CONTENTS

1.INTRODUCTION TO MICROSCOPE.
History of microscope.
What is a microscope?
Why use a microscope?
Interaction of light with matter.
Image formation
Lens aberrations :Chromatic aberration
Spherical aberration
Coma
Astigmatism
Field curvature
Distortion

2.The components of a microscope

3.The compound microscope
4.The dark ground microscope
5.The fluorescence microscope
6.The polarizing microscope.
7.The phase contrast microscope.
8.The interference microscope
9.The electron microscope.
10.The practical use of microscope.
11.Setting up the microscope.

HISTORY OF MICROSCOPE
BIRTH OF THE LIGHT MICROSCOPE
1590 Zacharias Janssen and his son 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.
1609, Galileo 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.

1632-1723 :ANTON VAN LEEUWENHOEK

The father of microscopy, Anton Van Leeuwenhoek of Holland, worked in a dry
goods store where magnifying glasses were used to count the threads in cloth.
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.
LEEUWENHOEKS MICROSCOPE

4 centuries of light
microscopy

400
years

Compound microscope

ROBERT HOOKE: The English father of microscopy, re-confirmed Anton van Leeuwenhoek's
discoveries of the existence of tiny living organisms in a drop of water. Hooke made a copy of
Leeuwenhoek's light microscope

19th century :CHARLES A. SPENCER :

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
marvelous instruments built by the American, Charles A. Spencer, and the industry he founded.

o1837- Andrew Ross made early microscope , He introduced a

triangular bar shaped limb and y-shaped foot.
o1843-Parvell and Lealand introduced small portable microscope.
o1873-Ernst Abbe published his work on theory of magnified and
resolution about microscope principals

ERNST ABBE

WHAT IS A MICROSCOPE?
Micro=small, Scope=to view
Microscope is an instrument used to produce an enlarged, well defined image
of the objects too small to be observed with the naked eyes.
It is built based on properties of light. It is most commonly used instrument in
the lab, yet least known about it by its users.
The term microscope strictly speaking includes not only instrument fitted with
separate objectives and eyepiece lenses, but also those composed of a single
lens.

These single lens microscope may be used either with or without

devices for holding them and the objects to be examined.
If the lens is fixed and there is a stage to carry the object under the
study, the instrument is usually called a simple or dissecting
microscope.
If no such mount then we speak of hand lens or magnifier.

HAND LENS

WHY USE A MICROSCOPE?

Objects of interest to us exists in a very wide range of sizes
but the unaided human eye is unable to see the smaller ones
because of its limited performances.
Microscopes are of value in fighting diseases for it happens
that many parasites are small and many cellular components are
smaller still, and to see both is to begin to understand both
better.

Microscope is an essential instruments which is used:1.To visualize fine details of an object.

2.To provide an magnified image of an object.
3.To measure features (length, areas, angles)in the object.
4.As an Analytical tool to measure optical properties(e.g. Reflectances, refractive
index)
5.Printed circuits in calculators, radios, televisions and computers require low
power microscope to help in there designing.
6.Amateur microscope still often make useful contributions to science
especially in natural history.
7. Again many surgeries now are carried out with such precision
that microscopes are needed in there execution.
.In all the above cases there is interaction between light, which forms the image,
the object itself and eye-brain complex of the observer.

INTERACTION OF LIGHT WITH MATTER.

Light is a form of energy ,like any other form of energy follows certain rules.
Knowing these rules we can design instruments to utilize this energy to our
advantage ,such an instrument is microscope, one of the principal tool
in laboratory.
PROPERTIES OF LIGHT:1)Light radiates in all directions from its sources.
2)Each radiating ray unless something interferes travels in a straight line to
infinity.
3)It is frequently shown as a sine curve representing waves of energy.

AMPLITUDE:-Refers to strength of the energy

or brightness of the light. When the light travels
through the medium ,amplitude decreases to a
greater or lesser degree depending on medium.

WAVELENGTH:-It is the distance between the

apex of one wave and the next wave. And is
measured in nanometers. wavelength
determines the colour.

REPRESENTATION OF LIGHT
RAY SHOWING WAVELENGTH
AND AMPLITUDE

FREQUENCY:-It is the number of waves per second.

COHORENT:-Individual rays of identical frequency from same source.
NON-COHORENT:-Rays from different sources or of different frequencies.

ABSORPTION: Is a process where by amplitude of light which

passes through an object is reduced in comparison with that of
which passes around it.

REFLECTION :Occurs when light

strikes the surface. In case of a
smooth surface angle of reflection is
equal to angle of incidence then it is
known as specular reflection.

SPECULAR REFLECTION FROM

SMOOTH SURFACE

If the surface is rough ,diffuse

reflection returns the light at all
possible angles. Reflection plays a
major role in microscopic study of
opaque objects .

REFLECTION FROM ROUGH SURFACE

RETARDATION AND REFRACTION:Media through which light is able to pass will slow down or retard the speed of the light in
proportion to density of medium.
Higher the density ,greater the degree of retardation .
Rays of light entering sheet of glass at right angles are retarted in speed but there direction is
unchanged.

a)rays passing from one medium to another ,perpendicular

to interface ,are slowed down at the same moment .b) rays
passing at any other angle to the interface are slowed down
and deviated.

If light enters glass at any other angle deviation of direction will occur in addition
to the retardation this is called refraction.
A curve lens exhibits both retardation and refraction ,the extent of which is
governed by :a)Angle of incidence -angle at which light strikes the lens.
b)Refractive index-density of glass.
c)Curvature of lens.

Rays passing through a curved lens exhibit both retardation and refraction

Angle to which rays are deviated within the glass or other transparent medium is called
angle of refraction and ratio of sine values of the angle of incidence (i) and refraction
(r)is refractive index(R.I)
Greater the R.I ,higher the density of the medium
R.I of air 1.00
R.I of water 1.30 .
R.I of glass 1.5.

Refraction of light

Light passing from one medium into a

more dense medium is refracted towards
the normal, And
when passing into less dense medium is
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 and no light
will pass through.

Total internal
reflection

POLARISATION OF LIGHT:-A light beam normally contains waves of

many different frequencies with all possible phase relationships, and
vibrating in all possible planes .If the vibration of the light waves are
restricted to one single plane, then the light is called plane polarized.
DIFFRACTION:-Is the scattering that occurs when an beam passes an
edge in an object.Diffraction appears as fanning out or apparent bending
of a beam,allowing the light to extend into the shadow areas.The amount
of such bending is related to wavelenght ,being larger for longer
wavelenght.Thus it is not possible to produce an absolutely sharp image
of any object because diffraction limits the resolution.

