopes

http://en.wikipedia.org/wiki/Scanning_probe_microscopy

http://en.wikipedia.org/wiki/Scanning_probe_microscopy

http://en.wikipedia.org/wiki/Electron_microscopes

http://en.wikipedia.org/wiki/Electron


http://en.wikipedia.org/wiki/Electromagnetic_waves

http://en.wikipedia.org/wiki/Wave





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rastering the probe across the sample, and by measuring the

interactions between the sharp tip of the probe and the sample, a

micrograph is generated. The exact nature of the interactions

between the probe and the sample determines exactly what kind of

SPM is being used. Because this kind of microscopy relies on the

interactions between the tip and the sample, it generally only

measures information about the surface of the sample.

Some kinds of SPMs are:

Atomic force microscope, Scanning tunneling microscope, Electric

force microscope , Magnetic force microscope (MFM) & Near-field

scanning optical microscope. 7

Optical microscopes, through their use of visible wavelengths of

light, are the simplest and hence most widely used type of

microscope. There are two basic configurations of optical microscope

in use, the simple (one lens) and compound (many lenses).

a) Single-lens Microscope

Consists of one lens only, which produces an enlarged image. It is

much like an ordinary magnifying glass.

http://en.wikipedia.org/wiki/Micrograph

http://en.wikipedia.org/wiki/Atomic_force_microscope

http://en.wikipedia.org/wiki/Scanning_tunneling_microscope

http://en.wikipedia.org/w/index.php?title=Electric_force_microscope&action=edit


http://en.wikipedia.org/wiki/Magnetic_force_microscope

http://en.wikipedia.org/wiki/Near-field_scanning_optical_microscope






http://en.wikipedia.org/wiki/Magnetic_force_microscope



http://en.wikipedia.org/wiki/Scanning_tunneling_microscope

http://en.wikipedia.org/wiki/Atomic_force_microscope




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b) Compound Microscope

Consists of objective and eyepiece. In its simplest form—as used by

Robert Hooke, for example—the compound microscope would have a

single glass lens of short focal length for the objective, and another

single glass lens for the eyepiece or ocular. Modern microscopes of

this kind are usually more complex, with multiple lens components in

both objective and eyepiece assemblies. 2, 8, 9

Various compound light microscopes are:

Bright field light Microscope (standard compound light

microscope)

Dark-field Microscope

Phase Contrast Microscope

Interference Microscope

Polarizing Microscope

Fluorescence Microscope

Stereomicroscope

Confocal Microscope

http://en.wikipedia.org/wiki/Robert_Hooke

http://en.wikipedia.org/wiki/Focal_length

http://en.wikipedia.org/wiki/Objective_%28optics%29

http://en.wikipedia.org/wiki/Eyepiece

http://en.wikipedia.org/wiki/Phase_contrast_microscope

http://en.wikipedia.org/wiki/Fluorescence_microscope

http://en.wikipedia.org/wiki/Fluorescence_microscope

http://en.wikipedia.org/wiki/Phase_contrast_microscope


http://en.wikipedia.org/wiki/Objective_%28optics%29

http://en.wikipedia.org/wiki/Focal_length

http://en.wikipedia.org/wiki/Robert_Hooke



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Chapter- IV

Fundamentals of

the Light Microscope

Basic optics has been unchanged over 300 years. Light

radiates in all directions from its source. Each ray, unless it is faced

with any interference in its path, travels in a straight line to infinity.

Properties:

Amplitude refers to the strength of the energy or brightness of

the light. When light travels through any medium, the amplitude

diminishes to a greater or lesser degree depending upon the medium.

The distance between the apex of one wave and the next is the

wavelength and is measured in nanometers. Wavelength determines

color.



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The number of waves per second is referred to as the frequency. The

frequency of a light wave remains constant. Individual rays of

identical frequency from the same source are said to be coherent.

Representation of a light ray showing wavelength and amplitude.

The amplitude( brightness)diminishes as light gets further from the

source because of absorption into the media through which it passes.



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These rays may combine or interfere with each other in an

observable way. Rays from different sources or of different

frequencies are said to be non-coherent. 1, 5, 10, 11

LENS:

A lens is the name given to a piece of glass or other

transparent material, usually circular, having the 2 surfaces ground &

polished in a specific form in order that rays of light passing through it

shall either converge or diverge.

A lens is called positive when it causes light rays to converge to

form a real image or it is negative in which case light rays passing

through will diverge or scatter & positive or real images will not be

seen. Positive lenses are thicker at the centre than at the periphery,

whereas negative lenses are thinner at the centre. Although the

shapes vary considerably, the characteristics remain the same.

In principle, a real image of any desired magnification can be

obtained from a single positive lens, but in practice this is

cumbersome because of the long lens-image distance. One or more

lenses can be used to magnify the image in stages (total

magnification equaling the product of the magnifications of each

lens). The image formed by one lens constitutes the object for the



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subsequent lens, whether or not a real intermediate image is formed.

5, 6

Various type of Lenses

Important phenomenon:

1. Retardation: Media through which light is able to pass will slow

down or retard the speed of the light in proportion to the density

of the medium (fig a).The higher the density, the greater the

degree of retardation.

Denser medium Denser medium

(a) (b) (c)



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2. Refraction: when light enters a sheet of glass at right angles it is

retarded in speed but its direction is unchanged. If the light enters the

glass at any other angle (fig b), a deviation of direction will occur in

addition to the retardation and this is called refraction. 1, 2, 3

A curved lens will exhibit both retardation and refraction (fig c). The

extent of which is governed by:

(a) The angle at which the light strikes the lens-the angle of

incidence.

(b) The density of the glass-its refractive index.

(c) The curvature of the lens.

The angle to which the rays are deviated within the glass or other

transparent medium is called the angle of refraction and the ratio of

the sine values of the angles of incidence (i) and refraction (r) gives a

figure known as the refractive index (RI) of the medium. In simple

words it is the ratio of the velocity of light in air to velocity of light in

that substance.

The greater the RI the higher is the density of the medium. The RI

of most transparent substances is known and is of great value in the

computation and design of lenses. Air has a refractive index of 1.00,

Water- 1.30 and glasses a range of values depending on type but



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averaging 1.5.

As a general rule light passing from one medium into a denser

medium is refracted towards the normal. And when passing into a

less dense medium refracted away from the normal. The angle of

incidence may increase to the point where the light emerges parallel

to the surface of the lens. Beyond this angle of incidence, total

internal reflection will occur & no light will pass through.



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Focus/image formation:

The word focus originally meant burning place, & was used to

indicate the point at which a lens concentrated the sun‘s rays to form

a sharp image having the power to burn. Parallel rays of light entering

a simple lens are brought together by refraction to a single point. The

principal focus or focal point is where a clear image will be formed of

an object. The distance between the optical center of the lens and the

principal focus is the focal length.

A real image is formed by rays passing

through the lens from the object, and can

be focused on a screen.

A virtual image is viewed through the lens.



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In addition to the principal focus, a lens also has other pairs of points,

one either side of the lens, called conjugate foci such that an object

placed at one will form a clear image on a screen placed at the other.

The conjugate foci vary in position, and as the object is moved nearer

the lens the image will be formed further away, at a greater

magnification, and inverted. This is the 'real image' and is that formed

by the objective lens of the microscope.

Virtual image formation



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If the object is placed yet nearer the lens within the principal focus,

the image is formed on the same side as the object, & is enlarged,

the right way up, and cannot be projected onto a screen. This is the

'virtual image' and is that formed by the eyepiece of the microscope.

1, 5, 9, 11, 12

Lens aberrations:

White light is composed of all the spectral colors and on

passing through a simple lens; each wavelength will be refracted to a

different extent, with blue being brought to a shorter focus than red.

This lens defect is chromatic aberration and results in an unsharp

image with colored fringes.

Spherical and chromatic aberration. a) Diagram to illustrate the spherical

aberration of parallel light rays passing through a biconvex lens; b) Diagram to

illustrate the chromatic aberration of a ray of light passing through a biconvex



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lens elements of different glass. 5

E.g. Fluorite, and of differing shapes

Chapter- V

Componenents of

the Microscope

Illuminating

• Mirror/light source

• Condenser

• Iris diaphragm

• Filters

Mechanical

• Nose piece

• Object stage

• Adjusting apparatus Components of Microscope

Table: Some linear measures commonly used in microbiology

1 inch – 2.54cm.

1 cm – 10mm.1 mm - 1000µ

1 µ - 0.001mm. = 0.00003937 or 1/ 25,400 inch =1000mµ

1mµ = 0.001 µ =10.0 Angstrom (A)

1A = 0.001 µ = 0.0000001mm. =1/254,000,000 inch



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Optical

• Objectives

• Eyepieces

• Body tube

1) The microscope proper, incorporating the body tube with the

objective at one end & eyepieces at the other

2) The stand, which includes the supporting, adjusting & illuminating

apparatus 1

Illuminating Apparatus & Illumination:

1) The sub stage:

Below the stage, & usually attached to it, is an adjustable sub stage

which can be moved up & down by a rack & pinion.

The sub stage consists of:

a) the condenser

b) an iris diaphragm

c) a filter carrier

d) a mirror

a) The condenser: Light from the lamp is directed into the first



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major optical component, the sub stage condenser, either

directly or by a mirror or prism. The main purpose of the

condenser is to focus or concentrate the available light into the

plane of the object. Within comfortable limits, the more light at

the specimen, the better is the resolution of the image. 1

The substage condenser gathers light from the microscope light

source and concentrates it into a cone of light that illuminates the

specimen with uniform intensity over the entire viewfield. It is critical

that the condenser light cone be properly adjusted to optimize the

intensity and angle of light entering the objective front lens. Each time

Paraboloid condenser

(Bausch and Lomb )



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an objective is changed, a corresponding adjustment must be

performed on the substage condenser to provide the proper light

cone for the numerical aperture of the new objective. 1, 5, 11

Illumination of microscopic object without and with substage condenser. The

condenser focuses all the light from the mirror on the object.



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Condenser height is controlled by a rack and pinion gear system that

allows the condenser focus to be adjusted for proper illumination of

the specimen. Correct positioning of the condenser with relation to

the cone of illumination and focus is critical to quantitative microscopy

and optimum photomicrography. 10 This is achieved by placing a slide

on the stage & viewing it through the 10X objective. After the radiant

field diaphragm is stopped down, the condenser is moved up /down

until the leaves around the edge of the diaphragm are in sharp focus

& the condenser is centered so that the circle of light is in the center

of the field of view. At this point, the leaves of the radiant field

diaphragm are opened until they just disappear from the field of view.

The condenser aperture diaphragm must now be adjusted. For best

viewing, the aperture should be closed slowly until the sharpest

image is obtained. 14

A critical factor in choosing substage condensers is the

numerical aperture performance that will be necessary to provide an

illumination cone adequate for the objectives. The condenser

numerical aperture should be equal to or slightly less than that of the

highest objective numerical aperture. Therefore, if the highest

magnification objective is an oil-immersion objective with a numerical



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exclusively for either spherical (aplanatic) or chromatic

(achromatic) optical aberrations. Achromatic condensers usually

contain three to four lens elements and are corrected in two

wavelengths (red and blue) for chromatic aberration. Aplanatic

condensers are well corrected for spherical aberration (green

wavelengths) but not for chromatic aberration. The highest level

of correction for optical aberration is incorporated in the aplanatic-

achromatic condenser. This condenser is well corrected for both

chromatic and spherical aberrations and is the condenser of

choice for use in critical color photomicrography with white light. 1,

5

3. When the objective is changed, for example from a 10X to 20X,

the aperture diaphragm of the condenser must also be adjusted to

provide a new light cone that matches the numerical aperture of

the new objective. This is done by turning the knurled knob on the

condensers.



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an opening of variable size for regulating the illumination. The

intensity of illumination should always, if possible, be reduced by

using light absorbing filters, or a variable resistance, not by closing

the diaphragm & never by rackiserng down the condenser.

Care must be taken to guarantee that the condenser aperture is

opened to the correct position with respect to objective numerical

aperture. When the condenser aperture diaphragm is opened too

wide, stray light generated by refraction of oblique light rays from the

specimen can cause glare and lower the overall contrast. On the

other hand, when the aperture is made too small, the illumination

cone is insufficient to provide adequate resolution and the image is

distorted due to refraction and diffraction from the specimen. 11

C) A filter carrier:

The filter carrier is usually a recessed metal ring, pivoting on a screw

to facilitate the easy removal of filters.



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d) The mirror:

The 2 sided mirrors are plane on one side & concave on the other. 1

2) Illumination & source of light:

Early microscopists relied on oil lamps and natural

sunlight to provide an external source of illumination for their primitive

microscopes. Daylight, which was formerly used for illumination

seldom, gives adequate lighting because the weather is too variable.

For this reason electric lamps are used. The objectionable yellowness

of artificial illumination can be eliminated with the use of blue glass

filters.11 Incandescent tungsten-based lamps are the primary

illumination source used in modern microscopes, with the exception

of those intended for fluorescence microscopy investigations. These

lamps are thermal radiators that emit a continuous spectrum of light

extending from about 300 nanometers to upward of 1200-1400

nanometers, with a majority of the wavelength intensity centered in

the 600-1200 nanometer region. Their design, construction, and

operation is simple consisting of an enclosed glass bulb filled with an



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inert gas and containing a tungsten wire filament that is energized by

a DC electric current. 7

Small Microscope

Lamp with day lightglass filters

The color temperature and luminance of these lamps varies

with the applied voltage, but average values range from about 2200 K

to 3400 K. When these lamps are used in photomicrography with

color film, the microscopists must use a lamp voltage that produces a

color temperature matching that of the film emulsion, usually

somewhere in the range between 3150 K and 3250 K. Often, the

color temperature must be fine-tuned for photomicrography by

http://www.olympusmicro.com/primer/lightandcolor/colortemp.html

http://www.olympusmicro.com/primer/lightandcolor/colortemp.html



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inserting filters into the light path that balance the illumination for the

color temperature of the film emulsion. 15

The source of illumination should be:

- uniformly intense

- should completely flood the back lens of the condenser with light

when the lamp iris

diaphragm is open & make the object appear as though it were self-

luminous

(1) Uniform intensity of illumination is most difficult to obtain since

the solid sources of light-tungsten arc or carbon arc-present

great difficulties if used over long periods. The difficulty is

overcome by using a closely wound filament with a diffusing

screen, although for routine work with a monocular microscope

a 60 watt pearl bulb will suffice. Kohler illumination may be

used.

(2) The source of light should be sufficient to enable its rays when

directed by the plane side of the mirror to flood the back lens of



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the condenser uniformly. The high intensity type of lamp has an

optical axis & must be correctly aligned for use, & the distance

from the microscope at which it is used adjusted so that the

lens magnifies the lamp image to the correct size, built-in light

source has been so adjusted. Where separate, the lamp & the

microscope should be connected so that accidental movement

of one or the other will not upset the alignment.

(3) The object will behave as if self-luminous if the opal bulb or the

image of the lamp condenser is focused in the object plane with

the substage condenser.5

There are 2 universally recognized methods for correct illumination.

(1)Nelson method or Critical illumination:

Critical illumination often is used with simple equipment & a

separate light source. The light source should be homogenous & no

amplifying condensers used. The light source is focused in the same

plane as the object, when the object is in focus, by racking the

substage condenser up or down.



41

(2) Kohler illumination:

With modern filament lamps the image of the filament causes

uneven illumination which is unacceptable. For this method to be

used the light source does not have to be homogenous, but a lamp

condenser is essential to project an image of the lamp filament on to

the substage iris diaphragm. In this system the lamp condensing lens

which is evenly illuminated functions as the light source. This method

must be used with compound lamps.

Image of the light source is focused by a lamp collector. The

image of the field or lamp diaphragm will now be focused in the object

plane & the illumination is even. The image of the light source & the

aperture diaphragm will in turn be focused at the back focal plane of

the objective & can be examined with the eyepiece removed. Poor

resolution will result unless the illumination is centered with respect to

the optical axis of the microscope. 1



42

For photography & all the specialized forms of microscopy it is

best to use Kohler illumination, where an image of the light source is

focused by the lamp collector or field lens in the focal plane of the

substage condenser.

Technique:

1. External light source:

1) The lamp should be positioned opposite the microscope, & a

blue daylight filter inserted in the filter carrier to absorb the

excess yellow given by artificial light.

2) Position the lamp so that the light strikes the center of the

mirror, & adjust the mirror so that the light is directed upwards

into the condenser. Modern microscopes have in-built,

condensing lenses & mirrors.

2. Internal source:

3) With a compound lamp focus the condensing lens so that an

image of the source of light is formed on the substage iris

diaphragm; if necessary hold a piece of white paper at this

position so that the image is visible.



43

4) Focus on an object on the stage & ensure that the field is

evenly illuminated.

5) With the object in focus, rack the substage condenser up or

down until a sharp image of the lamp iris diaphragm appears.

6) Center the image of the field diaphragm using substage

centering controls.