FLUORESCENCE:-Is the name given to the process by which

energy from light in the shorter wavelength regions of the spectrum
(green,blue,&uv)is absorbed by an object and then almost
immediately re-emitted as a light of longer wavelength

Some objects are intrinsically fluorescent (auto-fluorescence) and

thus appear self luminous under the microscope if ultra violet light
is used . Such auto-fluorescence is very weak ,and the fluorescent
dyes may be added to the specimen so as to produce higher
contrast images with a much brighter fluorescence.

TYPES
OF
MICROSCOPE
There are two main types of microscopy:1.Light microscopy(0.2 resolution)
- Visible light Routine microscopy
-Ultraviolet Fluorescence microscopy
2.Electron microscopy(0.2nm resolution)
-Beam of electrons in an electromagnetic field are used to visualize the specimen

y microscope were simple microscope, but with advancement of science

pound microscope were built
Simple microscope:-It consists of a single lens are a magnifying glass.
Compound microscope:-it consists of two or more lenses.

TYPES OF MICROSCOPE:The following are the types of microscopes employed :1.Optical or light Microscope
2.Compound Microscope
3.Phase contrast Microscope
4.Dark field /dark ground Microscope
5.Interference Microscope.
7.Polarisation Microscope.
8.Electron Microscope.

TYPES OF MICROSCOPE

IMAGE FORMATION:-

Parallel rays of light entering a simple lens are brought together by refraction to a single point,
the principal focus or focal point, where a clear image of an object will be formed. The
distance between the optical center of the lens and the principal focus is the focal length.
In addition to the principal focus ,the lens also has other pairs of points ,on either side of the
lens called conjugate foci such that the object placed at one will form a clear image on a screen
placed at the other. The conjugate foci vary in the position ,and as the object is moved nearer to
the lens the image will be formed further away ,at a greater magnification and inverted. This is
the real image and is formed by the objective lens of the microscope.

Formation of real image

If the object is placed yet nearer the lens within the principal focus the image is formed on
the same side of the object and is enlarged the right way up and cannot be projected on to
the screen. This is the virtual image and is that formed by the eye piece of the microscope
level of the real image projected from the objective.This appears to be at a distance of
approximately 25cm from the eye around the object stage level.

LENS:-Lens is the name given to the piece of glass or other transparent material
,usually circular having the 2 surfaces ground and 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 concentrate or converge to form a
real image.
It is called negative ,in which case light rays passing through will diverge or scatter
and positive or real image will not be seen.
These two types can easily be differentiated since positive lenses are thicker at the
centre than at the periphery, whereas negative lenses are thicker at centre .

TYPES OF LENS

POSITIVE LENS

NEGATIVE LENS

Types of simple lenses

Lenses are classified by the curvature of the two optical
surfaces. A lens is biconvex (or double convex, or just convex) if
both surfaces are convex. If both surfaces have the same radius of
curvature, the lens is equiconvex.
A lens with two concave surfaces is biconcave (or just concave).
If one of the surfaces is flat, the lens is plano-convex or planoconcave depending on the curvature of the other surface.
A lens with one convex and one concave side is convex-concave
or meniscus. It is this type of lens that is most commonly used in
corrective lenses.

If the lens is biconvex or plano-convex, a collimated or

parallel beam of light travelling parallel to the lens axis
and passing through the lens will be converged (or
focused) to a spot on the axis, at a certain distance behind
the lens (known as the focal length). In this case, the lens
is called a positive or converging lens.

If the lens is biconcave or plano-concave, a

collimated beam of light passing through the lens
is diverged (spread); the lens is thus called a
negative or diverging lens.

Plano-concave lens:
Plano-concave glass lenses are manufactured with one concave
surface and one plane surface. These lenses have negative focal
lengths.
These lenses diverge collimated incident light.
They form virtual images which are seen through the lens.
They are often used to expand light or to increase focal lengths in
existing systems.

Bi-convex lens:
Bi-convex lenses are manufactured with an identical convex surface on both sides
of the lens.
Bi-convex lenses have positive focal lengths and form both real and virtual
images.
Aberrations such as coma, distortion, and chromatic aberration almost exactly
cancel out at unit conjugate ratios due to the symmetry.
Recommended for virtual imaging of real objects, and for positive conjugate
ratios from approximately 0.2 to 5. Values are wavelength sensitive.
Antireflection coatings can be ordered as an option.

LENS ABERRATIONS:These are the defects which impair the ability of a lens to produce an image
which is an exact copy of an object.
There are six basic types of aberrations:1) Spherical aberrations
2) Chromatic aberration
3) Coma
4) Astigmatism
5) Curvature of field
6) Distortion

In the objective lenses used in the modern microscopes only spherical aberration is
significant, since it is often introduced into the image by the incorrect use of the or by using
unsatisfactory preparation.
CHROMATIC ABERRATION:-When light is spilt into its component parts ,each part
vibrates to a different degree producing different colour to the eye. These
colours(vibgyor)are known as primary spectrum and are seen in the rainbow, or through a
spectroscope.
Red has the longest wavelength with vibration of 0.7,blue 0.45,and violet 0.35.It will
be seen that vibration of red is twice the length of those of violet. (results in unsharp image
and colored fringes)
Since light rays cannot vibrate as easily in a dense medium as in a
rare medium, It follows that various colours will be affected by lens to different degrees the
colours with shorter wavelength such as blue,violet being affected to a greater degree
than those having longer wavelength such as red and orange.

Path of white light through a simple planoconvex lens showing

chromatic aberration. Blue rays are focused closer to the lens
than either the green or red rays.

CORRECTION OF CHROMATIC ABERRATION:It can be corrected by using two component lens.

A positive lens is combined with a negative lens made of glass producing a greater chromatic
aberration,but with a same refractive index.
Negative lens corrects the chromatic aberration in the positive lens and only partially
neutralises its magnifying power.This type of lens is known as an achromatic lens.

The addition of concave lens of higher dispersion glass to the

plano-concave lens corrects the chromatic aberration.

If flourspar is incoporated in the glass of the achromatic lens, three colours can
be bought to a single focal point,and amount of chromatic aberration visible in
the image will be negative.such lenses are known as apochromatic lens.

SPHERICAL ABERRATION:- It is a defect of single lens, due to the fact that it

has a curved surface. Since the angle at which light rays enter and leave the lens
vary with each part of the lens, Those rays passing through the periphery will be
refracted to a greater degree than those travelling through the central area.

CORRECTION OF SPHERICAL ABERRATION:-

This fault could be minimised by using only central area of the lens but since microscope objectiv
must have a shorter working distance and high magnification,
a large angle of light is required from each point of object. Hence it can be corrected by using
powerful positive lens and partially neutralising its magnifying power
with a negative lens made of glass having a greater relative aberration.