7) Open the field diaphragm until its circle of light is just larger

than the field of view. This reduces glare to the minimum.

8) Remove an eyepiece & adjust the substage iris diaphragm until

two-thirds of the back focal plane of the objective is illuminated.

Replace the eyepiece. The microscope is now ready for use.

For critical microscopy & photomicrography, the field diaphragm may

need to be centered each time the objective is changed. 5, 16

One cardinal rule for the microscopists is always to rack the

objective down near the object before looking through the eyepiece &

then to focus on the object by racking the objective up & away from

the object. This will avoid damaging the object or the front lens of the

objective, & is particularly important when using oil-immersion

objectives, which have very short working distances. This is good



44

practice even when using objectives with safety retracting front

lenses.

Central & oblique illumination: depends on the direction in

which light enters the microscope. To obtain central illumination the

mirror should be so adjusted that the light from the source is reflected

directly up the tube of the microscope. This is easily done by

removing the ocular & looking down the tube while adjusting the

mirror. The ocular is then replaced & the light reduced as much as

desired by means of the diaphragm.

In simple instruments oblique illumination is obtained by

swinging the mirror to one side so that the light enters the microscope

obliquely. In more complicated instruments it is obtained by means of

a rack & pinion, which move the diaphragm laterally. If the light is

oblique, an object in the center of the field appears to sway from side

to side when the fine adjustment is turned back & forth. The amount

of light admitted is also important which is regulated by the

diaphragm.

To see color & study the outline of an object use central illumination.

To study surface contour, use oblique light of a strength suited to the

color/opacity of the object. 2



46

e.g. From 0-80, & the other from 80-110 to avoid confusion in the

readings. Opposite these graduations will be the smaller vernier

scale, marked from 0-10. These 10 graduations, being equal to 9 in

the main scale, enable each of the latter to be subdivided by 10. 5

A stage can be classified according to design and functionality. In the

simplest case, the Plain stage consists of a rectangular or square

design containing several clips to hold the specimen slide. The

circular graduated stage is one of the most versatile and useful

designs for all types of microscopy and photomicrography. These



47

stages rotate 360°, permitting complete rotation of the samples and

great ease in fine-tuning the composition of view fields for

photomicrography.

Specialized Microscope Stages:

There are a wide variety of microscope stages that are designed for

specific purposes:

a) Inverted Microscope stage

b) Micromanipulators - It is often necessary to manipulate the

specimen while it is being observed under the microscope. This is

the case in many tissue culture and in vitro fertilization experiments

as well as genetic implantation procedures that require close

observation of the sample during the experiment.

c) Universal Stage - This stage permits tilting of a thin specimen at

any angle for measuring the optical structure of a birefringent crystal.

7



48

2) Body tube:

The body tube is attached to the limb of the microscope

which in turn, is attached to the base either directly or by a hinged

joint. A carrier or nosepiece for a number of objectives is usually fitted

at the lower end of the body tube. It rotates on a central pillar, & is

designated by the number of objectives it carries as double, triple or

quadruple nosepiece. The nose piece should bring each objective

into its correct position i.e., to say, centered on the optical axis, & at

the correct tube length. An increase in magnification is simply a

matter of rotating the nose piece, which is optically better than

changing the eyepiece since a large aperture is being used. The oil-

immersion lenses are, of course, an exception since the body tube

needs to be raised to place oil on the slide. The depth of the nose

piece will affect the tube length & this is generally 18mm in depth, the

actual length of the body tube being only 142mm.



50

focused at the lower focal plane of the eyepiece. This is achieved by

using 4 prisms. The lower central prism consists of 2 prisms

cemented together, at the interface of which there is a semi-silvered

surface: this silvering is a special process, fine grains of silver being

deposited so that alternate light rays are differentially treated, one

being reflected to the right & the other passing into the upper prism.

The light rays passing through the semi-silvered surface to the

upper prism travels through a greater thickness of glass than those

that are reflected- having the effect of retarding them-& this is



51

Body tube

compensated for by making the right hand prism with an extra

thickness of glass. Eyepieces with the prism attached, can be easily

moved together or apart, & the interocular distance adjusted to suit

individual requirements.

With a binocular body on a microscope, the optical tube length

may be increased from 160-240mm, & since the objectives are

corrected for the shorter tube length, a compensating lens is

incorporated to overcome this factor; the lens is also necessary to re-

focus the virtual image for the new tube length. The increase of tube

length also has the effect of increasing magnification, & binocular

attachments may have their magnifying factor engraved on them

which, since the tube is usually increased by one half is x1.5.

Magnification changers may be cited in the body tube above the

objective on a rotating mount. The magnification increase is engraved

at each position, for e.g. X1.25, X1.5. 5

Adjustment apparatus:



53

The type and quality of the objective has the greatest influence on the

performance of the microscope as a whole.

Within the objective there may be lenses and elements from 5

to 15 in number, depending on image ratio, type and quality. The

main task of the objective is to collect the maximum amount of light

possible from the object, unite it and form a high quality magnified

real image, some distance above. Magnifying powers, or more

correctly, object-to-image ratio of objectives is from 1:1 to 100:1 in

normal biological instruments.

2

Field NumberUIS2

Country originProduct nameCompany

SpecificationMagnification

Immersion medium / NA

Mechanical tube length /Thickness of cover glass



54

The ability of an objective to resolve detail is indicated by its

numerical aperture and not by its magnifying power. The numerical

aperture or NA is expressed as a figure, and will be found engraved

on the body of the objective. The figure expresses the product of two

factors and can be calculated from the formula.

NA = n x sin u

Where n=refractive index of the medium between the cover glass

over the object & the front lens of the objective & u=angle between

the optical axis of the lens & the outermost ray which can enter the

front lens.

In practice the maximum NA attainable with a dry objective is 0.95

Water immersion objective-1.30

Oil immersion objective -1.50 1, 5, 13

Magnification: is the increase in the size of the image of an object

.The power of a microscope is described with a number followed by

the letter "X". For example, if through a microscope you can see

something 25 times larger than actual size, its magnification power is

25X. The actual power or magnification of an optical microscope is

the product of the powers of the ocular (eyepiece), usually about 10X,

and the objective lens being used. Dependent on:

http://en.wikipedia.org/wiki/Magnification






56

Maximum

magnification

Field of view: the area visible through the microscope lenses. Field of

view decreases as magnification increases.

Resolution - Ability to distinguish closely spaced points as separate

points.

Resolution Limit - Smallest separation of points which can be

recognized as distinct.

Resolving Power - Resolution achieved by a particular instrument

under optimum viewing conditions. 8

Limit of resolution:

As mentioned, the value for resolution may be determined in

one of two ways. It can be measured as the smallest distance

between two points, which allows us to see the points as distinct.

With this measurement, resolution increases as the distance

decreases-that is, there is an inverse correlation between the limit of

resolution and what the eyes actually resolve.

resolving power of the eye

resolving power of the

microscope



57

0.61 X λ

Limit of Resolution = ---------------

NA

To change this to a direct correlation, one need only use the

reciprocal of the limit of resolution. Resolution is the reciprocal of the

limit of resolution. For measures of resolution then, as the value

increases, resolution increases. Consequently, most microscopists

today use resolution rather than limit of resolution to measure the

quality of their lenses.

The reason for a dichotomy between magnification and

resolution is the ability of the human eye to distinguish two points. It is

necessary that two points are about 0.1 mm apart when held 10" from

the face in order for us to detect them as two objects. If they are

closer than 0.1 mm, we will perceive them as a single object. If two

objects are 0.01 mm apart, we can not detect them unless we

magnify an image of them by 10X.

Unfortunately, a lens can magnify an image without increasing

the resolution. Several artifacts can be inherent in the lens design



58

which causes the objects to become blurry at the edges. Thus, even

though they can be made to appear 0.1 mm apart, the edges are so

blurry that we lose the ability to see them as two objects.

Resolution can be increased in three ways:

The easiest method is to increase the angle of light incidence, by

altering the position and/or design of the sub stage condenser.

Second, the refractive index can be maximized by using specially

manufactured lenses, and by controlling the medium through

which the light travels, i.e. using immersion oil with lenses

designed for this purpose.

The third method is to decrease the wavelength of light used.

For practical purposes, the wavelength has a larger effect on

resolution than either changes in the angle of incidence or the

refractive index. For maximum resolution, all three properties must be

optimized. 1, 5, 7

In practice, magnification can be increased in 2 ways:

Using a high power objective: As a rule this is the best way,

because resolving power is also increased, but it is often

undesirable because of the shorter working distance & because



59

the higher power objective often gives greater magnification

than is desired or cuts down the size of the real field too much.

Using a high power ocular: This is the simplest method. It has,

however, certain limitations. When an ocular that is too strong

is used, there results a hazy image in which no structural detail

is seen clearly (Empty magnification).

Types of objectives:

The objective lens is, at its simplest, a very high powered

magnifying glass i.e. a lens with a very short focal length. This is

brought very close to the specimen being examined so that the light

from the specimen comes to a focus about 160 mm inside the

microscope tube. This creates an enlarged image of the subject. This

image is inverted and can be seen by removing the eyepiece and

placing a piece of tracing paper over the end of the tube. By careful

focusing a rather dim image of the specimen, much enlarged can be

seen. It is this real image that is viewed by the eyepiece lens that

provides further enlargement.



60

All objectives are engraved with the information needed to obtain

their maximum performance as well as any possible limitations. Such

an engraving might read:

Plan 40/0.65

160/0.17

with indication that it is a planachromat; 40X magnification at a tube

length of 160mm, has a NA of 0.65 & should be used with a

coverglass of 0.170±0.01mm in thickness.

Achromatic Objectives:

It is the most commonly used objective. Modern well corrected lenses

of this type are more than adequate for routine microscopy

Apochromatic Objectives:

They are used in conjunction with highly corrected aplanatic or

achromatic condenser & compensating eyepieces. Must always be

used for photomicrography.

Flourite Objective (Neoflour):

Flourite/semi apochromatic objectives have flourite incorporated into

the lens system to give better colour correction. They represent a

quality of image mid way between that of achromat & apochromat.

Plan objectives:



61

Many type of PLAN objectives, such as Plan Apochromat / Plan

Fluorite / Plan Achromat. They are used to give a perfectly flat field,

with the whole field in focus at the same time. Planapochromat

objectives are mainly used for photomicrography. Planachromats are

used for cytology screening.

Polarizing Objective:

They are strain-free objectives & are used for polarizing microscope.

Phase Objectives:

They contain a phase plate for use in phase-contrast microscopy.

They have a designated phase with a number which refers to the

matching annulus.

Dry type objective:

It is the most commonly used objective with a range of magnification

from low to high power (1.25-100X) . 1, 5

Oil immersion objective:

When the rays of light emerge from the upper surface of the

condenser, some are reflected beyond the scope of the objective &

lost. Others are reflected away from the underside of the glass slide

on which the objective is mounted, & lost. Others are refracted &

reflected from its upper surface. Others are lost by refraction &



62

reflection in the object & at the surface of the objective lens. A

considerable part of these various losses & distortion of the image

can be prevented by eliminating the optical effect of these surfaces.

This is done by placing a clear, colorless fluid (immersion oil), having

the same refractive index as glass, between condenser & slide &

between slide & objective lens. For high power microscopy the

objective lens is made for oil-immersion. Immersion oil in effect can

increase the NA of a lens because it brings in more light rays. 4, 13

Water immersion objective:

– For brain slice specimen

– Use water as medium

Effect of immersion oil on light rays in

compound microscope. Light rays enter the

condenser from below. Light ray A shows

path of light if oil is placed only between

slide and objective lens (the common

practice).Broken line A shows loss of rays

A if oil is placed between slide and

objective, as above, and also between slide

and condenser(the practice in dark field

microscopy). Broken line B shows loss of

rays B if oil is not used. Arrows R,R,R

show additional loss of light by reflection

fromtop and bottom surfaces of slide if oil



63

Cover glass thickness:

Most objectives are designed for use with a cover glass

protecting the object. Oil immersion objectives do not have cover

glass restrictions since they will have the same RI as the immersion

oil. The cover glass thickness is only important if high power dry

objectives are being used. A figure giving the correct cover glass

thickness should be found engraved on the objective between 0.11-

0.22mm, usually this is 0.17 mm. 1, 5

Eyepiece:

Eyepieces are the final stage in the optical path of the

microscope. Their function is to magnify the image formed by the

objective within the body tube and present the eye with a virtual



64

image, apparently in the plane of the object being observed. Usually

this is an optical distance of 250 mm from the eye. In most

microscopes, the eyepiece is a compound lens, which is made of two

lenses one near the front and one near the back of the eyepiece tube

forming an air separated couplet. In many designs, the virtual image

comes to a focus between the two lenses of the eyepiece, the first

lens bringing the real image to a focus and the second lens enabling

the eye to focus on the now virtual image.

In all microscopes the image is viewed with the eyes focused at

infinity. Headaches and tired eyes after using a microscope are

usually signs that the eye is being forced to focus at a close distance

rather than at infinity.They may be used to correct residual errors in

the objective lenses & may be either under corrected or

overcorrected.

Undercorrected: when a blue ray of light will be refracted to a greater

degree than the red, this can be identified by the blue fringe that is

seen around the edge of the field diaphragm

Overcorrected: when the reverse is the case & an orange fringe may

be seen at the edge of the field diaphragm.



65

The eyepieces are classified as positive & negative eyepieces.

1. Negative Eyepiece:

Focus is within the lenses of the eyepiece, composed of 2

lenses-lower field lens & upper lens. Lower lens collects the image

that would have been formed by the objective (virtual image plane) &

cones it down to a slightly smaller image at the level of the field

diaphragm within the eyepiece. Upper lens then produces an

enlarged virtual image which is seen by the microscopists.

2. Positive Eyepiece:

Focus is outside the eyepiece lens system. Used as a simple

microscope. Diaphragm is outside the eyepiece, from which the

virtual image is focused & magnified by the eyepiece.



66

Types of Eyepieces available:

Huygenian Eyepiece:

They were originally designed by Huygens for the telescope. They

are most commonly used eyepieces. They are Negative, Undercorrected &

are best suited for use with achromatic objectives.

Ramsden Eyepiece:

These are positive oculars. Most of the compensated eyepieces are

of Ramsden type, having doublet or triplet lenses instead of single

lens. It is preferred for micrometer eyepieces as they impart less

distortion to scales.

Wide Field Eyepieces:

These lenses give a large field of view. They are valuable in

biological laboratory.

Size of the “real fields”

(actual areas seen through themicroscope) with variousobjectives and occulars and

the tube length of 160mm.The

size differs slightly with

microscopes of differentmakes.



67

High-Eye point Oculars:

They were introduced primarily for spectacle wearers. With normal

eyepieces, the distance between the top of the eyepiece & the exit

pupil is so small as to prevent the wearing of the glasses, but the high

eye point of these special oculars make this possible. It is advised

that the rubber guards supplied with such eyepieces be used to

prevent the scratching of the spectacle lens.

Compensating Eyepieces:

They were originally intended for use with apochromatic objectives

only but now are recommended for use with all modern objectives.

English speaking countries mark them ‗comp‘ & the German by the

letter ‗K‘.5

Extensions of the optical microscope:

Most modern instruments provide simple solutions for micro-

photography and image recording electronically. However such

capabilities are not always present and the more experienced

microscopist will, in many cases, still prefer a hand drawn image



68

rather than a photograph. This is because a microscopist with

knowledge of the subject can accurately convert a three dimensional

image into a precise two dimensional drawing . In a photgraph or

other image capture system however, only one thin plane is ever in

good focus.

Creating careful and accurate micrographs requires a

microscopical technique using a monocular eyepiece. It is essential

that both eyes are open and that the eye that is not observing down

the microscope is instead concentrated on a sheet of paper on the

bench besides the microscope. With practice, and without moving the

head or eyes, it is possible to accurately record the observed details

by tracing round the observed shapes by simultaneously "seeing" the

pencil point in the microscopical image. Practising this technique also

establishes good general microscopical technique. It is always least

tiring to observe with the microscope focussed so that the image is

seen at infinity and with both eyes open at all times. 7



69

Chapter- VI

Care of the Microscope

Microscopes get less attention than they deserve

because any deterioration is usually so gradual as to pass unnoticed

in day-today use. There are, in fact, only two places where sudden

catastrophic failure may occur. One of these is the lamp bulb which

will burn out with no warning. The other is the nosepiece clip which

breaks after long use, usually giving warning by gradually losing its

springiness so that the nosepiece rotates too freely. Whenever a



70

microscope is in constant use, the user is strongly recommended to

keep a spare lamp bulb and also a nosepiece clip with suitable

screws ready to hand.

When the performance deteriorates gradually, three possibilities

should be considered. These are: dirty lenses, misalignment so that

the optical axis is not straight and incorrect focusing of the light

source. If these faults are sought and corrected periodically, the effort

will be amply repaid by the optical and aesthetic rewards obtained. 12

Everything on a good quality microscope is unbelievably

expensive, so be careful.