COMA: This is an aberration associated with points which lie off the axis and
which as the consequence of the coma are imaged as a conical or comet shaped blur.
Coma is the most objectable aberration but is not found in todays objective lenses.
A lens corrected for the coma has equal magnification in its different zones for points in
the field which are off axis.
When a lens is corrected for coma throughout ,say about two-third of its diameter and is
nearly free of coma for reminder ,and in addition is free of spherical aberration ,then it is said to
be aplanatic.

Coma in the lens

ASTIGMATISM:-This aberration often affects the oblique rays passing through the
lens. The effect is that the image of an off axis point is not reproduced as such but its
appearance differs according to the focal point chosen.

Image formation by astigmatic lens.

FIELD CURVATURE:- Here instead of image lying parallel with that of the lens
it falls upon a surface which has a spherical curvature. This defect if present will
be troublesome with high aperture objectives of short focal length, when only
either the center of the field of the view or peripheral areas will be in sharp focus.
Curvature of the field may be partially corrected for the medium power
objectives by use of a special eye piece.

DISTORTION:-This distortion results if the magnification of the lens varies across

the diameter of the field of the view . Greater magnification at the center of the field
results in so called barrel distortion while the converse gives pin cushion or
negative distortion

Differences between simple and

compound microscope.

Simple microscope

Invented by leeuwenhoek.
It consists of a single lens or a
magnifying glass.
PRINCIPAL:-a lens of short
focal length is used to produce
an enlarged image of an
illuminated object at a short
distance. the lens fixed in a
frame is adjustable to view the
object. shorter the focal length
larger is the image.
Source of power is sunlight.

Compound
microscope.

Invented by hooke.
It consists of two or more
lenses.
PRINCIPAL:- if a short focal
length is used to produce an
enlarged image of an
illuminated object at a short
distance, then another lens can
be so fixed that it would
produce a further enlargement
of that image.
Source of power is electricity.

COMPONENT PARTS OF A
MICROSCOPE
A.
(i)
(ii)
(iii)

SUPPORT SYSTEM
Base
Pillars
Handle/Limb

B. FOCUSSING SYSTEM
(i) Course adjustment screw
(ii) Fine adjustment screw
C. THE STAGE
(i) Fixed Stage
(ii) Mechanical stage

D. OPTICAL SYSTEM
(i) Body Tube
(ii) Nose piecea.Fixed
b. Revolving
(iii) Objective lenses
(iv) Eye piece
E. ILLUMINATION SYSTEM
(i) Source of light
(ii) The mirror
(iii) The condenser

TYPES OF MICROSCOPE
1. SIMPLE MICROSCOPE [LEEUWENHOEK] [250X]
>Hand held magnifiers[5x-20x]
>Supported magnifiers{watch makers eye glass, dissecting
microscope, binocular headband
magnifier , spectacle supported lenses}
>micro film readers{for large histo specimens }

2. HIGH POWER SIMPLE MICROSCOPE [HORACE DALL][450X]

3. LOW POWER STEREO MICROSCOPE
4. MACROSCOPES
5. COMPOUND MICROSCOPES
6. DARKGROUND MICROSCOPES
7. FLOURESCENCE MICROSCOPES
8. POLARIZING MICROSCOPES
9. PHASE CONTRAST MICROSCOPES
10. INTERFERENCE MICROSCOPES
11. ELECTRON MICROSCOPE

COMPOUND MICROSCOPE

It employs two separate lens system

It is one in which real magnified image produced by one lens
(or lens system)called the objective is further magnified by another
lens i.e the eye piece,which typically forms a final magnified virtual
image for observation of the users eye .

Two types:monocular
binocular

COMPONENTS OF MICROSCOPE
[MONOCULAR]

A.SUPPORT SYSTEM

Base or foot ->heavy u-shaped or horse shoe shaped base or foot

which supports the microscope on work table and
provides stability

Pillars

->two upright pillars project up from the base and are

hinged to the handle.The hinge joint allows the
microscope to be tilted at suitable angle for
comfortable viewing of histological slides

. Handle or limb->The microscope is held by its limb while moving it.

It is further attached to the body tube.

B. THE FOCUSSING SYSTEM

1.
2.
.

.
.
.
.
.
.
.

Coarse adjustment screw-heads

Fine adjustment screw-heads
The screw heads are employed for raising or lowering the optical
system with reference to the slide under study
till it comes into focus.
They help to place the objective lens at its optimal working length
The screw heads work on a double sided micro meter mechanism
They are attached to the limb of the microscope
Coarse screw head moves the optical system up or down rapidly
through a large distance via a rack and pinion arrangement
Fine screw head needs several rotations to move the tube through a
small distance
It is graduated in 1/50ths ,each division corresponds to 0.002mm
movement of the tube
It is used for accurate focusing

C. THE STAGE
1.Fixed stage

It is a square platform with an aperture in the centre , fitted to the

limb below the objective lens

The slide is placed on it and centred over the aperture

The converging cone of light emerging from the condenser passes

through the aperture and then through the specimen into the
objective lens

The aperture is 1 to 1 inches in diameter

The stage is provided with simple metal springs to hold the object
or is fitted with the mechanical stage

2. Mechanical stage

It is a calibrated metal frame fitted on the right edge of the fixed stage

There is a spring mounted clip to hold the slide in position

Two screw heads move the slide from side to side and forwards and
backwards

The standard mechanical stage takes a 3x1inch slide and moves over
an area of ~3 x 1 1/4 inches so that the whole slide may be
examined

Special stages are available to take very large slide and petridishes

Circular rotating stages are also available like in polarizing microscope

The vernier scale on the frame indicates the degree of movement so

tha the slide can be replaced in much same position on reexamination
immediately

D.OPTICAL SYSTEM

Form a magnified image of the specimen

1.
Body tube
.
It is attached to the limb and rarely contains a draw tube
.
Older microscope have inner graduated draw tube to increase the
tube length and magnification-now discarded due to loss of clarity
.
The distance between the upper ends of the objective lens and the
eye piece is called Tube length [16-17cm]
.
The distance between the upper focal length of the objective and the
lower focal length of the eye piece is called optical tube length
.
The distance between the objective shoulder and the specimen is
called Parfocalising distance of the objective
.
Three forms are available
->MONOCULAR
->BINOCULAR :- Inter pupillary distance can be adjusted for
observation
->COMBINED PHOTO BINOCULAR :- Has prism system
allowing 100% light to go either to eye piece or camera or it can
have beam-splitting prism dividing the light- 20% to eyes and 80%
to camera. This facilitates continuous observation during
photography

2. Nose piece
It is fitted at the lower end of the body tube.
It rotates on a central pillar and is designated by the number of
objectives it carries , for example , double,triple or quadruple
nosepiece.
It should bring each objective into center of the optical axis and at
correct tube length which is indicated by a click.
It is 18mm in depth.
Magnification can be increased by rotating the nosepiece.

3.Objective lenses
It is attached to the lower end of the body tube.
Three spring loaded objectives of varying magnifying powers are
commonly used.
Within the objective there may be lenses and elements from 5 15 in
number ,depending on image ratio,type and quality.
The main task of the objective is to collect maximum amount of light
possible from the object, unite it and form a high quality magnified
real image, some distance above.

Object to image ratios of objectives are from 1:1 to 1:100 in normal biological
instruments.
NUMERICAL APERTURE:-[NA]
The ability of an objective to resolve detail is indicated by its NA and not by its
magnifying power.
It allows the cone of light that can be collected from the object.
It is expressed as the product of two factors by a formula
NA= n x sin u
Where
n refractive index of the medium between the coverglass over the object
and front lens of the objective. For example , air, water or immersion oil.
u angle between the optical axis of the lens and the outermost ray which
can enter the front lens.

Greatest angle will be formed when the surface of front lens coincides with the
specimen ie u= 90 degrees.
Dry objective NA= 0.95
Oil immersion NA= 1.5
Air R.I =1.0
Water R.I = 1.3

EFFECTS OF HIGH NA:(i) it reduces the depth of focus ie the ability to focus on more than one
an object at same time.
(ii) it reduces the flatness of the field , so that the edges are out of

layer of

focus.

 RESOLUTION

It is the smallest distance between two dots or lines that can be seen as
separate entities.

The angle formed by the images of these two points or dots at the eye
is called as the VIEWING ANGLE.

It is about one minute of arc when calculated for an object at 250mm

from the eye , the conventional NEAREST DISTANCE OF
DISTINCT VISION.

Resolution = 0.61 lambda / NA

RESOLVING POWER

Ability to resolve the detail of an object that can be measured

As NA of an objective increases the resolving power increases but working distance ,
flatness of field and focal length decreases

TOTAL MAGNIFICATION

It is a product of magnification values of objective and eyepiece, providing the system

is standardized to an optical tube length of 160mm or 16cm
optical tube length
magnification of eyepiece
focal length of objective

----------------------------- x

brightness = (NA)(NA)
-------------------------------------(magnification) (magnification)

Low magnification gives bright images since brightness is inversely proportional to

magnification.

LOW POWER LENS [LP] [10X , NA-0.25]

Focal length is 16mm

This lens magnifies the image 10 times
It provides a larger area of the slide for viewing
Has a yellow ring etched on its surface

HIGH POWER LENS [HP] [45X , NA-0.65]

Focal length is 4mm

Magnifies 45 times
Has a blue ring etched on its surface

OIL IMMERSION LENS [100X , NA 1.3]

Focal length is 2mm

Immerged in oil magnifies 100 times
Has a black ring etched on its surface

SCANNING OBJECTIVE LENS [3X or 4X] [NA 0.1]

Focal length is 40mm

Very low power lens
Allows scanning of much larger area on slide
Has a red ring etched on its surface

TYPES OF OBJECTIVES
1.
2.

3.

4.
5.
6.

Achromatic which is a combination of positive and negative

lens, most commonly used for routine microscopy.
Apochromatic they are used in conjunction with a highly
corrected achromatic condenser and compensating eyepiece.
These objectives are used for microphotography.
To get maximum light with high power objectives having NA
>1 , oil immersion condensers should be used with NA equal
to that of objective and immersion oil between the condenser
and the slide as well as between the objective and the slide.
These are highly corrected for other lens aberrations
Flourite objective [neoflour] flourite glass is incorporated to
give better colour correction. Quality of image is between
achromat and apochromat
Planochromat gives a perfect flat field recommended for
photomicrography and cytology screening
Polarizing objectives strain-free objectives for use in
polarizing microscope
Phase objectives contain a phase plate used in phase
contrast microscope

COVERGLASS THICKNESS
is not neccesary for oil immersion lenses because of same R.I.
For dry lenses No.1 coverglass is used[0.12-0.22mm]
And then the collar setting is done to change the contrast.

PARFOCAL SYSTEM
Switching from LP to HP requires only a little turn of fine adjustment to get a sharp image.

4. Eye piece

Function is to magnify the image formed by the objective within the body
tube and present the eye with a virtual image apparently in the plane of the
object being observed, usually this is an optical distance of 250mm from
the eye.

Available magnifications are 5x , 8x , 10x , 6x , 15x.

5x is the tallest , 10x is the shortest.

Eyepiece has two lenses
EYE LENS on top
FIELD LENS at the bottom

Field lens collects the divergent rays of the primary image and passes these
to eye lens which further magnifies the image

Pointer eyepiece has a small pin mounted in it which is used to point out a
particular cell or object in the field.

 Field of view in mm= field of view number

------------------------------------magnification of the
objective
Actual diameter of the specimen can be calculated.

TYPES OF EYEPIECES

1.

Negative focus is within the lenses of the eyepieces.

2.

Positive focus is outside the eyepiece lens system.( like in simple

microscope)
Huygenian originally designed by huygens for the telescope , most
commonly used.
they are negative , under-corrected (ie when a blue 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
chromatic abberation) . Best suited for use with achromatic objectives.

3.

4.

Ramsden they are positive eyepieces .

compensated eyepieces (ie over corrected with an orange
fringe seen around the edge) are of ramsden type ;
having
doublet or triplet lenses.
They are used for micrometer eyepieces due to less
distortion to scales.

5.

Wide field eyepieces has large field of view for use in biological lab.

6.

High eye-point eyepieces for spectacle wearers .

The eyepieces are engraved with a diagram of a pair of spectacles
It is provided with a rubber guard to prevent scratching of the
spectacles.
12.5x high eyepoint , wide angle eyepiece is routinely used.

7.

Periplan eyepiece corrected the chromatic abberation and gave a flat field
of view. It has six lens elements.

8.

Compensating eyepiece used with apochromatic lenses . Designated as

K (German lenses) COMP (English lenses).

ILLUMINATION SYSTEM
1.
.
.
.
.

.
.
.
.

CONDENSER.
It is a system of lenses mounted in a short cylinder and lies beneath the stage
hence comprises of the substage.
It can be raised or lowered by a rack and pinion arrangement.
It concentrates light onto the object with uniform intensity over the whole
field.
It controls the aperture of the illuminating cone of light and matches it to the
NA of the objective.
TYPES.
Low power NA= 0.25
Medium power dry - NA=0.95
High power oil immersion-NA=1.4

 OTHER TYES.