Hold a microscope firmly by the stand, only. Never grab it by the

eyepiece holder, for example.

Hold the plug (not the cable) when unplugging the illuminator.

Since bulbs are expensive, and have a limited life, turn the

illuminator off when done.

Always make sure the stage and lenses are clean before putting

away the microscope.

Never use a paper towel, a kimwipe, or any material other than

good quality lens tissue or a cotton swab (must be 100% natural

cotton) to clean an optical surface. Be gentle. May use an



72

deposits at this site may include immersion oil and occasionally a thin

film or streak of mounting medium (balsam or D. P. X.) from a newly

mounted slide; these transparent films may not be obvious until the

lens is viewed with a magnifying glass in a good light.

The top of the condenser may collect dust and also minute

chips of glass; these are broken from the edges and corners of slides

by stage clips that are allowed to spring sharply into place. Because

of the likelihood that these glass chips will scratch the lens surface,

the condenser must be cleaned by blowing or gentle brushing before

being rubbed with even the softest tissue. The mirror collects dust. It

may be cleaned with no special precautions except when a surface-

aluminized or surface-silvered mirror is provided, as it may be when

the illumination is built in. In this case, special care is needed to avoid

scratching the delicate metallic coating; gentle mopping with tissues

soaked in alcohol and then with dry tissue is the most that is

permissible.

When cleaning any of the optical components of the micro-

scope, it is essential to avoid all forms of fibrous or starchy textiles; a

soft camel-hair brush will remove dust particles, and a piece of lens

tissue or well-washed soft and thin cotton may be used to remove



73

grease. The surface may be moistened by-condensation from the

breath, or by clean water. Obstinate grease marks can usually be

removed successfully with diluted alcohol; stronger grease solvents

(e.g. xylene) should be handled with caution since they may soften

the cement in which the lenses are mounted.

Sometimes the microscope image is marred because dust

particles appear to be superimposed on it. As an aid to the location of

this dust, the best plan is to proceed as follows. First move the slide

to make sure that the dust is not there. Secondly rotate the eyepiece;

if the dust particles rotate they are on one of the eyepiece lenses.

Thirdly, if the dust is still undetected, alter the focus of the condenser;

if the image of the dust vanishes it is on the condenser, mirror, or

lamp; if it persists it is in the objective. Fourthly: if the mirror is

adjustable, move it slightly; if the dust moves it is on the lamp or the

mirror itself. More rarely the microscope image appears to have a

fibre or thread superimposed upon it. This can usually be located in

the way already described, but occasionally the fibre will be found to

be caught in the edge of an iris diaphragm so that it projects into the

path of light. Gently opening and closing the iris diaphragms will

readily locate such a fibre.



74

When cleaning the optical system of a microscope, the com-

ponents should be dismantled as little as possible. It is better to

return an unsatisfactory component to the supplier, or to call for the

services of an expert, than to venture into unfamiliar territory. Such

items as high-power objectives and, above all, binocular prism

systems should not be dismantled by the inexperienced. Dust or

opacity in a compound lens or prism is very rarely due to a fault

between the various glass elements. When, however, attention to the

accessible surfaces does not remove the dust or opacity, the defect is

probably attributable to crystallization of the cement between two

elements, or the growth of fungi within the lens. Faults like these can

only be remedied by an expert.

The mechanical parts of the microscope also need to be

cleaned from time to time, but once again it is better not to dismantle

unfamiliar components. In general, it is comparatively easy to

dismantle and reassemble nineteenth-century and early twentieth-

century microscopes, since these were assembled by hand from

blocks of solid brass. Many modern microscopes, however, include

mechanical components that were assembled with the use of special

tools. Most modern iris diaphragms, for instance, should be



75

dismantled only by the expert. If the microscope is protected against

dust when not in use, it will need cleaning and lubricating only

occasionally. A piece of rag soaked in xylene is useful for removing

dirty oil, but all the xylene must be wiped away before new oil is

applied.

The parts requiring lubrication are: bearings that house rotating

axis (e.g. the coarse adjustment spindle), pivots (e.g. stage clips and

many fine adjustments), and slides (e.g. those permitting the

condenser mounting to slide up and down on the microscope stand).

The actual teeth of cogwheels, or rack and pinion mechanisms, do

not need lubricant since this collects dust and grit which is likely to

grind away the surfaces of the teeth until they fail to mesh firmly with

each other. In choosing a lubricant, the best plan is to follow the

instructions of the microscope manufacturer. In general we have

preferred to use light machine oil at frequent intervals. The alternative

is thin grease; this is particularly popular for old microscopes, where it

may confer a temporary improvement in performance by reducing the

play in the worn mechanical stage or other moving part. Grease does

not need to be renewed as often as oil; this sometimes produces a

false sense of security so that the microscope receives no attention



76

for a long time. It is then found that every trace of grease has been

squeezed out of the working parts and has dried up in gummy brown

nodules along their edges. 12

Daily cleaning routine

The microscope should be dusted daily, & the outer surface of

the lenses of objectives polished with lens tissue or cotton wool.

The top lens of the eyepiece should be polished to remove dust

or fingermarks, & the microscope set up for correct illumination.

Rotation of the eyepiece will show if any dust is still present, in

which case the eyepiece may need to be dismantled & both

lenses cleaned.

The substage condenser & the mirror are cleaned in a similar

manner: dust on the condenser will be apparent when this is

racked up & down, since it will come in & out of focus.

A little attention to cleaning the microscope daily will, by the

removal of chemically-active & sharp pieces of grit & foreign

matter, prolong the life of the instrument & make the weekly

cleaning task a short & simple one.



78

Chapter- VII

Micrometry

The standard unit of measurement in microscopy is a

micrometer, which is a 0.001mm. To measure microscopic object an

eyepiece micrometer scale is used inconjunction with a stage

micrometer. The eyepiece micrometer scale is usually a disc on

which is engraved an arbitrary scale. This is placed inside the

huygenian eyepiece, resting on the field stop. Eyepiece micrometers

may be purchased with the scale permanently in position; these are

usually Kellner eyepieces which have a focal plane below their



79

bottom lens. They give a sharp image of the scale & have a greater

eye clearance; they are an advantage for general work if spectacles

are worn. The stage micrometer consists of a 3X1 inch slide on which

a millimeter scale is engraved in 1/10 & 1/100 graduations.5

Graticule - a network of fine lines, dots, cross hairs, or wires in the

focal plane of the eyepiece of an optical instrument. Most "whole

world" graticules are laid out from -180 to 180 degrees Longitude and

from -70 to 70 degrees Latitude in spacing of 10 degrees.

The Difference between Graticules and Grids

Graticules are always expressed in geographic coordinates (latitude

and longitude) while grids are expressed in the native X and Y

coordinates of the coordinate system of the component. For

components using the Latitude / Longitude "non-projection", both

graticules and grids will appear as a grid of horizontal and vertical

straight lines. In projected coordinate systems, graticules will be

created as curved lines (if necessary) to parallel the curved form of

meridians of longitude or parallels of latitude in the projection. Grids,

however, will always appear as a grid of horizontal and vertical

straight lines. 12

An object may be measured by the following method:



80

-insert a micrometer eyepiece scale & place the stage micrometer on

the stage

-select the objective to be used when measuring the object, & focus

on the stage micrometer scale

-determine the number of divisions of the eyepiece scale equal to an

exact number of divisions of the stage micrometer scale

-Remove the stage micrometer, focus on the object to be measured &

determine the number of eyepiece divisions exactly covered by the

object.

Micrometer eyepiece with a movable scale

Calculate the size of the object as follows, assuming that 100

eyepiece divisions were equal to 10 small stage divisions, & that the

diameter of the object was exactly covered by 12 eyepiece divisions. 5

100 stage divisions=1mm=1000µm

100 eyepiece divisions=10 stage divisions



81

Therefore 100 eyepiece divisions=100µm

Therefore 1 eyepiece division=1µm

Therefore 12 eyepiece divisions=12µm

The diameter of the object, therefore, was 12 µm.

Chapter- VIII

Alignment of Light Microscope for Bright Field

Turn on transformer for tungsten light source and set at appropriate

level

Set condenser setting to bright field

Center lamp

Place centering disk (paper, plastic, frosted glass or centering

aid) over opening in stand below stage



83

Adjust sub stage condenser with centering screws until image

of field diaphragm is centered

Open field diaphragm until it is just outside field of view

Check focus of lamp filament (when aligning after bulb change)

Open sub stage aperture diaphragm

Remove eyepiece

Loosen lamp lock screw on lamp housing

Rotate bulb and move back and forth until illumination is most

intense and even

Adjust sub stage aperture diaphragm

Adjustment will vary for specimen

Start with aperture wide open

Close diaphragm slowly until image has best contrast

Use neutral density filter(s) or adjust transformer rheostat to adjust

brightness of illumination during viewing. 1, 5, 7, 16

Bright Field Microscopy Applications:

Bright field microscopy is best suited to viewing stained or naturally

pigmented specimens such as stained prepared slides of tissue



85

The interpretation of ground sections under the optical

microscope is complicated by both the thickness and crystalline

nature of the material. Often the material on the slide is 150 microns

thick and this means that there is a superimposition of features. The

presence or incorporation of cellular and organic material in the

mineralized tissue will alter the refractive index and consequently

influence its optical appearance.

The mineralized tissues do not take up stain as readily as the

soft tissues and the view under the optical microscope depends upon

the differences in refractive indices between the various structures.

To view these sections its better to use less light in the microscope by

closing down the diaphragm controls and not by reducing the lamp

intensity. 1

Advantages:

Simplicity of setup with only basic equipment required.

No sample preparation required, allowing viewing of live cells.

Limitations :

The technique can only image dark or strongly refracting objects

effectively.



86

Compound optical microscopes are limited in their ability to resolve

fine details by the properties of light and the refractive materials

used to manufacture lenses (to approximately 0.2 micrometre).

Out of focus light from points outside the focal plane reduces

image clarity.

Live cells in particular generally lack sufficient contrast to be

studied successfully, internal structures of the cell are colourless

and transparent. The most common way to increase contrast is to

stain the different structures with selective dyes, but this involves

killing and fixing the sample. Staining may also introduce artifacts,

apparent structural details that are caused by the processing of

the specimen and are thus not a legitimate feature of the

specimen.

Optical microscopes have a focal point, either chosen or fixed,

where the image is clear. This covers a two-dimensional area only.

A single optical image cannot capture all the details of a three-

dimensional shape in focus. 1, 5, 6, 12

Oblique Illumination:

This uses sideways (oblique) illumination; either by covering

part of the light source to give asymmetric lighting, or even an

http://en.wikipedia.org/wiki/Micrometre

http://en.wikipedia.org/wiki/Staining_%28biology%29

http://en.wikipedia.org/wiki/Artifact_%28observational%29

http://en.wikipedia.org/wiki/Focus_%28optics%29

http://en.wikipedia.org/wiki/Focus_%28optics%29

http://en.wikipedia.org/wiki/Artifact_%28observational%29

http://en.wikipedia.org/wiki/Staining_%28biology%29

http://en.wikipedia.org/wiki/Micrometre



87

external light source being shone sideways in the sample. This gives

the image a 3D appearance and can highlight otherwise invisible

features. 17

Reflected Light Microscope:

In brightfield reflected light microscopy, proper use of the two

variable diaphragms: the aperture iris diaphragm (closer to the light

source) and the field iris diaphragm (closer to the specimen), enable

the use of the highly desirable Kohler illumination. These diaphragms

are in the opposite of their respective positions in transmitted light,

the aperture diaphragm now being closer to the light source. In these

reflected light systems, the objective serves a dual function: on the

way down as a matching well-corrected condenser properly aligned;

on the way up as an image-forming objective in the customary role of

an objective projecting the image-carrying rays toward the eyepiece.

In a transmitted light system, changing the objective requires an

adjustment in the numerical aperture of the condenser to match that

of the new objective. However, in reflected light, the objective and

condenser numerical apertures change simultaneously with a new



88

objective. Conjugate planes are similar to those described for

transmitted light, with images of the light source being formed in the

back focal plane of the objective and within the aperture diaphragm

iris opening. This serves to reduce the complexity of establishing the

conditions of Koehler illumination in reflected light microscopy. 7

Inverted Light Microscope:

To observe cultured cell, living cell & Laboratory dish.



89

Chapter- IX

Dark Field Optical Microscopy



90

So far the microscope has been shown as suitable for the

examination of stained preparations. Staining aids the formation of

images by absorbing part of the light (some of the wavelengths) and

producing an image of amplitude differences and color. Occasions

arise when it is preferable or essential that unstained sections or

living cells are examined. Such specimens and their components

have refractive indices close to that of the medium in which they are

suspended and are thus difficult to see by bright-field techniques due

to their lack of contrast.

Stars can be readily observed at night primarily because of the

stark contrast between their faint light and the black sky. Yet stars are

shining both night and day, but they are invisible during the day

because the overwhelming brightness of the sun "blots out" the faint

light from the stars, rendering them invisible. During a total solar

eclipse, the moon moves between the Earth and the Sun blocking out

the light of the Sun and the stars can now be seen even though it is

daytime. In short, the visibility of the faint star light is enormously

enhanced against a dark background.

This principle is applied in darkfield (also called darkground)

microscopy, a simple and popular method for making unstained



91

transparent specimens clearly visible. Such objects often have

refractive indices very close in value to that of their surroundings and

are difficult to image in conventional brightfield microscopy. Dark-

ground microscopy overcomes these problems by preventing direct

light from entering the front of the objective and the only light

gathered is that reflected or diffracted by structures within the

specimen. This causes the specimen to appear as a bright image on

a dark background, the contrast being reversed and increased.

In this microscope, oblique light is achieved by using a modified

or special condenser to form a hollow cone of direct light which will

pass through the specimen but outside the objective. Dark-ground

condensers may be for either dry, low-power objectives, or for high



92

power oil immersion objectives. Whichever is used, the

objective must have a lower NA than the condenser.

In order to obtain this condition it is sometimes necessary to

use objectives with built in iris diaphragm or, more simply, by

inserting a funnel stop into the objective. Perfect centering of the

condenser is essential, & with the oil immersion systems it is

necessary to put oil between the condenser & the object slide in

addition to the oil between the slide & the objective. As only light

diffracted by the specimen will enter the objective, a high intensity

light source is required. 1, 5

An excellent type of microscope lamp suitable both for ordinary work

& the darkfield illumination.



93

DF condenser with lamp attached.

Objectives & Condensers:

Low-power objectives work at some distance from the object &

therefore darkground illumination is obtained by simply inserting a

small circle of black paper in the filter carrier.



94

High-power objectives, having a much shorter working distance

require a special condenser which will accurately focus a hollow cone

of light at an acute angle. This angle is so acute that if oil is not used

between the condenser & slide the light rays are reflected back into

the condenser (total internal reflection). Immersion oil must be used

between the object & the objective to ensure that maximum amount

of reflected light from the object enters the objective. To get the best

results the condenser must be accurately centered & focused.

Because of the very acute angle of the light required, very few

darkground condensers can be used with an objective having a

numerical aperture more than 1.0. The most convenient is 2mm

objective having a NA 1.3 which is incorporating an iris diaphragm,

since this can be closed just sufficiently to stop any direct light.

The Fixed-focus type of darkground condenser is most

common, but this can only be used with extra-thin glass slides &

coverslips. Focusing darkground condensers are available which will

allow a variety of slides & coverslips to be used.

Most bright field microscopes can be converted for dark-ground

work by using simple patch stops, made of black paper, placed on top

of the condenser lens or suspended in the filter holder.



95

The actual size will vary, depending upon several factors including

the proximity of the stop with respect to the condenser aperture

diaphragm, the numerical aperture of both the objective and the

condenser, the degree of aberration correction for the condenser, and

the field number of the eyepiece. Also important in determining the

stop size is the diameter of the condenser back lens, the

magnification power of the eyepiece (smaller magnifications require

slightly larger stops), and the type of mounting medium. Stop size

varies proportionally to the refractive index of the mounting medium:

higher refractive index requires a larger stop. A dry mount will also

need a smaller stop than an aqueous suspension. 1, 5, 7, 11, 16

Parabolid Condenser Cardioid

Condenser

Abbe condenser with DarkField sto



96

Rheinberg illumination is a special variant of dark field

illumination and is named after its inventor, Julius Rheinberg. In this

variant transparent colored filters are inserted just before the

condenser so that light rays at high aperture are differently colored

than those at low aperture, using a dark color for the center disc and

a contrasting lighter color for the periphery. This system reduces the

glare of conventional dark ground and reveals the specimen in, say,

red on a blue background.

Variable intensity dark ground is obtained by making the Rheinberg

discs from polarizing filters, the center being orientated at right angles

to the periphery. This allows good photomicrography 1, 5

Requirements:

Thin slides and cover glasses should be used and the

preparation must be free of hairs, dirt and bubbles.

Dark field microscopy uses a carefully aligned light source to

minimise the quantity of directly transmitted light (ie.



97

unscattered light) entering the image, and only collected light

scattered by the sample. This is done by confining the

illumination to a ring of light.

For DF various adjustments must be much more accurate than for a

BF. The most frequent causes of failure to secure a satisfactory DF

with brilliantly lighted objects that appear to be self-luminous are:

1. Use of an objective with too wide an aperture. When the regular

oil immersion is used, its aperture must be reduced by means

of the stop provided by the makers.

2. Failure to focus & to center the condenser accurately. Very

slight readjustments of the condenser/mirror after the

examination is begun may remedy matters, provided the slide is

not too thick to permit accurate focusing.

3. Inclusion of air bubbles in the preparation/in the oil above/below

the slide. It is generally necessary to remove the oil & apply

again.

4. Inclusion of too many microscopic objects in the field. This may

be remedied by diluting the fluid to be examined/by reducing

the thickness of the preparation by means of slight pressure on

the coverglass. 2



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5. Applications:

Dark-ground illumination is particularly useful for spirochetes,

flagellates, cell suspensions, flow cell techniques, parasites and auto

radiographic grain counting, and is commonly used in fluorescence

microscopy. Many small structures are more easily visualized by

dark-ground techniques due to increased contrast, although

resolution may be inferior to bright-field microscopy.

Spirochetes visualized by dark ground microscopy.

Spirochetes are much thinner than most bacterial cells (

approximately 0.1mm in diameter compared with 1mm

for Escheria coli), but they appear larger when viewed

by dark ground illumination.



99

Advantages:

Clearly shows even transparent objects in the sample.

Simplicity of setup with only basic equipment required.

No sample preparation required, allowing viewing of live cells.

Limitations:

The main limitation of dark field microscopy is the low light levels

seen in the final image. This means the sample must be very strongly

illuminated, and can cause damage to the sample.

Low apparent resolution due the blur of out of focus objects.

Chapter- X

Phase Contrast Microscopy

Phase contrast is a widely used technique that shows

differences in refractive index as difference in contrast. Unstained

and living biological specimens have little contrast with their



101

different RIs, through which light acquires small phase differences

and these form the image. Unstained cells are similar to diffraction

gratings as their contents also differ very slightly in RI.



102

Figure describing optical principle

Two rays of light from the same source, having the same frequency,

are said to be coherent, and when recombined, will double in

amplitude or brightness if they are in phase with each other

( constructive interference). If however they are out of phase with

each other, destructive interference will occur.

Suppose a ray is 1/2 lambda out of phase with the other, then

they cancel each other out. This is maximum destructive interference

and no light is seen, resulting in maximum contrast. However if one

ray is brighter than the other (increased amplitude) and is still 1/2

lambda out of phase then the difference in amplitude can be seen

while maintaining maximum interference. This last position is that

which occurs in the phase contrast microscope.

To achieve phase contrast the microscope requires

-Intense light source is required to be set up for Kohler illumination

-Modified objectives and condenser

-And relies on the specimen retarding light by between 1/8-1/4λ 1, 2, 3,

4, 5, 9

The microscope equipment for phase contrast



103

The microscope condenser usually carries a series of annular

diaphragms made of opaque glass, with a clear narrow ring to

produce a controlled hollow cone of light. The required ring diameter

increases with the numerical aperture, i.e. high apertures require the

maximum diameter (e.g. 0.9 in air or 1.3 with oil immersion).

Phase contrast requires special objectives which are equipped

with a phase ring near the pupil. They are easy to recognize by the

green inscription ―Ph1‖, ―Ph2‖ or ―Ph3‖. Each objective requires a

different size of annulus, an image of which is formed by the con-

denser in the back focal plane (BFP) of the objective as a bright ring

of light. The objective is modified by a phase plate which is placed at

its BFP.

A positive phase plate consists of a clear glass disc with

circular trough etched in it to half the depth of the disc. The disc in the

objective has special optical properties: it first of all reduces the direct

light in intensity, but more importantly, it creates an artificial phase

difference of about a quarter wavelength i.e., The light passing

through the trough has a phase difference of 1/4λ compared to the

rest of the plate. The trough also contains a neutral-density light-



104

absorbing material to reduce the brightness of the direct rays, which

would otherwise obscure the contrast obtained.

The phase stops must be centered once after they have been

inserted in the condenser so that the image of the phase stop in the

objective pupil corresponds exactly with the position of the phase ring

in the beam path. Centering is performed using two small wrenches

on the turret disk of the condenser. Again, look into the objective pupil

and bring the bright image of the condenser phase stop into

coincidence with the phase ring of the objective. This is clearly shown

in the figure below: On the left side, the phase stop of the condenser

(bright) is not aligned, while it is in perfect congruence with the phase

ring of the objective on the right



105

Phase contrast microscope accessories: Rotatable turret type

condenser, a set of 4 objectives with phase altering patterns in the

rear focal planes, a green filter, & a centering telescope.

To be particularly precise, a centering telescope for the setting

can be used. This small accessory looks like an eyepiece and is also

inserted into the tube instead of an eyepiece. When it is focused on

the pupil of the objective, the aperture diaphragm and the phase

stops can be conveniently controlled. 1, 3, 5, 9

Each combination of annulus and objective phase plate will

require centeration. When the hollow cone of direct light from the

annulus enters the specimen some will pass through unaltered while

some rays will be retarded (or diffracted) by approximately1/4λ. The

image of an object in phase contrast can be influenced by

appropriately selecting the retardation of the main beam through the

phase ring in the objective.

The direct light will mostly pass through the trough in the phase

plate while the diffracted rays pass through the thicker clear glass

and are further retarded. The total retardation of the diffracted rays is

now1/2λ and interference will occur when they are recombined with



106

the direct light. Thus an image of contrast is achieved revealing even

small details within unstained cells. 1

Light waves A (solid lines), are transmitted through the object & pass

through the phase-altering ring on the phase plate. At this point they

acquire a one-quarter-wave-length advance over light waves, B

(broken lines), which do not pass through the object but are partly

diffracted around it. Waves (B) do not pass through the phase altering

ring on the phase plate. The resultant interference/resonance effects



107

of the 2 portions of the light form the final image. Altered phase

relations in the illuminating rays, induced by otherwise invisible

elements in the specimen, are translated into brightness difference

(contrast) by the phase-altering plate; hence, phase contrast.

Depending on the retardation selected, objects with a higher

refractive index than their surroundings appear either brighter or

darker than their surroundings. This is also called ―positive‖ or

―negative‖ phase contrast. Today, ―positive‖ phase contrast is

standard, where the darkness of objects increases with their

refractive index. This simulates absorption to the observer‘s eye in

areas where a higher refractive index becomes locally effective. 4

Advantages:

This is a quick and efficient way of examining unstained

paraffin, resin and frozen sections.

Studying living cells and their behavior.

In the interference microscope the retarded rays are entirely

separated from the direct or reference rays allowing improved



108

image contrast, color graduation and quantitative

measurements of phase change (or 'optical path difference'),

refractive index, dry mass of cells (optical weighing) and section

thickness. 1, 5, 9,10

Central eosinophil & RBCs as seen in 3 different microscopes:

Bright Field Dark Field Phase Contrast

Limitations:

Contrast is excellent; however it is not for use with thick objects.

In phase contrast microscopy, the specimen retards some light

rays with respect to those which pass through the surrounding

medium. The resulting interference of these rays provides



109

image contrast but with an artifact called the 'phase halo' which

obscures detail.

Chapter- XI

Differential Interference Contrast Microscopy

Differences in optical density will show up as differences in

relief, which is manifested in the "visibility" of boundaries, and



111

by side form two fans of rays which will cross and if coherent, will

observably 'interfere'. If each ray is regarded as a wave it can be

seen that phase conditions of increased amplitude and extinction are

bound to occur at points where the waves cross and interfere. The

result of this in the microscope is a series of parallel bands,

alternately bright and dark across the field of view.

With white light, bands of the spectral colors are seen, because

the wavelengths making up white light are diffracted at different

angles. With monochromatic light the bands are alternately dark and

light, and of a single color. The same effect can be shown if separate

beams of coherent light are reunited. This phenomenon is known as

'interference'. Early microscope models split a light beam into two

parts, each traversing two sets of perfectly matched optics, one beam

passing through the specimen (measuring beam) and the other acting

as a reference beam. The beams were widely separated and suitable

only for large specimens and interference fringe measurements. Later

models used a double beam system, where the separation is

produced by birefringent materials and is close enough to require

only one objective.



112

If the two paths are equal and in the same phase, the

interference bands can be seen running straight and parallel across

the field. If into one beam path an object is introduced that causes

some shift in the phase, this will be seen as a displacement in the

interference bands. When using monochromatic light, each interval

comprising one dark and one light band is one wavelength wide, and

thus the distance in nanometers is known. Displacement of the bands

is measured with a micrometer eyepiece and with this information,

coupled with either the RI or object thickness, the measurements

referred to earlier can be determined.1

The equipment comprises:

A polarizer

A condenser with a modified Wollaston prism &

A beam splitting slide consisting of a modified Wollaston prism

oriented at 45 degrees to an attached analyzer, mounted in an

adjustable carriage & accommodated in the analyzer slot

between the objective & the eyepiece.

The system consists of a special prism (Nomarski prism,

Wollaston prism) in the condenser that splits light in an ordinary and

an extraordinary beam. The spatial difference between the two

http://en.wikipedia.org/wiki/Nomarski_prism

http://en.wikipedia.org/wiki/Wollaston_prism

http://en.wikipedia.org/wiki/Wollaston_prism

http://en.wikipedia.org/wiki/Nomarski_prism



113

beams is minimal (less than the maximum resolution of the objective).

After passage through the specimen, the beams are reunited by a

similar prism in the objective. In a homogeneous specimen, there is

no difference between the two beams, and no contrast is being

generated. However, near a refractive boundary (say a nucleus within

the cytoplasm), the difference between the ordinary and the

extraordinary beam will generate a relief in the image. Differential

interference contrast uses polarised light to work properly. Two

polarising filters have to be fitted in the light path, one below the

condenser (the polarizer), and the other above the objective (the

analyser). The prism below the condenser acts as a compensator.

Every interference fringe of the upper prism is correlated with an

interference fringe of the same order but opposite sign in the

compensator. The first birefringent prism in the condenser separates

the beams and after passing through the object they are recombined

by the second identical prism at the back of the objective.

A different pair of prisms is required for each magnification.

This produces 'interference contrast' and together with rotation of the

polarizers enhances the three-dimensional effect in the image.

http://en.wikipedia.org/wiki/Polarised_light

http://en.wikipedia.org/wiki/Polarised_light



114

Nomarski in 1952 modified the Wollaston prisms so that the lateral

separation is less than the resolving power of the microscope

producing excellent 3D colored images from unstained specimens.

Additionally only one such prism is required at the objective level for

all magnifications.

Two types of double-beam systems have been used. One

involved focusing the reference beam below the object-the 'double

focus' system-and the other involved a lateral displacement of the

reference beam called 'shearing' where the separation of the beams

is very small. This latter system is illustrated using polarized light and

Wollaston prisms.

The basic difference between the interference microscope &

the phase contrast microscope is that the former does not rely on

diffraction by the object for interference, but generates mutually

interfering beams which produce the contrast. It is this feature which

enables very small phase changes to be seen & measured. 1, 3, 5

Advantages:

-Contrast is very good. Individual parts of living cells may be studied

with maximum detail.



115

-condenser aperture can be used fully open, thereby reducing the

depth of field and maximising resolution.

-This system permits enhanced visualization of immunocytochemical

preparations.

-As a highly accurate optical balance, it may be used for estimating

dry mass down to 1X10-14 g. 1, 5

Chapter- XII

Polarized Light Microscopy

The use of polarized light in microscopy has many useful and

diagnostic applications. Numerous crystals, fibrous structures (both



117

an intensity or color effect, for example, by reduced glare when

wearing polarized sun glasses.

Substances or crystals capable of producing plane polarized light are

called birefringent. Light entering a birefringent crystal such as calcite

is split into two light paths, each determined by a different refractive

index (RI) and each vibrating in one direction only (i.e., polarized) but

at right angles to each other. The higher the RI the greater the

retardation of the ray, so that each ray leaves the crystal at a different

velocity. The high RI ray is called slow and the low RI ray is called

fast. There is also a phase difference between the rays, so that if theyC



119

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.

Polaroid discs:

Invented by Land in 1932 ‗Polaroids‘-glass or celluloid covered

discs with the ability to polarize light were first made available in

place of Nicol prisms. They act as a single crystal of heraphite which

is not only birefringent, but has the ability to absorb the ordinary ray

(which would be refracted out of Nicol prisms), only the extraordinary

ray being transmitted. Polaroids are made by suspending

ultramicroscopic crystals of heraphite in nitrocellulose. All the crystals

in the suspension are oriented so that their optical paths are aligned.

This suspension when mounted between 2 glass plates or celluloid

sheets acts as a single crystal.

There will be a direction within a birefringent crystal along which

light may pass unaltered: this is called the optic axis. Substances

through which light can pass in any direction and at the same velocity

are called isotropic and are not able to produce polarized light.

Knowledge of RI and polarization measurements identifies many



120

crystalline structures and is particularly useful to the material scientist

but is of limited use to the histologist.

Some substances and crystals can produce plane polarized

light by differential absorption and give rise to the phenomenon of

dichroism. Such crystals suspended in thin plastic films and

orientated in one direction have replaced the bulky and expensive

Nicol prisms. These thin films totally absorb the slow rays and are

pleochroic (absorbing all colors equally), and are the most useful in

microscopy as they occupy very little space and can be used with any

microscope. 1, 2, 4, 5

Components:

Condensers:

Polarized light microscopy requires a condenser that is similar

to that used in conventional brightfield microscopy, typically an

achromat with a numerical aperture between 0.90 and 1.35.



122

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.



123

Polarizing Filter

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.

The human eye is not able to distinguish any difference

between polarized and natural light although when looking through a



125

• A slot to allow the insertion of compensators/retardation plates

between the polarizers, which are used to enhance optical path

differences in the specimen. 7

Two phenomenon detected in polarized light are interesting to

the histologist. The first is birefringence. When a birefringent

substance is rotated between two polarizers which are crossed, the

image appears and disappears alternately at each 45° of rotation. In

a complete revolution of 360° the image appears four times and four

times it is extinguished completely. When one of the planes of vibra-

tion of the object is in a parallel plane to the polarizer only one part

ray can develop, and its further passage is blocked by the analyzer in

the crossed position. At 45°, however, phase differences between the

two rays which can develop are able to combine in the analyzer and

form a visible image.

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 centeration are provided, which are critical on a polarizing



126

microscope. Centeration of the objective and stage ensures that the

center of the stage rotation coincides with the center of the field

Birefringence in polarized light

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.

Some birefringent substances are also dichroic, which is the

second of the phenomena useful to the histologist. Only the polarizer

is used and if no rotating stage is available the polarizer itself can be

rotated. Changes in intensity and color are seen during rotation. The

color changes in a rotation of 90°, and back to its original color in the

next 90°. This is due to differential absorption of light depending upon

the vibration direction of the two rays in a birefringent substance.

Weak birefringence in biological specimens is enhanced by the



128

dispersed in a liquid, gas/solid; they can give rise to birefringence

even if separately either or both are isotropic. Tests for form

birefringence depend upon causing media of varying RI to penetrate

between the particles when, at the appropriate RI, form birefringence

will disappear. (Examining objects mounted in a variety of Mountants

with differing RI, e.g. water, glycerol, HSR…)

Strain birefringence

When a dielectric substance is subjected to mechanical stress,

the bonds within the substance can be distorted & give rise to a

pattern which will result in birefringence. This is most simply

demonstrated by twisting clear plastic (Perspex) between crossed

Polaroids when a birefringent spectrum of color is produced.

Similarly, glass or elastic tissue fibres under stress show

birefringence.

Sign of birefringence

If the slow ray (higher RI) is parallel to the length of the

crystal or fiber, the birefringence is positive. If the slow ray is

perpendicular to the long axis of the structure, the birefringence is

negative. The sign of birefringence is diagnostically useful and is

determined by the use of a compensator (birefringent plate of known



129

retardation) either above the specimen or below the polarizer at 45°

to the direction of polarized light. Rotate the compensator or the

specimen until the slow direction of the compensator is parallel to the

long axis of the crystal or fiber. The field is now red and if the crystal

is blue the birefringence is positive. If the crystal is yellow, the slow

direction of the compensator is parallel with the fast direction of the

crystal and the birefringence is negative. Quartz and collagen exhibit

positive birefringence while Polaroid discs, calcite, urates and

chromosomes are negative. Simple compensators can be made from

mica or layers of sellotape.

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. 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



131

1. Artifacts: Formalin pigment, sutures, starch.

2. Crystals: Talc, pyrophosphate, silica.

3. Lipids: Myelin.

4. Bone structure: osteoid seams, woven bone.

5. Teeth structure: Enamel striations, dentinal structures.

6. Protein: Collagen, amyloid, keratin.

7. Miscellaneous: Muscle striations, Charcot-Layden crystals,

hydatid hooklets. 5

Chapter- XIII

Fluorescence Microscopy



132

Fluorescence is a member of the ubiquitous luminescence family of

processes in which susceptible molecules emit light from

electronically excited states created by either a physical (for example,

absorption of light), mechanical (friction), or chemical mechanism.