Abbe two lens illuminator- uncorrected, shows extensive zone of confusion, no

sharp focus.
max NA=0.5
It is cheap, has long working distance, illuminates large area of the object.

Abbe three lens or aplanatic- corrected for spherical abberation but not for
colour.
NA=0.65
when used with oil NA=1.2
It is commonly used but expensive

Achromatic condenser- most expensive, chromatically and spherically corrected.

NA upto 1.4 when used with oil immersion apochromatic objective.

 IRIS DIAPHRAGM

It is employed to limit the angle of cone of light passing through the object so
that it will just fill the front of the objective.
The size and volume of the cone of light is altered by this diaphragm.
If closed too much image becomes too contrasting and if it is wide open image
will suffer GLARE.

FILTERS

The intensity of illumination is reduced by the filters.

Coloured filters.
Colour correction filters.
Neutral density filters-to reduce brightness
Heat filters used in high intensity tungsten halogen lamps.
Light absorbing filters.

FILTER CARRIERS

It is usually a recessed metal ring, pivoting on a screw to

facilitate the easy removal of filters.

 MIRROR.

It is fitted 4 inches below the stage.

Two sided mirror is used with plane and concave surfaces.
Concave surface converges the reflected rays, used with low power objectives.
The plane mirror must always be used with condenser so that the lens is filled
with light.
Planeused with natural light.
Concave with artificial light.

SOURCE OF LIGHT.

All the light that reaches the eye should come from the specimen under study.
Light from any other part of the slide will tend to obscure the details, this is
called GLARE.
So only a small area of the slide is illuminated.
Daylight may be diffuse, reflected and scattered by atmosphere.

Artificial sources of light.

1. Electric filament lamp
2. External flourescent tube
3. Tungsten filament bulbs.
4. Mercury or xenon vapour discharge tubes.
5. High intensity lamp with a condenser is usually used.

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
Should make the object appear as though it was self luminous.

TYPES OF ILLUMINATION:-

Critical or nelsonion or source focused

illumination.

Homogenous light source is used to get this kind of

illumination.
No lamp condenser used
Normally employed with a base light source.
Light source be focused on the object plane by racking the
substage condenser up and down.
Light source must be large.
Used in routine visual microscopy. Easy to set up.

 Kohler illumination.

Non homogenous light source is used.

Lamp condenser or collecter lens is essential.
A Field iris is placed in front of the collecter to focus the light from the collecter
onto the substage iris.
It is used with compound lamps .
Used in photomicrography.
Hence image obtained from the field iris will be found in the primary image
plane and retina of the eye.

 TRANSMITTED ILLUMINATION

Used For thin preparations where much light passes through

without being absorbed.
In normal practice light provided by the condenser forms an
axial cone of rays which occupy most of the aperture of the
condenser and objective.
This is called BRIGHT FIELD ILLUMINATION. Works well with
stained preparations.
To obtain extra contrast for finer details the cone of rays is
deliberately made off axis . This is called OBLIQUE
ILLUMINATION.
EPI ILLUMINATION
Used for thick specimens where light cannot pass through
them or if they are intensely colored.
Light is incident on them from above .
Used in low power stereomicroscopes.
Light reaches the tube at right angles and is reflected onto the
objective at 45 degrees.

 BINOCULAR MICROSCOPE.

Light rays emerging from the objective are equally divided by

the two eyepieces.
Modern binoculars incorporate 4 prisms and the eye receives 2
parellel beams.
Lower central prism 2 prisms cemented at interface by
semisilvered surface.
They are differentially treated one being reflected to the right
and the other passing into upper prism.
The light rays passing through semisilver surface to upper
prism travel through great thickness of glass than those that
are reflected, having effect of retarding them.
Compensated by right hand prism with an extra thickness of
glass.
Slight variation of focus is present on right eyepiece usually
and is adjusted by using adjustment screws for focussing.
Here the optical tube length may be increased from 160 to 240
mm, so a compensating lens is incorporated to overcome this.
This lens re- focuses the virtual image for the new tube length

Eyepiecies can be easily moved together or apart and the inter pupillary
distance can be adjusted to suit individual requirements.

ADVANTAGES
Minimum amount of eye fatigue.

 SETTING UP THE MICROSCOPE.

The bench on which it is mounted should be free of vibration.

The microscopist works with his back to the window, a light screen, the back
and sides of which are finished with a flat black paint to minimize back scatter
of light.

TECHNIQUE
The lamp should be positioned opposite the microscope and a blue day light
filter inserted to absorb the excess yellow light given by artificial light.
Position the lamp so that light strikes the center of the mirror and adjust mirror
so that light is directed upward into the condenser .modern microscopes have
in-built lamps, condensing lens and mirror.
Focus on an object on the stage and ensure that the field is evenly illuminated.
With the object in focus , rack the condenser up and down until a sharp image
of lamp iris appears.
Center the image of field iris using substage centering controls.

Open the field iris until its circle of light is just larger than the
field of view, this reduces the GLARE.
Remove an eyepiece and adjust the iris until twothird of the
back focal plane of the objective is illuminated. Replace the
eyepiece. The microscope is ready to use.

 CLEANING AND MAINTENANCE.

Microscope is a complicated and delicate apparatus and requires experience to

maintain it.
Prisms and back lenses of the objectives should never be touched and cleaning
should confined to blowing off the dust with a rubber bulb fitted with a small
bore metal tube, since the slightest disalignment of the prisms will cause eye
fatigue.
Lenses should be wiped only with FRESH LENS TISSUE or COTTON
WOOL, to avoid scratching. Adjustment screws and condenser are also cleaned
in the same manner. ISO- PROPANOL can also be used.
Immersion oil should be removed immediately after use or removed with cotton
wool damped with XYLENE or ETHER ( highly volatile ).
Eyepiece can be dismantled to remove dust and finger marks.
IF IN DOUBT LEAVE WELL ALONE
seek professional help than risking
damage to the valuable instrument.

 MICROMETRY:

The standard unit of measurement in microscopy is a

micrometre ( um ) which is 0.001 mm.
To measure the microscopic objects an eyepiece micrometer
scale is used in conjunction with a stage micrometer.
The micrometer scale is placed inside the eyepiece , the stage
micrometer consists of a 3x1 slide on which a mm scale
engraved in 1/10 and 1/100 graduations .
OBJECT IS MEASURED AS:
Insert the eyepiece and stage micrometers.
Select the objective to be used and 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 scale.
Remove the stage scale, focus on the object and determine
the number of eyepiece divisions exactly covered by the
object.

 CALCULATION OF THE SIZE OF THE OBJECT:

Assuming that 100 eyepiece divisions were equal to 10 small stage divisions
and object had covered 12 eyepiece division
100 stage divisions = 1mm = 1000 um
100 eyepiece division = 10 stage division
therefore 100 eyepiece division = 100 um
1 eyepiece division = 1um

Therefore 12 eyepiece division = 12 um

therefore the diameter of the object was 12 um.

DARK GROUND MICROSCOPE

For an object to be examined microscopically, it must be visible.

Visibility is dependent on contrast, the dark background increases the contrast.

Most objects examined microscopically are naturally transparent , but in general

they reflect or scatter the light rays, and as in dark-ground illumination , oblique
light is thrown upon them which do not enter the objective, they will appear as self
luminous objects on a dark back ground.

Dark ground illumination is a valuable technique for increasing the contrast of

unstained specimens.

Direct light is not allowed to take part in the formation of the image, this is done by
either blocking the zero-order light in back focal plane of the objective or more
usually by illuminating the specimen with a hollow cone of light of such obliquity
that all the light falls outside the acceptable angle of objective.

A central OPAQUE PATCH STOP of the correct size is fitted in the front focal plane of
the condenser , this ensures that all the light falling on the object is too oblique to enter the
objective.

Light diffracted by the specimen, emerges in a cone over 180 degrees , some of this will enter
the objective and form an image in which features scattering light will appear bright.

Patch stops are more effective with objectives of NA <0.65.

For high NA objectives, reflecting condensers are used instead , oiled to the slide.

Even with such condensers it may not be able to obtain an adequate darkgrounds when a
high-aperture, oil-immersion objective is used .This problem may be solved by restricting the
NA by a small iris diaphragm built into its back focal plane.

Darkgrounds reflecting condensers need care in their centration and focusing so that the
specimen is located exactly at the focal point of rays.

Objects examined by dark ground illumination give a misleading impression of size ; fine
particle appear to be much larger than they are , owing to their light scattering properties

This factor is of advantage when examining fine structures such as spirochetes

which are clearly visible by this method , yet when stained [Giemsas stain] are
difficult to see.
Preparations must be as thin as possible .
If single object is to be examined in mass of light reflecting material, the contrast
will be lost.
Extraneous refractile material such as air bubbles, red blood cells and oil droplets
should be avoided .

SETTING UP THE DARK GROUND MICROSCOPE

METHOD:Make a thin preparation , using a thoroughly clean thin slide and cover slip and
taking care not to have air bubbles
Place the lamp of microscope and focus the image of the source on the plane surface
of the mirror
Direct , or adjust , light through the condenser so that it is evenly distributed
Rack the condenser down , place a drop of emulsion oil on the top lens of condenser
and on the lower side of the slide ; place the slide on the stage and slowly rack up
the condenser until the two surfaces of the immersion oil meet without air bubbles
[air bubbles would reflect light in all directions]
Focus on object with LP objective such as 16mm
With the centering screws , adjust the condenser until a point of light is in center of
field usually adjusted according to small circles engraved on the upper surface
Place a drop of immersion oil on cover slip and focus the object with HP oilimmersion objective.
if an iris diaphragm is incorporated in the objective it is adjusted to give max
performance

After use,the oil should be carefully cleaned off both the condenser and objective

1.
2.
3.
4.

ERRORS OCCURING IN SETTING UP

The slides or coverslips are too thick
Preparations has to many air bubbles
Condenser is not properly focused or centered
Light is not sufficiently intense

PHASE
CONTRAST
MICROSCOPE

INTRODUCTION :Phase contrast microscopy is an optical microscopy

illumination technique in which small phase shifts in the light
passing through a transparent specimen are converted into
amplitude or contrast changes in the image
This type of microscope , which has been produced commercially
only since 1946, was devised by Zernicke , and converts slight
differences in refractive index into degrees of lights and shades.
This enables unstained objects to be observed in great detail , and
since such cell constituents like mitochondria , granules , nucleoli
and so on may be seen clearly, it represents a great advance in
microscopy.

Phase is only useful on specimens that are colorless and transparent and usually
difficult to distinguish from their surroundings. We call these specimens "phase
objects".
Examples of phase objects include cell parts in protozoans, bacteria, sperm tails
and other types of unstained cells. This phase contrast technique proved to be such
an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in
1953

Image above with regular bright

field objectives.
Notice the air bubbles at three
locations, some cells are visible at
the left side

Same image with phase contrast

objectives.
White dots inside each cell are the
nuclei.

OPTICAL PRINCIPALS :If a diffraction grating is examined under the microscope ,

diffraction spectra are formed in the back focal plane (BFP) of the
objective due to interference between direct and diffracted light rays.
The grating consists of the alternate strips of material with slight
different RI s , through light acquires small phase difference and these
form the image.
Unstained cells are similar to diffraction grating as their content also
differs slightly in RI s.

Two rays of light from the same source having the same frequency , are said to be
COHORENT , and when recombined will double in amplitude or brightness.
If they are in phase with each other
INTERFERENCE

it is called CONSTRUCTIVE

If however they are out of the phase with each other , DESTRUCTIVE
INTERFERENCE takes place .
The following figure represents the wave form of a light ray.

TECHNICAL DETAILS :To achieve phase contrast the microscope requires modified objective and
condenser and relies on the specimen retarding the light by between 1/8 th and
1/4th lambda.
An intense light source is required to be set up this microscope.
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.
Each objective requires different size of annulus , an image of which is formed by
the condenser in the BFP of the objective as a bright ring of light.

The objective is modified by a phase

plate which is placed at the BFP .
A positive phase plate consists of a
clear glass disk with circular through
etched into it, to the half the depth of
the disc.
The light passing through the trough
has a phase difference of 1/4th lambda
compared to the rest of the plate.
The trough also contains a neutraldensity light absorbing material to
reduce the brightness of the direct
rays,which would otherwise obscure
the contrast obtained.

It is essential that the image of the bright annular

ring from the condenser is centered and
superimposed on the dull trough of the objective
phase plate .
This is achieved by using either a focusing
telescope in place of the eyepiece or a Bertrand
lens situated in the body tube of the microscope.
Each combination of annulus and objective phase
plate will require centeration. when hollow cone of
the direct light from the annulus centers the
specimen , some will pass through unaltered while
some will be retarded or diffracted by
approximately 1/4th lambda

The direct light will mostly pass through the trough in

the phase plate while the diffracted light pass through
the thicker clear glass and further retarded.
The total retardation of the diffracted rays are now
1/2th lambda and interference will occur when they
are recombined with the direct rays.
Thus the image of contrast is achieved revealing the
small details with in the cell.