Generation of luminescence through excitation of a molecule by

ultraviolet or visible light photons is a phenomenon termed

photoluminescence, which is formally divided into two categories,

fluorescence and phosphorescence, depending upon the electronic

configuration of the excited state and the emission pathway. 7

Fluorescence is the property of some atoms and molecules to

absorb light at a particular wavelength and to subsequently emit light

of longer wavelength (lower energy than the original exciting light due

to loss of certain amount of energy in the form of heat before the

electron returns to its ground state) after a brief interval, termed the

fluorescence lifetime. The process of phosphorescence occurs in a

manner similar to fluorescence, but with a much longer excited state

lifetime. In fluorescence microscopy, the exciting radiation is usually

in the ultra-violet wavelength (ca 360 nm) or blue region (ca 400 nm),

although longer wavelengths can be used.



134

range for instance Tungsten Halogen filament lamps produce enough

to be useful. 1

HBO High Pressure Mercury Lamps: The choice of a suitable

source depends upon the type of work to be performed and for

routine observation purposes it is better to use the Mercury Vapor

burners. These operate on alternating current and their starting

equipment is not so costly. Emits a spectrum whose characteristics

are ideal for excitation in the near UV, Violet or Green range, their

background emission is also sufficient for blue excitation. However,

mercury vapor burners are preferable because of their higher energy

output if the work involved includes two-color fluorescence (e.g. FITC

plus TRITC) or where the requirement is for high magnification or

techniques where a low-level of fluorescence is expected, e.g.

membrane marker methods,. Most fluorescent microscopes now are

equipped for mercury vapor lamps because of this versatility. 1, 5

Types: HBO 50 (widely used for incident light fluorescence)

HBO 100 W/2

HBO 200 W/4



135

XBO High Pressure Xenon Lamps: operate on direct current which

requires rectifiers to be included with the starter equipment if they are

to be used on normal mains supply. Xenon burners on a DC supply

can be stabilized and are therefore suitable for fluorimetry or the

measurement of fluorescence emission. Emit a spectrum similar to

daylight giving an intense illumination. 1, 5

Types: XBO 75 W/2

XBO 150W/1

XBO 450

The two types of lamps differ in their emission curves that is to

say the mercury lamps at some wavelengths reach very high

amplitudes whereas at other parts of the wavelength range the

emission is low. The curve in general has a very spiky profile; xenon

on the other hand has a smoother more continuous curve.

Fortunately the peaks in the mercury vapor emission coincide with

the excitation wavelengths of the more widely used Fluorochromes.

Because they contain gas at high pressure these burners must

be handled with great care and housed in strong protective lamp

houses. Heat and infrared waves are filtered out before the light from

the source begins its journey. A record of the use of HBO & XBO



136

lamps should be kept & should certainly be replaced if it becomes

dim or flickers. At one time all fluorescence systems used the

transmitted light route common to normal light microscopy; nowadays

the incident route is widely used. 1

Filters

Preparations for fluorescence may contain other fluorescing

material in addition to that in which one is interested. It is necessary

therefore to filter out all but the specific excitation wavelength to avoid

confusion between the important and the unimportant fluorescence.

Basically there are three categories of filters to be sorted out:

exciter filters, barrier filters and dichromatic beamsplitters (dichroic

mirrors), which are usually combined to produce a filter cube or block.

Proper selection of filters is the key to successful fluorescence

microscopy.

Exciter filters permit only selected wavelengths from the

illuminator to pass through on the way toward the specimen. Barrier

filters are filters which are designed to suppress or block (absorb) the

excitation wavelengths and permit only selected emission

wavelengths to pass toward the eye or other detector. Dichromatic



137

beamsplitters (dichroic mirrors) are specialized filters which are

designed to efficiently reflect excitation wavelengths and pass

emission wavelengths. They are used in reflected light fluorescence

illuminators and are positioned in the light path after the exciter filter

but before the barrier filter. Dichromatic beamsplitters are oriented at

a 45 degree angle to the light passing through the excitation filter and

at a 45 degree angle to the barrier filter.

Fluorescence filters were formerly almost exclusively made of

colored glass or colored gelatin sandwiched between glasses.

Besides the possibility of non-specific and auto-fluorescence, there

may also be materials that are excited at more than one excitation

wavelength. So it is better to employ filters of a narrower band trans-

mission that have their transmission peaks closer to the excitation

maximum of the fluorochrome, such as FITC. As a result of more

sophisticated filter technology, interference filters have been

developed that consist of dielectric coatings (of varied refractive

indices and reflectivity) on glass. These filters are designed to pass or

reject wavelengths of light with great selectivity and high

transmission.



138

Most of today's exciter filters are the interference type; some

barrier filters are also, for special needs, like the interference type.

Dichromatic beamsplitters are specialized interference filters.

Sometimes short pass filters (SP) and long pass (LP) filters are

combined to narrow the band of wavelengths passing through such a

combination. Narrow band filters are often of the 'interference' filter

type, and are vacuum-coated layers of metals on a glass support.

They have a mirror-like surface, and must be inserted in the beam

with the reflective face towards the light source. The better quality

filters are carefully selected for their transmission characteristics, and

only a few are finally judged suitable. For this reason, they are

expensive. Careful handling to avoid corrosive fingermarks and

scratches is essential.

Attachment may be as follows:

-placed in a filter carrier below the condenser

-inserted in fitted slides carrying several filters, in front of the

collecting lens of the illumination system & protected by the heat

absorbing KG 1 filter.



139

Exciter filters:

First, 'dyed in the mass glass' filters, with such designations as

UG 1 and BG 12; these are broad band filters and transmit a wide

range of wavelengths, the width of the range depending upon the

composition and thickness of the filter. Modern exciter filters are

designated by letters & numbers indicating the type & their

wavelength of maximum transmission. For ex: G 405 (G=dyed in the

glass filter). Today, most exciter filters are of the interference type.

Barrier filters:

Block (suppress) shorter wavelengths and have high

transmission for longer wavelengths. Barrier or suppression filters are

placed before the eyepiece to prevent short wavelength light from

damaging the retina of the eye. They must however allow the

fluorescing color to pass; otherwise a negative result may be

obtained. Barrier filters are colorless through yellow to dark orange

and of specific wavelength transmission. An orange filter (cut-off 510

nm) is particularly suitable for FITC conjugates.

For example a K.470 filter will block all wavelengths below 470

nm (a prefix K is used by Leitz for their barrier filters for transmitted

light fluorescence). Colored barrier filters may alter the final color



140

rendering of the fluorescent specimen and for this reason all filters

used in the system must be recorded when reporting results.

Attachment may be as follows:

-inserted into the eyepiece by removal of the top lens, or they may be

screwed into the bottom of the eyepiece.

-inserted in the body tube by means of specially fitted slides

-incorporated in a rotary filter changer which is fitted below the

binocular attachment.

Dichromatic Beamsplitters:

These filters are always the interference type. The coatings are

designed to have 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 cube through the reflected light fluorescence illuminator.

Their function is to direct the selected excitation (shorter

wavelengths) light through the objective and onto the specimen. They

also have the additional functions of passing longer wavelength light

to the barrier filter, and reflecting any scattered excitation light back in

the direction of the lamphouse. 1, 5



142

Filters in fluorescence microscopy

A combination of three blocks: (1) for UV light excitation (FITC); (2)

for broad-band blue light excitation (FITe), and (3) for green light

excitation (TRITe) is the most useful in routine laboratory

applications. This allows the use of (2) for most routine FITC work,

with the alternative use of (1) to distinguish between specific and

autofluorescence in FITC preparations; (2) plus (3) gives suitable

two-color combination of FITC and TRITC preparations. 1



143

Condensers for fluorescence microscopy

Bright-field condensers are able to illuminate the object using

all the available energy but they also direct the rays beyond the

object into the objective. Not only is this a potential hazard to the

eyes of the observer but it can set up disturbing auto fluorescence in

the cement and component layers in the objective itself. In

consequence most systems employ a dark ground condenser which

does not allow direct light into the objective and in addition is more

certain to give a dark contrasting background to the fluorescence. At

the same time it should be realized that only about one-tenth of the

available energy is used, limited by the design of the condenser.

Fluorescent light emission is in most cases very poor in relation

to the amount of energy absorbed by fluorochromes or fluorophores

with an efficiency ratio somewhere between 1: 1000 and 1: 100 at

best. So any system that reduces the available energy to any extent

should be well considered before being put into use.



144

Condenser in fluorescence microscopy

Objectives:

Objectives too must be carefully chosen. It has already been

noted that auto fluorescence is a hazard with bright-field illumination

and for that system. Only the simpler achromat objectives are

preferred to apochromat as they rarely fluoresce & their color

correction is usually adequate. With dark-ground illumination the



145

range of objectives is considerably widened and more elaborate

lenses with higher apertures and better 'light gathering power' are

possible.

The early fluorescence microscope utilized transmitted light

illumination (diascopic fluorescence). A primary filter to select the

excitation light wavelengths was placed in the light port of the

microscope and a secondary barrier filter was positioned above the

microscope nosepiece to block residual excitation light and to select

emission wavelengths reaching the eye or camera.

Disadvantages:

The numerical aperture of the higher magnification oil or water

immersion objectives has to be reduced by a built-in iris

diaphragm (with consequent loss of light intensity and

resolution) in order to prevent excitation light from entering the

objective directly.

The darkfield method is also very wasteful of light, since the

excitation light irradiates much of the specimen outside of the

field of view being observed, thus reducing the usability of

excitation intensity.



146

Incident light fluorescence: The trend today in fluorescence

techniques is in incident illumination or lighting from above and

through the objective down to the object. A number of advantages are

gained over the transmitted route.

Incident fluorescence illumination



147

In principle the excitation beam after passing the selection

filters is diverted through the objective on to the preparation where

fluorescence is stimulated. This fluorescence travels back to the

observer by the normal route. Dichroic mirrors have been produced to

divide and divert the beam. These mirrors have the property of being

able to transmit light of some wavelengths and reflect other

wavelengths .By selection of the appropriate mirror, the wavelength

desired is reflected to the object: the remainder passes through to be

lost. At the same time, visible fluorescent light collected by the

objective in the normal way can pass to the eyepiece and any

excitation rays bouncing back (from slide and coverglass) are

reflected back along their original path to the source thus being

prevented from reaching the observer.

Since the objective in this system also acts as a condenser, the

illumination and objective numerical apertures are one and the same,

optically correct and at their most efficient condition. Fluorescence is

stimulated on the observer's side of the preparation and is therefore

more brilliant not being masked by covering material or section

thickness.



148

The use of dichroic mirrors in these systems has made possible

much brighter images, since up to 90% of the exciting energy can

reach the preparation and 90% of the resultant visible light can be

presented to the eye. In addition new objectives, both of oil and water

immersion types, in low and high powers have been developed. As

immersion objectives they have higher numerical apertures, and can

gather more light avoiding much of the lost stray light reflected from

coverslips. The use of low magnification eyepieces is now more

widely accepted improving fluorescence techniques far beyond

anything hitherto possible. The dichromatic filter sets or clusters,

comprising the exciter filter, dichromatic beam splitter & barrier filter

are situated in a special holder sited above the objective in line with

the illuminating beam. 1

Microscopic preparations:

Microscope slides: these should be of even thickness. Special UV

transmitting slides may be purchased, but unless a quartz condenser

is used it is pointless to employ them. Optical glass will only transmit

light of 300nm & over, & at this range thin glass slides have an

adequate transmission.



149

Section adhesives: Thinly applied routine section adhesives do not

interfere with the preparations.

Mountants: cleared preparations may be mounted in HSR (Harleco

synthetic resin) or Depex. Flourmount will probably give the best

results.

Aqueous mounts: these may be mounted in Apathy‘s media with the

exception of acridine orange or fluorescent antibody stained

preparations.

Fluorescent antibody preparations: these are mounted in glycerin to

which 10% phosphate buffered saline (pH 7.1) has been added.

Acridine orange stained preparations: these are mounted in buffer

only. 5

Autoflourescence:

The ability of some naturally occurring compounds to fluoresce

is on occasion a great advantage in identification. Autoflourescent

material can present a great hazard to the inexperienced

microscopists, because dependent on its structure; it may fluoresce

any color & thus appear to have been stained by the technique

employed. For this reason unstained smears, identically prepared in



150

all other respects, should always be used as controls of fluorescent

stains.

Preparation of the material:

Unfixed smears or cryostat cut sections of unfixed tissue should

be used. It may be found subsequently that fixation does not interfere

with the specific fluorescence; 95% alcohol ethyl /ether-alcohol are

usually satisfactory. Formalin should be avoided if possible as it tends

to increase the blue Autoflourescence of tissue.

Specific Autoflourescence:

Tissue: generally tissues fluoresce a bright blue, although this may be

absorbed by use of a yellow or orange filter.

Elastic fibres: fluoresces a brilliant blue while unstained, & may be

easily seen even in a H&E stained section.

Ceroid & riboflavin: these fluoresce in shades of yellow.

Lipids & lipochromes: shades of yellow.

Vitamins: many vitamins are fluorescent in shades of yellow, green &

blue.

Porphyrins: this group & chlorophyll are among the very few

compounds with an intense red fluorescence. This characteristic has

been made use of by adding a drop of concentrated H2SO4 to blood



151

stains; the H2SO4 takes the iron out of the hemoglobin forming

haematoporphyrin which gives a brilliant red fluorescence.

Nissl substance: bright yellow in formalin fixed unstained tissue.

5-HT: golden yellow fluorescence after formalin treatment.

Drugs: Tetracycline- bright yellow fluorescent foci in malignant

tumors. This antibiotic is used to show areas of new bone formation

in tetracycline fed animals.

Hydrocarbons: the carcinogenic compounds, in particular, have been

found to be strongly fluorescent. 3:4 benzpyrene has been used by

Berg to demonstrate even the finest lipid granules. 5

Applications:

In contrast to other modes of optical microscopy that are based

on macroscopic specimen features, such as phase gradients, light

absorption, and birefringence, fluorescence microscopy is capable of

imaging the distribution of a single molecular species based solely on

the properties of fluorescence emission. Thus, using fluorescence

microscopy, the precise location of intracellular components labeled

with specific fluorophores can be monitored, as well as their

associated diffusion coefficients, transport characteristics, and

interactions with other biomolecules.



152

Number of compounds are flourescent to some degree, only

relatively few give sufficiently brilliant flourescence that they may be

detected in small quantities by their autoflourescence, or used as

flourescent dyes. One particularly powerful method is the combination

of antibodies coupled to a fluorochrome as in immunostaining.

Examples of commonly used fluorochromes are fluorescein or

rhodamine. Some compounds & dyes, while brilliantly flourescent as

pure compounds, may lose their power to flouresce when bound to

other structures. This is known as Quenching of flourescence. This is

sometimes a useful property, since non-specific flourescence can be

quenched to give greater contrast.

Immunofluorescent methods are extensively used in the

detection of antibodies in serum. Frozen sections are usually required

for their application to biopsy material, but Mera et al. (1980) have

described their successful application to paraffin sections of skin

following trypsinization. Immunofluorescence has made an important

contribution to diagnosis in two main areas, renal glomerular disease

and certain skin diseases. Immunofluorescent examination of bone

marrow aspirates is also of value in lymphoproliferative disorders



153

such as myeloma. Immunofluorescent methods are also potentially

valuable in the specific tissue diagnosis of infective disorders. 1

Immunofluorescence stain and oral blistering diseases:

IMF is a helpful and confirmatory test and, at times, a

necessary test for skin and mucosal immune diseases such as lupus

erythematosus, Bullous mucous membrane pemphigoid, Pemphigus

vulgaris, linear IgA, lichen planus and other chronic immune

diseases. These are important diseases to recognize and properly

diagnose, especially since some, such as pemphigus vulgaris, can be

life threatening.

IMF can yield specific histology more often than a standard

H&E. For example, BMMP can be diagnosed through use of the H&E

stain evaluating the clean separation of the surface epithelium from

the connective tissue below the basal cell layer, but the IMF stain

yields a much more specific linear deposit of IgG, C3 and sometimes

IgA along the basement membrane, making a diagnosis much more

reliable and definitive.



154

Immunofluoroscence staining for diagnosis of oral blistering disease



155

Chapter- XIV

The Confocal Microscope

In fluorescence microscopy using conventional epifluorescence

microscopes, the fluorochrome present in the field of view will be

excited whether in or out of focus. The effect is that the out-of-focus

fluorescence will reduce the contrast and resolution of the image.