This is quick and efficient way of examining the

unstained paraffin, resin and frozen sections, as
well as studying living cells and their behavior.

Setting up your microscope for phase contrast

observations

Shown at left is the phase contrast kit

used on the National Optical 160
series microscopes.
It consists of 4 objective lenses, a
centering telescope and Zernike phase
condenser lens. The long adjustment
screws on the phase condenser push in
to engage set screws for proper
alignment of the phase ring.
There is a thumb wheel on the
opposite side used to dial in the
proper setting to match the power of
the objective lens.

Not all phase contrast

microscopes are the same but
generally they rely on similar
techniques to set up the
system for optimum results.
In the system shown at the
left, the phase condenser has
five settings that we spin with
our thumb ( 10X, 20X, 40X,
100X and BF) BF is "bright
field", no phase.

Next, we remove one of the

eyepiece lenses and insert the
centering telescope in its place. The
set screw is used to focus the
centering telescope. When looking
through the telescope, we will see
two rings. They may or may not be
concentric. By turning centering
adjustment screws on the
condenser, we align the rings so that
they become concentric.

Above, what we might see before

alignment of the phase condenser

Once aligned and optimized for

phase contrast microscopy

We then remove the centering telescope and replace the

eyepiece lens. Specimen is put back on the stage and its ready
for phase observations. When we change objectives, we should
go through this procedure again (although we may discover that
the alignment remains consistent with all objectives).

THE POLARIZING
MICROSCOPE

PROPERTIES :THE TWO MAIN PROPERTIES

USED IN POLARISING
MICROSCOPE :1. POLARIZATION :- Produced

by the polarizing crystals like

nicols prism .These devises
are placed in the substage.
2. BIREFRINGENCE :-

produced by birefringent
object. these devices are
placed in the stage.

WHAT IS POLARIZED LIGHT?

Transverse wave light whose vibration possess direction is called
polarized light.
Light from an ordinary light source (natural light)
that vibrates in random directions is called Non polarized light

NATURAL LIGHT

POLARISATION OF LIGHT
A light beam normally contains waves of

many different frequencies with all

possible phase relationships, and vibrating
in all possible planes .If the vibration of
the light waves are restricted to one single
plane, then the light is called plane
polarized.

HOW TO PRODUCE POLARIZED LIGHT ?

By using device like NICOLS PRISM polarized light can be obtained.
NICOLS PRISM:

Nicol devised a prism from which light rays, having passed through ,
would emerge vibrating in a single plane , that is , as a POLARIZED
LIGHT.
The single direction in which the light is vibrating when it emerges is
known as OPTICAL PATH of the prism.
HOW IS THE PRISM MADE ?

The prism is composed of a crystal of iceland spar , cut to a particular

shape , slit in half and the halves cemented together with canada balsam .

HOW IS POLARIZATION BOUGHT ABOUT IN

THIS CRYSTAL ?
On entering the prism , the light ray (A) is divided into two rays (B and C )
which are refracted differently , ray C being refracted to one side .owing
the difference in the refractive index between the canada balsam and
calcite spar crystals and the cement , ray C on meeting the surface CB-CB
at a greater angle than ray B , is totally reflected out of the prism .
Ray B passes through the prism and emerges vibrating in the direction of
the optical path of the prism only and is POLARIZED LIGHT.

2) BIREFRINGENCE :If a dot is drawn on the sheet of the white card is viewed through a block of glass
laid on top , only one dot will be visible from above.
If the block of glass is replaced by a polished block of crystal ,two dot will be
visible .such a crystal is described as being BI-REFRINGENT or ANISOTROPHIC.
IT has split light ray from the dot into two rays which emerge from the crystal at
different points.

THIS SPLITTING OF LIGHT RAYS BY CERTAIN MATERIALS IS

DUE TO THEIR UNEVEN OPTICAL DENSITY.
Light when entering a dense medium is retarded in speed .
Further , being unevenly dense medium ,the crystal will retard the two
rays to different degrees ,and since refraction is partly depended on density
,the two rays will be refracted or bent to different degrees.
This is known as DOUBLE REFRACTION or BIREFRINGENCE.
A ray of light entering such a crystal will be converted into two rays
which will emerge at different points , and the emergent light will be
POLARIZED; that is all the vibrations in one ray will be in one single
direction .
Examples of birefringent materials found in histologic sections are :- Talc
crystals , hair, myelin, silica, and collagen fibers

TYPES OF BIREFRINGENCE :Certain crystals or tissue structure show more than one index of
refraction for a given wavelength of light and are therefore said to be
doubly refracting or birefringent.
This is due to some sort of asymmetrically oriented spatial
arrangement of particles .
These particles carry resonating chargers capable of interacting with
the oscillations of light wave .
Birefringent material may show one or more than one type of such
arrangement , the more common types may be summarized as
follows :1.Intrinsic or crystalline birefringence.
2.Form birefrigence.
3.Strain birefringence.
4.Positive and negative birefringence.

INTRINSIC OR CRYSTALLINE BIREFRINGENCE :This refers to type of anisotropy due to an asymmetric

alignment of chemical bonds , ions or molecules .
Many crystal display this type of birefringence , it is
common in biological object such as collagen and muscle
fibers and chromosomes.
Intrinsic birefringence in a specimen is independent of the
refractive index of the immersion medium which is probably
due to the fact that the orientated elements are of close
structure between which the medium does not penetrate.

STRAIN BIREFRINGENCE :When a substance is subjected to mechanical stress , the bonds

with in the substance can be distorted and gives rise to a pattern
which will result in birefringence .
This is more simple demonstrated by twisting the plastic
(perspex) between the crossed polariods when birefringent
spectrum of colors is produced.
Similarly glass or elastic tissue fiber under stress shows
birefringence.

POSITIVE AND NEGATIVE BIREFRINGENCE:In a polarizer , slow and fast rays are separated by
birefringence material .
If the slow ray (high R.I ) is parallel to the length of the
crystal or fiber, the birefringence is positive.
If slow ray is perpendicular to the long axis of the structure ,
the birefringence is negative.
In a collagen fiber the slow axis of transmission is parallel to
the long axis of the fiber , the fiber is thus said to show
positive birefringence with respect to its long axis .
A chromosome shows negative birefringence with respect to
its long axis.

POLARIZING MICROSCOPE:Has polarizer , is placed beneath the substage

condenser and is held in a rotatable graduated
mount ,and can be removed from the light path
when not required.

The other is called analyzer , is to be placed

between the objective and the eyepiece and is also
graduated for measurement to be taken.

A circular rotating stage would also be present for

rotation of the specimen.