Confocal microscopy is an imaging technique used to increase

micrograph contrast and/or to reconstruct three-dimensional images

by using a spatial pinhole to eliminate out-of-focus light or flare in

specimens that are thicker than the focal plane. The Confocal

Principle and Microscope Design

"Confocal" is defined as "having the same focus." What this

means in the microscope is that the final image has the same focus

as or the focus corresponds to the point of focus in the object. The

object and its image are "Confocal." The microscope is able to filter

out the out-of-focus light from above and below the point of focus in

the object. Normally when an object is imaged in the fluorescence

microscope, the signal produced is from the full thickness of the

specimen which does not allow most of it to be in focus to the

observer. The confocal microscope eliminates this out-of-focus


http://en.wikipedia.org/wiki/Contrast_(vision)

http://en.wikipedia.org/wiki/Reconstruct

http://en.wikipedia.org/wiki/Lens_flare

http://en.wikipedia.org/wiki/Focal_plane

http://en.wikipedia.org/wiki/Focal_plane

http://en.wikipedia.org/wiki/Lens_flare

http://en.wikipedia.org/wiki/Reconstruct

http://en.wikipedia.org/wiki/Contrast_(vision)




159

Chapter- XV

Electron Microscope

A light microscope, even one with perfect lenses and perfect

illumination, simply cannot be used to distinguish objects that are

smaller than half the wavelength of light. White light has an average

wavelength of 0.55 micrometers, half of which is 0.275 micrometers.

Any two lines that are closer together than 0.275 micrometers will be

seen as a single line, and any object with a diameter smaller than

0.275 micrometers will be invisible or, at best, show up as a blur. To

see tiny particles under a microscope, scientists must bypass light

altogether and use a different sort of "illumination," one with a shorter

wavelength.

The fundamental advantage of transmission electron microscopy

(TEM) is the vast improvement in resolution it offers over that

possible with conventional light microscopy.



160

The physical basis for this benefit lies in the formula:

R = 0.61λ

NA

Where: R, the resolution, represents the capacity of the optical

system to produce separate images of objects very close together; λ

is the wavelength of the incident illumination, and NA is the

numerical aperture of the lens.

Thus for any given lens, resolution is directly related to the

wavelength of the source radiation. For example the limit of resolution

for a standard microscope using white light is around 200 nm

whereas a fluorescence microscope operating on shorter wavelength

ultraviolet light is capable of resolving objects around 100 nm apart.

Electron microscopy takes advantage of the wave nature of rapidly

moving electrons. Where visible light has wavelengths from 4,000 to

7,000 Angstroms, electrons accelerated to 10,000 KV have a

wavelength of 0.12 Angstroms.1 Although it has long been known that

electromagnetic radiations such as electron beams of high energy (60

kv/more) have very short wavelengths (around 0.5 µm), their use in

ordinary (optical microscopes) has been impossible because glass is



161

opaque to electrons. However, electrons are deflected from their line

of propagation by magnetic fields. This discovery made possible the

use of electron beam, thus forming electron images.

The first practical electron microscope was constructed by Knoll

& Ruska in Berlin in 1931. Improved commercial instruments first

came into general use around 1940. From these basic discoveries

the modern electron microscope has evolved. 4

Theoretically a beam of electrons accelerated to a potential of

100 kV would be capable of resolving approximately 0.001 nm.

Although flaws in lens design severely restrict this potential a

contemporary transmission electron microscope is capable of

regularly resolving structures of 0.2 nm or less. With this greater

resolving power the electron microscope is able to venture beyond

the histological appearance and reveal the substructure or

ultrastructure of individual cells. 1

THE TRANSMISSION ELECTRON MICROSCOPE

It is the standard or original form of electron microscopy. The basic

optical principle upon which the transmission electron microscope



162

operates is identical to that of the compound light microscope-lenses

are used to form magnified images. The difference lies in the

radiation used (light or electrons) and the means to focus that

radiation (glass or electromagnetic lenses).

Electron gun:

The device responsible for generating the beam of electrons is the

electron gun. The most important components of the gun are the

filament, Wehnelt shield and anode. Filament:

Electrons are normally generated by thermionic emission from a V-

shaped length of tungsten wire. Adjusting the bias resistance, and

thereby the voltage differential between the filament and the grid,

allows the beam current to be adjusted from a small de-focused

beam current, through a focused maximum current, to cut-off. Cut-off

is that point at which the more strongly negative fields of the grid

prevent any electrons from reaching the anode by reversing the

gradient completely around the filament.

While the plain tungsten wire filament is the most common

cathode material in use, there are several variations and different

materials used. Oxide and thoriated coatings have been explored to



163

increase the emissivity of tungsten. Such coatings have not found

much commercial use.

LaB6 cathode

In 1951, Lafferty2 established that the rare earths, and

particularly Lanthanum Hexaboride (LaB6), had high thermionic

emission characteristics and sufficiently low vapor pressures to be

desirable cathode materials for electron microscopy. The tip of the

rod is polished to a point, and then a small angled flat is usually

polished at the point. The flat provides a defined area for emission.

Without the flat, or if the cathode material evaporates past the flat,

emission occurs from a broad undefined area around the point and

resolution is decreased.

LaB6 cathodes provide around an order of magnitude higher

brightness than tungsten cathodes. Longer cathode life is also an

advantage, but they are expensive.

Wehnelt shield:

The filament is completely covered by an apertured electrode

known as the Wehnelt shield (or Wehnelt cap). This carries a higher

negative voltage (the bias voltage) than the filament-the like charge



164

deflects and drives the electrons that emanate from the filament

towards the shield aperture. The electron cloud that forms is referred

to as the effective electron source. As the filament current is

increased and more electrons are given off, the bias voltage

increases commensurately, forcing the electrons into a circle (or

'spot') of decreasing size but increasing brilliance. Optimal efficiency

occurs at just beyond filament saturation point. At higher excitation

levels the increasing bias voltage prevents electrons from reaching

the shield aperture and brightness decreases.

Elect ron microsc ope



165

Components of Elect ron Microscop e



166

Anode:

The anode, an apertured disk, is positioned a short distance

away from the shield.

As the anode is kept at zero potential, electrons are attracted away

from the effective source and accelerate through the anode aperture

and into the column. The speed at which the electrons move

depends on the voltage difference between the effective source and

the anode: this voltage is thus referred to as the accelerating voltage.

Since the wavelength of the emergent electron beam is inversely

proportional to the accelerating voltage, the resolving power of the

microscope is directly affected by the operation of the electron gun.

This suggests that to gain maximum resolution it is necessary to

operate at the highest accelerating voltage possible. However, it is

also the case that as electron speed increases there is a

corresponding reduction in electron scatter as the beam passes

through the specimen, giving lower contrast in the final image. The

general view is therefore that the microscope should be operated at

the highest accelerating voltage that allows structures in the

specimen to be clearly discriminated. For most situations this will be

between 80-120 kV.



167

Classical vs. electron optics

1) Classical optics: The refractive index changes abruptly at a

surface and is constant between the surfaces. The refraction of light

at surfaces separating media of different refractive indices makes it

possible to construct imaging lenses. Glass surfaces can be shaped.

2) Electron optics: Here, changes in the refractive index are gradual

so rays are continuous curves rather than broken straight lines.

Refraction of electrons must be accomplished by fields in space

around charged electrodes or solenoids, and these fields can assume

only certain distributions consistent with field theory. There is a

serious disadvantage in that they cannot be shaped to correct for

chromatic aberration and other errors. In practice, electronic lenses

are difficult to manufacture and typically display spherical and

chromatic aberrations as well as astigmatism.

Spherical aberration is caused by electrons at the periphery of

the lens being focused closer to the lens than those in the central

region. This problem is minimized simply by using apertures to block

peripheral electrons from entering the lens in the first place.

Apertures made from molybdenum or platinum need to be cleaned

regularly, unlike those made from gold foil which is self-cleaning.



168

Chromatic aberration is due to electrons of different speed

(and thus wavelength) being focused at different planes. It is

primarily a function of the electrons losing speed as they pass

through the section itself and it gives rise to blurred images. The

effect is minimized by using thin sections and high accelerating

voltages combined with balanced lens currents.

Astigmatism is the result of asymmetry in the magnetic field

and reflects flaws in lens construction. It is also exacerbated by

contamination of the lens or other components in the electron

pathway. The problem is predominantly overcome by positioning

additional small electromagnets ('stigmators') around the primary

lens. The supplementary magnetic fields generated compensate for

the discrepancy in the primary field. Regular checking and

adjustment of the stigmators is necessary to maintain optimal image

quality.

Lens arrangement and image formation

The arrangement and function of lenses in the transmission

electron microscope is also analogous to that in the compound light

microscope. Electrons generated by the electron gun are collected

by a condenser lens system which is responsible for determining the



169

beam diameter (spot size) and maximizing specimen illumination.

The electrons that pass through the specimen (transmitted electrons)

then enter the imaging system which consists of the objective,

intermediate and projector lenses. The fundamental roles of these

lenses are to focus, magnify and direct the beam onto the viewing

screen or image recording unit (camera or digital imaging system).

The inherent difficulty associated with locating and re-locating

ultrastructural features, as well as the fragility of grids and sections

necessitates the use of some form of image recording mechanism.

Photographic film specifically manufactured for TEM remains the

most common system in use as it provides excellent levels of

resolution and image clarity. More recently developments in high-

resolution digital cameras have allowed images to be captured and

stored electronically. An additional advantage is that digital cameras

can operate at very low illumination levels thus minimizing beam-

induced specimen damage. By interfacing with the appropriate

software digital images with low contrast (that occur at higher

accelerating voltages) can be enhanced and various forms of

morphological and densitometric analyses can be performed.



170

Transmission electron microscopes produce two-dimensional

images. 1, 2, 3, 4

Transmission Electron Microscope

Tissue preparation for Transmission Electron Microscopy:

Although accelerated at great speed, the electron beam is only

capable of penetrating around 100nm. Thus in order to obtain a high

quality image & optimize the resolution of the instrument it is

necessary to section the tissue to a thickness of around 80nm.

Sectioning at this level requires tissues to be embedded in extremely

rigid material like synthetic resins. Resins are also capable of

withstanding the vacuum in the electron microscope column & the

heat generated as the electrons pass through the section. In most

circumstances, hydrophobic epoxy resins are preferred.



171

Flow chart illustrating the steps in the preparation of specimens for

diagnosis by electron microscopy .



172

Fixation - Primary fixation with Glutaraldehyde to stabilize the

proteins followed by osmium tetroxide which retains lipids.

Dehydration - replacing water with organic solvents such as

ethanol or acetone.

E mbedding - infiltration of the tissue with a resin such as

araldite or epoxy for sectioning.

Sectioning - produces thin slices of specimen, semitransparent

to electrons. These can be cut on an ultra microtome with a

diamond knife to produce very thin slices. Glass knives are also

used because they can be made in the laboratory and are

much cheaper.

Collection of sections- Ultra thin sections are mounted onto

specimen grids for viewing. Grids measure 3.05 mm in

diameter & are made of conductive material, commonly copper,

nickel or gold. A large range of patterns & mesh sizes are

available with 200 square mesh being commonly used.

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Staining - uses heavy metals such as lead, uranium or tungsten

to block electrons to give contrast between different structures,

since many (especially biological) materials are nearly

"transparent" to electrons (weak phase objects). 1

Applications:

A major application is to define tumor classification, when light

microscopy is equivocal & when proper therapy & prognosis

depend on accurate diagnosis. In general, poorly differentiated

neoplasms may be better defined.

Some examples of specimen grids: (from top left)

mesh(200 size); slotted (200 size); Parallel with

divider (200 size); mesh(50 size); hexagonal (7size); parallel (75 size); freeze fracture; single hole;

slotted; tabbed mesh(400 size); tabbed

mesh(75size)

Apparatus for application of plastic support films. Thewater level is raised over the level of the wire mesh, on

which grids are then placed. Approximately 0.2ml ofliquid plastic film is dropped onto the water

surfaceover the submerged gids and the solventallowed to evaporate.The water is then drawn off,

allowing the film of plastic to settle onto the grids.

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Ultrastructural diagnosis is useful especially in endocrine tumor.

Confirmation of small cell anaplastic carcinoma of neuroendocrine

type is enhanced.

Differential diagnosis of small cell tumors of possible Ewing‘s type

is supported, especially in pediatric age range (in which the

differential diagnosis includes neuroblastoma,

lymphoma/leukemia, embryonal rhabdomyosarcoma).

Other important applications include diagnosis of some spindle

cell tumors, distinction in occasional instances between carcinoma

& sarcoma & confirmation of amelanotic melanoma, in which it is

possible to identify premelanosomes.

Diagnosis of poorly differentiated leukemias, some lymphomas &

of histiocytosis-X is sometimes enhanced by electron microscopy.

EM is helpful in differential diagnosis between lymphoma &

undifferentiated carcinoma & sometimes in differential diagnosis

between primary & metastatic tumor.

Certain viral & infectious diseases, Metabolic/storage diseases.

Direct examination of specimens allows rapid identification of virus

particles & detection of viruses that are difficult or impossible to

cultivate (rotaviruses, hepatitis A virus). Fluid for examination is



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dried onto a copper grid & examined. About one million virus

particles per ml are needed if they are to be detectable. The

sensitivity can be increased by reacting the fluid with antiviral

antibody so that clumps of virus particles are visible. This is

known as immunoelectron microscopy, a technique analogous to

immunofluorescence in light microscopy. 17

Electron micrograph of a Bacteriophage without shadowing (A) &

with shadowing (B)



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Effects of wavelength on resolution: In each sketch the circles

represent wavelengths: in A-electrons, B-Visible light. In A the

projected image reveals all of the details of the form of the object. In

B image lacks detail.

Limitations:

Sampling errors

Expensive

Time consuming

Usually not useful in distinction between benign & malignancy.

Utility is diminished by crushing or drying.

Glutaraldehyde should be avoided since it contains a



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precipitate.

The role of EM has recently been somewhat eroded by rapid

advances in immunocytochemistry for the differential diagnosis

of tumors.

SCANNING ELECTRON MICROSCOPE

Introduction to scanning electron microscopy:

The scanning electron microscope works by bouncing

electrons off of the surface and forming an image from the reflected

electrons. Actually, the electrons reaching the specimen (the 1 °

electrons) are normally not used (although they can form a

transmitted image, similar to standard TEM), but they incite a second

group of electrons (the 2 ° electrons) to be given off from the very

surface of the object. Thus, if a beam of primary electrons is scanned

across an object in a raster pattern (similar to a television scan), the

object will give off secondary electrons in the same scanned pattern.

These electrons are gathered by a positively charged detector, which

is scanned in synchrony with the emission beam scan. Thus, the



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name scanning electron microscope with the image formed by the

collection of secondary electrons.

The SEM generally has a lower resolving power than the

TEM; however, it is particularly useful for providing three-dimensional

images of the surface of microscopic objects. Scanning electron

microscope resolutions are currently limited to around 25 Angstroms.

By correlating the sample scan position with the resulting signal, an

image can be formed that is strikingly similar to what would be seen

through an optical microscope. The illumination and shadowing show

a quite natural looking surface topography.

Electron beam generation

The electron gun in a scanning electron microscope is the

source for the electron beam used to probe the sample. Electrons are

emitted from a cathode, accelerated by passage through electrical

fields and focused to a first optical image of the source. The size and

shape of the apparent source, beam acceleration and current are the



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primary determining factors in the performance and resolution of a

scanning electron microscope. 4, 5, 18

Samples viewed under an electron microscope may be treated in

many ways:

Cryofixation - freezing a specimen so rapidly, to liquid nitrogen

or even liquid helium temperatures, that the water forms

vitreous (non-crystalline) ice. This preserves the specimen in a

snapshot of its solution state. An entire field called cryo-electron

microscopy has branched from this technique. With the

development of cryo-electron microscopy (CEMOVIS), it is now

possible to observe virtually any biological specimen close to its

native state.

Fixation - preserving the sample to make it more realistic.

Glutaraldehyde - for hardening - and osmium tetroxide - which

stains lipids black - are used.

Dehydration - replacing water with organic solvents such as

ethanol or acetone.

Embedding - infiltration of the tissue with a resin such as

araldite or epoxy for sectioning.

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extremely fragile "pre-shadowed" metal replica of the fracture

surface is released from the underlying biological material by

careful chemical digestion with acids, hypochlorite solution or

SDS detergent. The still-floating replica is thoroughly washed

from residual chemicals, carefully fished up on EM grids, dried

then viewed in the TEM.

Ion Beam Milling - thins samples until they are transparent to

electrons by firing ions (typically argon) at the surface from an

angle and sputtering material from the surface. A subclass of

this is Focused ion beam milling, where gallium ions are used

to produce an electron transparent membrane in a specific

region of the sample, for example through a device within a

microprocessor.

An important technique in Scanning electron microscopy is the

use of "shadowing." This involves depositing a thin layer of

heavy metal (such as platinum) on the specimen by placing it in

the path of a beam of metal ions in a vacuum. The beam is

directed at a low angle to the specimen, so that it acquires a

"shadow" in the form of an uncoated area on the other side.

When an electron beam is then passed through the coated

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preparation in the electron microscope and a positive print is

made from the "negative" image, a three dimensional effect is

achieved

Evaporation, Thin-film deposition, or sputtering of carbon, gold,

gold/palladium, platinum or other conductive material to avoid

charging of non conductive specimens in a scanning electron

microscope. 5, 7, 18

Modifications of SEM:

It is possible to focus the primary electrons in exactly the same

manner as a TEM. Since the primary electrons can be focused

independently of the secondary electrons, two images can be

produced simultaneously. Thus, an image of a sectioned material can

be superimposed on an image of its surface. The instrument then

becomes a STEM, or Scanning-Transmission Electron Microscope. It

has the same capabilities of a TEM, with the added benefits of an

SEM.

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SEM allows a good deal of analytical data to be collected in

addition to the formed image. As the primary electrons bombard the

surface of an object, they interact with the atoms of the surface to

yield even more particles and radiations other than secondary

electrons. Among these radiations are Auger electrons, and

characteristic X-rays. The X-rays have unique, discreet energy

values, characteristic of the atomic structure of the atom from which

they emanated. If one collects these X-rays and analyzes their

inherent energy, the process becomes Energy Dispersive X-ray

Analysis. Combining the scan information from secondary and Auger

electrons, together with the qualitative and quantitative X-ray

information allows the complete molecular mapping of an object's

surface.

Finally, the scanning microscope has one further advantage

that is useful in cell structure analysis. As the electron beam scans

the surface of an object, it can be designed to etch the surface. That

is, it can be made to blow apart the outermost atomic layer. As with

the emission of characteristic x-rays, the particles can be collected

and analyzed with each pass of the electron beam. Thus, the outer

layer can be analyzed on the first scan, and subsequently lower



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layers analyzed with each additional scan. Electrons are relatively

small, and the etching can be enhanced by bombarding the surface

with ions rather than electrons (the equivalent of bombarding with

bowling balls rather than BB's). The resultant Secondary Emissions-

Ion Scanning data can finally be analyzed and the three- dimensional

bit-mapped atomic image of an object reconstructed.

Scanning electron microscopes are often coupled with x-ray

analyzers. The energetic electron beam - sample interactions

generate x-rays that are characteristic of the elements present in the

sample. Many other imaging modes are available that provide

specialized information.

Because the signal is incoherent (unlike the conventional TEM

bright-field image), the resolution of the ADF image is higher than that

obtainable in the TEM by a factor of almost two. Another important

advantage of STEM is that any analytical signal, such as X-ray

fluorescence spectroscopy and electron energy loss spectroscopy

(EELS), can also be obtained at high resolution (0.1 nm in the very

best, aberration-corrected STEMs). Other signals include Auger



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spectroscopy, electron nanodiffraction and high-resolution secondary

electron imaging. 2, 4, 15, 18, 20

Disadvantages:

1. Real electron microscope images do not carry any color

information, they are greyscale.

2. Electron microscopes are expensive to buy and maintain. As they

are sensitive to vibration and external magnetic fields, suitable

facilities are required to house microscopes aimed at achieving high

resolutions.

3. The samples have to be viewed in vacuum, as the molecules that

make up air would scatter the electrons.

4. The samples have to be prepared in many ways to give proper

detail, which may result in artifacts purely as the result of treatment.

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Chapter- XVI

Stereomicroscope

It uses two separate optical paths with two objectives and two

eyepieces to provide slightly different viewing angles to the left and

right eyes. In this way it produces a three-dimensional (3-D)

visualization of the sample being examined.

Great working distance and depth of field here are important

qualities for this type of microscope. Both qualities are inversely

correlated with resolution: the higher the resolution (i.e.,

magnification), the smaller the depth of field and working distance. A

stereo microscope has a useful magnification up to 100×. The

resolution is maximally in the order of an average 10× objective in a

compound microscope, and often much lower.

A Colour CCD camera of moderate quality documents all

observations. Particularly in molecular biology and in gene

technology this new observation technique offers ideal conditions for

in-vivo and in-situ investigations of living organisms in real time.

Applications:

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The stereo microscope is often used to study the surfaces of

solid specimens or to carry out close work such as sorting, dissection,

microsurgery, and the like.

A stereomicroscope is a highly mobile and readily

maneuverable microscope, which can be used to examine any object

that is unable to be placed on the stage of a conventional

microscope. Equipped with a rollable floor stand, highly flexible

extension arms and fibre-optic light guide, the microscope can be

employed to examine objects virtually at any axes and angles, with

an impressive 3D effect, profound depth of field and richness of

contrast.

Ultraviolet & television color-translating microscopes:

One value of ultraviolet light is that it is passed by some parts of

a cell but is more/less completely absorbed by other parts, thus

creating contrasts between otherwise indistinguishable intracellular

structures. Unfortunately, direct vision by ultraviolet light is not

feasible & the object can therefore be studied only by means of

photographs.

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The ultraviolet television microscope permits continuous

observation of the object under ultraviolet illumination without the

intermediation (with consequent distortion, blurring & delay) of

photographs. 4

IMAGE ANALYSIS

The practice of histology and histopathology has traditionally relied

upon the subjective interpretation of microscopic preparations by a

highly trained individual. The accuracy with which such interpretations

can be made is the foundation of histology and of histopathology. It is

important to note that these interpretations are based on pattern

recognition, that is, overall arrangements of elements within the

specimen, a task for which the human visual system is well suited.

The human visual system is not well suited for quantitative functions,

such as assessment of linear measurements, areas, or density of

stain. The human eye is a remarkable sensor, but one that is highly

adaptable. It is able to alter its sensitivity depending on the brightness

of the object being viewed. The eye is also a non-linear sensor, with a

response to brightness that more closely approaches a logarithmic

response. These two characteristics preclude accurate assessment



189

of density of specimens viewed through a microscope.

Human observers do not accurately estimate physical distances,

and areas of specimens. The eye is reasonably good at comparisons,

and most microscopists will 'estimate' sizes based on some internal

specimen object, such as the diameter of red blood cells. Even with

such comparisons, length and size estimates made by microscopists

are neither accurate nor highly repeatable. It is the purpose of all

types of image quantitation to eliminate observer-to-observer

variation, and produce evaluations that are accurate and repeatable.

Morphometry is the general term used to describe the

measurement of size parameters of a specimen. Size is here defined

as length, height and area of an object of interest. These basic

measurements can be combined to provide additional

measurements, such as perimeter, smoothness, centers, etc.

TRADITIONAL APPROACHES

The history of development of the microscope is filled with

clever devices designed to assist in performing Morphometry of

specimens. One such device is the camera lucida, which is an optical



190

system that projects an image of the specimen onto a surface

adjacent to the microscope. This projected image can be used to

draw the specimen, or to measure portions of the image. Accurate

measurements within these projected images require calibration of

the projection, in a manner identical to that used to calibrate reticules.

Photographic approaches have eliminated the use of the

camera lucida in many laboratories, as convenient cameras have

become universally available for microscopes. As with projections, a

photographic system must be calibrated, using a stage micrometer. In

addition to the calibration of the photographic negative, the enlarging

process must also be calibrated for accurate measurements. For both

camera lucida drawings and photographs, areas are generally

determined using a device called a Planimeter. A planimeter is a

mechanical device that is used to manually trace the outline of

objects of interest. Using a set of 'x' and 'y' calibrated wheels, the

total area of the object is determined.

Stereology is a technique developed for analysis of metals and

minerals, where generally the properties being measured relate to

number, size and distribution of some particle in the sample. Being

based in geometry and probability theory, and using statistical math-



191

ematics, stereology makes specific assumptions about the object

being analyzed. Since the foundation of stereology is statistical the

general nature of the distribution of whatever is being measured

should be describable using some statistic. This condition may be

met under specific conditions, such as examining the distribution of

chromatin 'clumps' within a cell nucleus, where the only object being

examined is a single nucleus. For highly ordered structures, such as

gland elements within an organ, the very organization of the structure

implies that there is no statistical distribution. Stereo logy can make

estimates of some parameters of specimens such as area of a total

image occupied by some particular component. Note that this is an

estimate. Use of stereology to derive measures of the three-

dimensional structure of cell and tissue specimens may provide

misleading information, since the probabilities used in the

mathematics assume that the entire volume of the specimen is

accurately reflected in the portion measured. While one cannot

disagree that stereology has provided many useful insights into

microscopic specimens, modern techniques of measurement can

provide real measures of the specimen, without any assumptions of

the distribution pattern. The development of newer forms of



192

microscopy (confocal) has extended this direct measurement

capability to the third dimension. With the speed of modern image

analysis systems, there is little justification for performing an estimate

of a cell or tissue parameter when the actual parameter can be

accurately measured, often in less time than is required for the

stereological approach.

Electronic light microscopy

Electronic measurements of light transmitted through

microscopic specimens have a long history, and roughly parallels the

development of photometers, spectrophotometers, and light detecting

devices. Until recently (1980s), these devices simply detected light,

and did not produce images. To use these early devices to produce

images, the portion of the specimen visible to the light detector had to

be restricted, and the specimen or image moved across this restricted

area to generate an actual image. Many mechanisms were

developed to acquire images using such techniques (Weid 1966,

Weid and Bahr 1970). These mechanisms tended to be expensive,

since they required high precision, and were also slow as the image

had to be acquired a small area at a time, and then 'put together' or

reconstructed into a recognizable image. The majority of literature



195

During the decade of the 1980s a variety of solid state sensors

were developed for television purposes. One technology in particular,

the CCD (Charge Coupled Device) camera matured into a

significantly useful device for microscopic imaging work. CCD

cameras continue to evolve and are the technology of choice for most

photometric and imaging microscopic studies. Recently a new

technology has emerged in solid-state cameras: CMOS or

Complementary Metal Oxide Semiconductor cameras. These devices

promise rapid image acquisition, low cost and the potential for some

image manipulation within the camera detector itself.

Solid-state cameras, whether CCD or CMOS are available in

either monochrome or color versions. Color cameras may use two

different techniques to generate a color image.

1. In one approach there are three separate detector arrays, each

with a color filter in front of the array. A prism or mirror system is used

to split the image coming from the microscope into three separate but

identical images so each detector sees the same image. The color

filters are red, green and blue, since a red image, a green image and

a blue image can be combined to create a full color image. This type

of camera is called a three chip color - camera.



196

2. The second approach to color cameras uses a single detector chip,

and places a pattern of color dots over the individual pixels. Again,

these color dots are red, blue and green. The most common pattern

for these dots is the Bayer pattern. In the Bayer pattern, there are

actually four dots per 'repeat,' since for each red and each blue dot

there are two green dots. This type of camera is called a single chip

color camera.

Because the three-chip camera has three individual detectors,

and also a beam-splitting system to divide the image, these cameras

are more expensive than a single-chip camera. Essentially, a three-

chip camera is three separate cameras in one. The advantage of the

three-chip camera is that every pixel is 'real,' that is, it is generating a

true signal. The disadvantage is that there may be differences in

sensitivity of a 'red' pixel and a 'green' pixel that are seeing the exact

same spot of an image. Use of a three-chip camera for photometry

where various colors are examined requires careful calibration and

correction of any variation in output between the separate detector

chips.

The single-chip camera can produce excellent color images,



197

but must be used carefully for quantitative work, and is unsuitable for

photometry, because only one pixel out of four (two in the case of

green) is actually seeing the specimen at the point of maximum

absorption. The other pixels in the Bayer pattern are being

approximated, by assigning their 'red' value to the same value as the

one real 'red' pixel in the pattern. In addition to the approximation of

true signal for a given color, the Bayer pattern results in a real loss of

Bayer pattern

resolution at the sensor level. Since only one of every four pixels (for

red and blue) actually sees a red or blue portion of the specimen, the

true resolution of the single-chip camera is one-fourth the total

numbers of pixels in the array.

The three-chip color camera is essentially three monochrome

cameras, with each camera having a different colored filter in front of

the camera detector. Software is then used to combine the three

separate images into a full-color image. This suggests that it is

possible to use a monochrome camera to capture full color images. A



198

number of cameras provide a mechanism for doing this. Within the

camera itself, there is either an electronic filter, or a filter wheel

carrying glass filters. To capture an image, three sequential images

are taken, each through a different colored filter. These images are

then combined to produce the full-color image. It is possible to do this

same thing, using a simple monochrome camera. One would place a

red filter in the light path of the microscope, and capture a 'red'

image. The same thing would then be done for 'blue' and for 'green.'

The result would be three separate images of the same specimen, in

different colors, and when these three-color planes are combined

using software, the result is a full-color image.

Cameras used for imaging are also described in terms of signal

resolution per individual pixel. This signal resolution is commonly

described as bit depth or gray levels (for monochrome cameras). The

signal resolution is a specification that describes the number of

divisions of the signal between 0 (no signal) and maximum signal. A

common value is 256 levels, and these divisions are often also

described as gray levels. They are based on the digital progression

by powers of two, and therefore a 256-level signal corresponds to 8

bits of resolution (2 to the eighth power). Many modern cameras



199

provide 10 or 12 bits signal resolutions. With a 12-bit camera, 4096

gray levels can be obtained. As the signal resolution increases, the

susceptibility of the signal to perturbation increases.

It is important to note the differences between cameras used to

capture images through the microscope, and the same image, viewed

with the human eye. The human eye is a remarkable detector of light

and of color. However, it is a non-linear, highly adaptive sensor. In

addition, the resolution of the eye detector (retina) varies across the

surface of the retina. Under ideal conditions, most individuals with

excellent eyesight can distinguish between 30 and 35 brightness

levels (gray levels). This is a far cry from the 256 or higher number of

levels seen by a digital camera. Therefore, a solid-state camera can

always detect intensity variations that would be invisible to the human

observer. This translates to the ability to detect finer detail within an

image than can be resolved by a human observer.

The human eye adapts to light intensity, so the 30 or 35 gray

levels that are detected vary depending on the intensity of the light,

and the immediately preceding light exposure of the eye. This is one

of the reasons why individuals must 'dark adapt' prior to doing fluo-

rescence microscopy. The same phenomenon occurs in bright field



200

microscopy, but is seldom recognized. If an individual is asked to

assess the density or 'darkness' of a stain, the assessment will vary

depending on whether the individual has been in a dim environment

or a bright environment just prior to performing the assessment.

Color capture is another area in which a camera differs from the

human eye. While there is much that is still unknown as to the way in

which the eye-brain combination processes color, the camera

provides a fixed model. The construction of the camera itself is based

on the RGB (red, blue, green) model of color. There are many other

models of color, and those that incorporate intensity and saturation

information appear more intuitive to human users of image systems.

One common model that employs such a system is the HSI (hue,

saturation, intensity) model. Software programs are available that

permit images to be converted from one type of color space model to

another, and often such conversions are useful when one works with

full color images.

Photographic color film is balanced for the type of light used to

illuminate the scene. The type of light is specified by a 'color

temperature' number. . Specialty films intended for microphotography

may be balanced for 'tungsten' illumination, with a color temperature



201

of 3,200° Kelvin. The 'color temperature' of a light source is actually a

measure of the intensity of the various components of the light

source, in the red, green and blue regions of the spectrum.

Photographic film records all of these components simultaneously,

and there is little opportunity to 'correct' values, other than limited

adjustment during processing. With a solid-state camera and capture

software, the situation is different. Each of the image components

(red, blue and green) are available as individual images. They are

combined to produce the final image. Since the individual

components (color planes) are available, it is possible to 'color

correct' the image. This is generally done in the capture software, or

the camera itself.

Computers:

Computers suitable for image analysis range from RISC based

workstations to personal computers. A variety of sophisticated image

analytic tools (programs) are available for each of these platforms. In

addition to commercial offerings, there are a number of freeware or

shareware programs available for the personal computers. All of

these offerings include the variety of image analysis processes,



202

although there is little standardization of terminology for specific types

of processes. In general these software systems are organized as a

way to display an image. The image may either be captured from a

camera, or retrieved from storage. Once the image is available, the

user can select, through menus or tool bars, a variety of image

manipulation tools. When the tool is applied to the image, the results

can be seen immediately. Most systems also provide a mechanism to

back up or undo, in case the result was not satisfactory. Among the

best known of these is the program originally designed for the Apple

computers, named NIH Image. A recent addition to the list of

available image analytic programs is Image-I. This program is also

freely available, and because it is written in the JAVA language, can

run on any computer which supports JAVA.

All image analysis programs must provide mechanisms to

display images, read images from a source (camera or storage), and

ultimately save the image and any derived data to storage. In modern

computers, these functions are part of a GUI (graphical user inter-

face) that permits the user to actually see the image and the various

alterations to it during and after various image analytic or

manipulation steps.



203

As camera resolutions increase, they often exceed the display

capability of many computer displays. As an example, consider a

common camera resolution available today, 1024 x 1310. The actual

image from such a camera is larger than the common display res-

olution of many computers, which may be 800 x 600. Another

common display resolution is 1024 x 756. In both cases, the larger

image is displayed completely on the monitor. This is accomplished

within the display program, by simply reducing the image to fit within

the monitor resolution. Therefore the displayed image may not

accurately represent the 'real' image that has been captured and is

available for analysis. Some capture/display programs provide tools

to permit the user to display the image at actual resolution, even

though only a portion of the image is seen on the screen. Such

programs allow the user to scroll over the image in order to see the

entire image. Many output devices, such as printers, actually reduce

the size of the image, and therefore lose resolution as compared to

the original image.

It is important to realize that an image, to the computer, is

simply an array of values of the individual pixels. For 8-bit

monochrome images, this would be a sequence of numbers, with



204

values for each ranging between 0 and 255. Image file storage

formats specify the number of pixels per row, and the total number of

rows. This information is part of the 'header' information in the file

storage, and is required for proper display and analysis of the

images. The user ordinarily does not have to worry about this

information, since it is taken care of transparently by the image

software. Because the computer software considers the image to be

an array of X by Y dimension, any pixel in the image can be

individually addressed if its location is known within the array. In the

case of color images, the actual image is stored as a sequence of

colored pixels, i.e., red, blue and green. To extract the 'green' image,

one would read every third pixel from the file. There are a variety of

formats for image file storage, and the programmer should be sure to

verify the 'bit order' in use prior to attempting to extract specific

information.



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Image analysis by computers

IMAGE ANALYSIS PROCESSES

Point processes

Most forms of image analysis of microscope images start

with a category of operations classically defined as point processes.

These processes are relatively simple, yet are basic to most image

operations. A point process acts on an individual pixel within the

image, and may modify the value of that pixel depending on the previ-



206

ous value. A common use of a point process is to change the value of

each pixel to some other value, depending on the original value. Such

an operation may make use of a LUT (look-up table). A common use

for such an operation is in a pseudo-color operation, where a gray

scale image is divided into a number of 'levels,' i.e., all pixel values

between 0 and 20 might be colored 'red,' all values between 21 and

50 might be colored 'blue,' etc. Since the human eye recognizes color

variations much more easily than density variations, such a point

operation might well make interpretation of a gray scale image easier.

Point processes are used to change the overall intensity of an

image. Suppose an image is captured, and the background appears

too dark, by adding a constant to every pixel the result is an image

that looks brighter. Often, in the process of color balancing the

individual color planes of a color image, it is a point process that is

used to set the 'clear' or 'background' pixels to 'white'. Point

processes can also be used to convert an image to a negative of the

original image. This is quite useful if the image is analyzed with a

system different from the one used for capture. It is also a useful

function for many intermediate image manipulations, particularly



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where images may be combined with one another.

Image contrast stretching is another example of a point

process. In a contrast stretch (image equalization), the range of gray

levels in the image is expanded. In many specimens, the actual

image values cover a relatively narrow range of the total available

gray values. As an example, in a nuclear preparation stained for DNA

with the Feulgen procedure, the total gray levels represented by the

stained nuclei occupy only about 30 per cent of the total available

levels. By contrast, stretching these gray levels to cover the entire

range of available values (256 levels), additional details can often be

seen and/or measured in such contrast stretched images.

By far the most common point process in image analysis

is image thresholding. Image thresholding is used to segment an

image into areas that have some particular interest, such as a

particular staining pattern. The action of a threshold is simple. The

user selects a particular gray level. The point process then sets all

pixels with a value lower than this threshold value to '0,' and all

values above this threshold value to '1.' In other words, the image is

converted to a binary image. While this simple threshold is sufficient



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for some purposes, such as determining the total area in the image

that is above some level (generally, the area of the image that is

'stained' by whatever is being analyzed), the simple binary image is

more generally used to combine with the original image to produce

some type of 'mask.' A common implementation is to combine the

binary image with the original image in such a way that all '0' or

background pixels are left '0,' while all '1' pixels are left with their

original image value. Such an operation leaves the desired portions

of the image visible, with the remainder eliminated from the image. A

second threshold step is often performed, thresholding from the

opposite direction. After this step, a group of objects of 'medium' gray

level could be separated or segmented from both lighter and darker

objects. Another common implementation is to combine the two

threshold operations with a pseudo-color or LUT (look up table)

operation, and simply place a transparent colored mask over the

desired objects, leaving the entire original image visible.

Area processes:

Area processes use groups of pixels either to derive information

from the image or to alter the image in some specific manner. In



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general, area processes involve a small portion of the image, in a

two-dimensional matrix. The matrix is generally made up of an odd

number of 'row' pixels and an odd number of 'column' pixels (a

convolution kernel). It is the pixel in the center of this matrix that may

be altered after performing the area process. Many of the area

processes are often referred to as convolutions. Convolutions com-

monly are based on matrix sizes of 3, 5, 7 or sometimes larger

dimensions. In the area process, a convolution matrix is defined. The

convolution matrix is placed over the image, and each pixel over

which the convolution mask lies is multiplied by the number contained

within the convolution mask. All of these multiplied pixel values are

then summed, and the sum is used to replace the central pixel. The

mask is then moved one pixel further along, and the process

repeated. In practice, the 'changed' pixel is used to construct a new

image, since the process of convolution would fail if the image being

analyzed were being altered during analysis. In other words, the

convolution does not change the 'original' image, but creates a new,

modified image based on the original.

Area processes in general are often called spatial filtering

operations, since they yield information about the rate of change of



210

intensities within the image. In fact, it is these rates of change that are

exploited by many common convolution filters. Typical area

processes are those used for spatial filtering such as high pass and

low pass filters. A low pass filtered image will reduce the contrast of

an image. Such an operation is often useful to remove unwanted

noise spike within an image. A high pass filtered image increases

contrast within the image, and is often used to improve the ability to

detect edges or other structures within the image.

An important type of spatial filtering is edge detection. A variety

of convolution matrices are available to perform it. Often, some type

of edge detection is used to perform segmentation within an image,

particularly if the area to be segmented is close in gray value to other

structures within the image, and thresholding is difficult.

While area processes are extremely important in image

analysis, they are computationally intensive processes. As an

example, a point process need only look at each pixel in an image

one time. An area process, in the simplest case, must look at each

pixel times the size of the convolution matrix. For a convolution matrix



211

of 3 x 3, and an image of 1 million pixels, 9 million operations would

have to be performed. For matrices of 7 x 7, 49 million operations

would be required. High resolution, full-color images require a

considerable space for storage in the computers. There are various

methods of making images smaller (image compression), most of

these forms of compression are 'lossy,' that is, they discard image

information, and this information cannot be retrieved from the stored

image. Certain image storage formats allow a type of compression

that is based on sequences of image data where all the pixels are the

same (like large areas of background). This form of compression is

called run length encoding, and does not discard any image

information. However, for a typical image of a microscope specimen,

where there are few or no 'constant value' areas, run length encoding

may actually result in a larger image storage size than the original

image. As a practical matter, any image intended for future analysis

should not be stored in a compressed format, particularly in view of

the low cost of large capacity storage devices.

Frame processes:

Both point processes and area processes treat the image as a



212

series of pixels, and address each pixel in a specific manner. Frame

processes in contrast operate on the entire image. Often frame

processes use simple operations to add, subtract, multiply, divide, or

otherwise combine two images to produce a new third 'result' image.

A common use of a frame operation is to correct a microscope image

for uneven illumination. By collecting and 'temporarily' saving an

image of the 'background' (when no specimen is present), the

background image can then be subtracted from the specimen image.

This will effectively remove any debris or other image-degrading

elements that are inherent in the microscope and illuminator. A

common biological application for a frame process would be to detect

movement in a cell culture being observed at intervals. By tracking

these changes over time, a 'trail' can be mapped and applied over the

image to follow the movement of cells over time.

Geometric processes:

Geometric processes are quite different from the processes

discussed previously, since they are mainly used to reconstruct or

correct images. Geometric processes actually move pixels within the

image, and can therefore be used to correct defects such as geo-



213

metric distortion. Geometric processes are used to rotate images,

change scale, translate images and produce mirror images. It is

geometric processes that are used to interpolate images from 'real'

size to a size that can be displayed on the monitor in use. Geometric

distortion would include such image defects as a microscopic section

that was attached to the slide in a manner that distorted normal

morphology. In such a case, a geometric process could 'transform'

the image to straighten or otherwise return it to the shape believed to

be correct. Geometric processes are used to make the specimen

'look better,' and to prepare the image for display or output to a print

device.

These processes can be used to align 'stacks' of images, to

reconstruct three-dimensional models of specimens. Geometric

processes can also be used to create mosaic images. A mosaic

image is an image that is created from several smaller images. Using

a motor driven stage (scanning stage), an image system can travel

over a slide, each time moving the exact width of the previous image,

and collecting another image. Each collected image can then be

'added' to the previous one to create a large, mosaic image. With



214

appropriate software, the area where these small images join can be

a perfect, seamless match.

SPECIMEN PREPARATION FOR IMAGE ANALYSIS

The most common staining protocol for routine pathology is the

hematoxylin and eosin (H&E) stain. Interpretation of this stain is

straightforward for those trained in microscopic diagnosis. However,

this stain is quite difficult to use in image analysis. If we consider the

use of a monochrome camera, where all color is converted to shades

of gray, the problem becomes apparent. H&E stained specimens

when viewed as gray scale objects (like a black and white

photograph), lack the sharpness and clarity that a human observer

would detect when viewing the object in full color through the

microscope. Light microscope images depend on absorption of light

by the dyes used to stain the specimen. While the eye can readily

detect the differences between the blue to purple of hematoxylin from

the red of eosin, in a gray scale image there is little distinction

between these two colors. The basis for this lack of differentiation is

that the absorption curves of hematoxylin and eosin overlap over a



215

considerable portion of the visible spectrum (Fig. 33.4). This overlap

of absorption results in slowly varying shades of gray, rather than

abrupt transitions between the blue-purple of hematoxylin and the red

of eosin. This problem is not new to image analysis; it has plagued

photomicrography for many years. In the case of black and white

photography, one can improve the appearance of H&E stained

specimens by simply substituting some other acid dye for eosin that

does not have as great an absorption curve overlap with hematoxylin.

Dyes that work well for this are Orange G or Napthol Yellow S.

Since the digital cameras used for image analysis respond

differently than the eye, specimens intended for image analysis

require modifications of traditional staining methods. In the case of

specimens intended for analysis with monochrome (black and white)

cameras, the staining protocol must be optimized to permit the

camera to detect different portions of the specimen. Since essentially

all image analysis tasks begin with methods to segregate some

portions of the image from other portions, the staining methods

should not use dye combinations that produce any absorption over-

laps. It is also desirable that the density of the various stained

components should be significantly different so the resulting 'gray



217

greatly improve the ability to detect the stained components. For

objects that are stained red to magenta, a green filter will be found

quite useful. Often when the specimen is stained with a method that

utilizes a combination of dyes, a filter can be found that will enhance

the ability to segment the monochrome image to select the

component of interest.

As has been mentioned in the discussion of photometry through the

microscope, accurate measurement of the concentration of a material

in a stained specimen requires:

A. A stoichiometric relationship between the dye and the component

of interest

B. An absorbing dye

C. An intact object, rather than a sectioned object (unless the

measurement simply

determines concentration per unit volume).

DAB (diaminobenzidine) is not a stain that should be employed

when concentrations of a cell or tissue component are desired. Other

chromogens for peroxidase in immunostains are true absorbing dyes,

and are a more appropriate choice.

Every step of the process must be rigidly controlled, beginning



218

with specimen collection and fixation. The length of time in fixative

must be precisely defined also, since length of exposure to most

fixatives may alter the binding of the final stain to the specimen. If the

specimen displays any 'edge effect,' that is, increased or decreased

staining at the periphery of the specimen as compared to the center,

measurements must always be taken at a specified distance from the

periphery. While on the subject of fixation, it should be mentioned that

common fixatives may give very different results with particular

staining protocols. For a quantitative study, it is imperative that all

specimens included in the study be fixed in the same fixative.

Staining protocols that are routine for visual examination may

prove problematic for quantitative work. As the length of time a

specimen is exposed to a particular staining step decreases, the

percentage of error that can be introduced by the physical time

required to insert or remove the slides from the staining solution may

become a significant source of error. In other words, if the total time

in a particular staining step is only 1 minute, a variation of 10 seconds

amounts to almost a 20 percent variation in staining time. To reduce

the potential error introduced by short staining times, staining

methods should be modified by decreasing stain concentrations, and



219

then increasing staining times. As a general guide, any staining time

that is shorter than 10 minutes should be extended to between 30

minutes and an hour, by reducing stain concentration. This strategy

effectively controls errors introduced by the time required to

physically introduce or remove specimens from staining solutions. For

stains that require differentiation, often this is done with a series of

dips, with differentiation controlled by an experienced technician. This

type of differentiation should be optimized by changing the

concentration of the differentiation solution in order to extend the dif-

ferentiation steps to a defined time, preferably long enough to

eliminate the effect of short times. In addition to times in actual

staining solutions, stain results may also be influenced by various

dehydration sequences. Standardization of every step of a stain

protocol including deparaffinization, hydration, staining,

differentiation, dehydration, clearing, and cover slipping will greatly

improve variability of specimens analyzed with image analysis

systems.

For studies that purport to measure the total amount of a

material present, the intact object containing that material must be

present in the slide. For many cellular materials, this means that



220

whole cells, such as from tissue cultures, be used. Another strategy is

simply to disaggregate tissues, and select intact cells for analysis.

Sectioned material may be employed for many image analysis tasks,

but generally is not suitable for measuring the total amount of

material present in a given cell or tissue component. One possible

exception to this is the measurement of cell nuclei constituents.

However, to measure nuclei, the operator must be certain to include

intact nuclei in the section, that is, nuclei which have not been sec-

tioned on either their top or bottom surface. The difficulty with this is

that in most fixed and sectioned material the average size of cell

nuclei is approximately 7 microns in diameter. Since general practice

in many laboratories is to section at under 5 microns in thickness,

then all 'nuclei in the specimen will be sectioned. One possible way to

address this is to cut thicker sections, and while this will yield some

nuclei that are intact, there is the additional problem of overlap of

nuclei from top to bottom of the section.

Morphometry, or the measurement of size and arrangement of

cell or tissue constituents, can be done in sections. Such studies

must also be carefully controlled, since there will always be a range

of 'profiles' of a given object shape in the section. As an example,



221

imagine a perfectly round sphere in a section. If the measurement

being done is the total area of the sphere, then one would obtain

different values as the section passes through the sphere. The result

would be a series of measurements, with only one approaching the

true diameter of the sphere. Any measure taken in sections must

account for this spread of values which result from the sectioning of

spheroidal objects. Obviously, some objects may have shapes other

than spheroids, and this particular geometry must be taken into

account when establishing a measurement approach.

Applications:

Image analysis can be used to provide numerical assessment

of many details of microscope specimens. An example is the

thickness of an epithelial layer, or the depth of penetration of an

epithelial tumor into underlying tissues.

Image analysis can also be used to perform repetitive

measurements where many objects must be measured, or where the

measurement must be restricted to a particular orientation. An



224

goal – discriminating molecules that are only two to 25 nanometers

apart.

The scientists published the details of the new technique in the

August 10, 2006, edition of Science Express, an advance online

publication of the journal Science. The basic concepts behind their

new technique are simple: The researchers label the molecules they

want to study with a photoactivatable probe, and then expose those

molecules to a small amount of violet light. The light activates

fluorescence in a small percentage of molecules, and the microscope

captures an image of those that are turned on until they bleach. The

process is repeated approximately 10,000 times, with each repetition

capturing the position of a different subset of molecules.

Because the number of molecules captured in each image is

small, they are far enough apart to see each molecule individually

and thereby localize its center. When a final image is created that

includes the center of each individual molecule, it has a resolution

previously only achievable with an electron microscope. Unlike

electron microscopy, however, the new technique allows for more

flexibility in labeling molecules of interest.



225

A great feature of PALM is that is can readily be used with

electron microscopy, which produces a detailed image of very small

structures – but not proteins – in cells. By correlating a PALM image

showing protein distribution with an electron microscope image

showing cell structure of the same sample, it becomes possible to

understand how molecules are individually distributed in a cellular

structure at the molecular scale. Correlative PALM unites the

advantages of light and electron microscopy, producing a

revolutionary new approach for looking at the cell in molecular detail.

Nanoscale Microscope Sheds First Light On Gene Repair

Proteins called H2AX act as "first aid" to DNA, among other

roles. For the first time, scientists using the world's most powerful

light microscope (the only one of its kind in the Americas) have seen

how H2AX is distributed in the cell nucleus: in clusters, directing the

first aid/repair after DNA injuries to the region where it is really

needed. 7



226

Image by Photoactivated Localization Microscopy

3-dimensional 4Pi microscopy visualization of Hela H2AX (green)

and gamma-H2AX (red) chromatin clusters at 100 nm resolution

(center image) is shown in front of a traditional overview z-projected

data stack of HeLa cells stained for H2AX, gamma-H2AX and DAPI

taken with a confocal microscope.



227

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30 th october micrscope final

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