THE USE OF POLARIZING MICROSCOPE :It has been shown that certain crystal have power to convert a
single ray into two rays of light , which are vibrating in a single
plane at right angles to each other .
If such a crystal is placed on the stage of the microscope having a
nicol prism in the substage , the effect will be seen as below.

The light ray will be vibrating in the optical path of the nicol prism
(AB) ,except those that pass through the crystal ,which will be
vibrating from C to D or E to F.

If set of the rays CD were absorbed , the

crystals would have the effect of changing
the plane of vibration from AB to EF
.therefore ,a nicol prism placed above the
object ,having its optical path from A to B ,
will absorb most of the rays which have
passed the crystal , and it will appear DARK
ON LIGHT BACKGROUND.
If the upper nicol prism has its optical path
in the direction EF , then the rays AB will
be absorbed and the crystal will appear
LIGHT ON A DARK BACKGROUND.
This later method is the one normally used
in the laboratories

USES OF POLARIZING MICROSCOPES

A polarizing microscope is a special microscope that uses polarized light
for investigating the optical properties of specimens. Although originally
called a mineral microscope because of its applications in mineralogical
research.
Polarized light microscopy is perhaps best known for its geological
applications--primarily for the study of minerals in rock thin sections, but it
can also be used to study many other materials.
These include both natural and industrial minerals whether refined,
extracted or manufactured, composites such as cements, ceramics, mineral
fibers and polymers, and crystalline or highly ordered biological molecules
such as DNA, starch, wood and urea.
The technique can be used both qualitatively and quantitatively and is an
outstanding tool for materials science, geology, chemistry, biology,
metallurgy and even medicine.

PARTS OF POLARIZING MICROSCOPE :Compared to a typical microscope, a polarizing microscope has a new
construction with the following added units:
1.A polarizing condenser that includes a polarizer,
2.A rotating stage that allows the positioning of the specimen .
3.A strain-free objective for polarized light,
4.a centerable revolving nosepiece ,
5.An analyzer,
6.A Bertrand lens for observing the pupil of the objective,
7.A test plate,
8.A compensator, and
9.A eyepiece with crosshair.

Polarizer and analyzer

The polarizer should be placed below the condenser
and the analyzer should be above the
objective.
The polarizer is rotatable 360 with degree
gradations
indicated on the frame. The analyzer can
also rotate 90 or 360, and the angle of rotation
can be figured out from gradations as well. As, the
vibration direction of a polarizer
should go side to side relatively to the observant,
and go vertically for an analyzer.

Polarizing objective (strain-free objective)

A polarizing objective differs from ordinary
objectives
in a respect that it possesses a high light polarizing
capability.

Polarizing condenser
A polarizing condenser has the following two
characteristics:
1)Built-in rotatable polarizer,
2) Strainfree optical system, like the objectives.

Polarizing rotating stage

Rotating an anisotropy between
crossed nicols changes the brightness. For
this reason, in polarized light observation,
the specimen is often rotated to the
diagonal position (the position where the
anisotropy is brightest).

Bertrand lens
A Bertrand lens projects an interference image
of the specimen, formed in the objective pupil,
onto the objective image position (back focal
length). It is located between the analyzer and
eyepiece for easy in and out of the light path.

Centerable revolving nosepiece

Eyepiece with crosshair

This is an eyepiece which has a built-in focusing plate
containing a crosshair. By inserting the point pin
into the observation tube sleeve, the vibration
direction of the polarizer and analyzer can be
made to agree with the crosshair in the visual
field.

Test plate and compensator

The test plate is a phase plate used for verifying
the double refractivity of specimens, determining
the vibration direction of pieces, and for retardation
measurement; The compensator is a phase plate that can
change and measure the retardation.

THE
INTERFERENCE
MICROSCOPE

The interference microscope incorporates certain principles of both phase

contrast and polarising microscope , and enables one to see unstained objects in
great detail .
In phase contrast microscope specimen retards some light rays with respect to
those which pass through the surrounding medium.
The resulting interference of these rays provides image contrast but with an
artefact called PHASE HALO.
In the interference microscope it does not rely on the diffraction by the object for
interference , but generates mutually interfering beams which produce the
contrast .It is this feature which enables such small phase changes to be seen and
measured allowing improved contrast , color graduation and quantitative
measurement of the phase changed ( or optical path difference) , refractive
index, dry mass of the cells (optical weighing ) and section thickness. And the
retarded rays are entirely separated from the direct rays or reference rays.

The two rays which eventually combine to produce a final image are formed by a
plate of birefringent material at the face of objective, each point in the final image
is a compound made up of two superimposed mutually different views of the same
point of the object .
By using a polarized light and an analyser the phase relationship between two
rays can be adjusted and measured.

Whenever light passes across the edge of an opaque object the rays close to the edge
are diffracted , or bent away from the normal path.

If instead of a single edge, the rays pass through a narrow slit , then the rays the
edge of the beam will fan out on either side to quit wide angles.

Two slits closely side by side form two fan of rays which will cross and if cohorent
,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

dark and bright across the field of view .
With white light , bands of spectral colors are seen,because wavelengths
making white light are diffracted at different angles .
With monochromatic light the bands are alternately dark and bright , and of
single color .
The same effect can be seen if separate beams of cohorent rays are reunited.
This phenomenon is called INTERFERENCE.

Types of interference
microscopes
There are two types currently used in great Britan :1) The BAKERS interference microscope
2) DYSON interference microscope.

Early microscope models spilt 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 reference beam.
The beams were widely separated and
suitable only for large specimens and
interference fringes measurement.
Later models used a double beam system ,
where the separation is produced by
birefringent materials and is close enough to
require only one objective.

If the two paths are equal and in 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 of the interference bands .

When using monochromatic light , each interval consisting of one dark and one
light band is one wavelength wide, and thus distance in nanometer is known.
Displacement of the bands is measured in micrometer eyepiece and this
information , coupled either with R.I or object thickness , the measurement
referred to earlier can be determined.
Two types of double beam system have been used .
1) Focusing the reference below the object the double focus system
2) Involved a lateral displacement of the reference beam called SHEARING
where the separation of the beam is very small .

USES :As an infinitely variable phase contrast microscope with which individual parts
of the cells may be studied with great detail; for this purpose little is to be
gained over a good phase contrast microscope.
As a highly accurate optical balance , it may be used for estimation of dry mass
down to 110-14g.
The discovery that an increase in R.I of 0.0018 is a result of 1 percent increase
in the solid substances contained in the cell , and the fact that the refractive
index of the cell components can be estimated from the phase differences
between them and the reference area (usually the fluid in which they are
suspended ) has made this possible. It is this aspect of interference microscope
that is at present being developed by many research workers.

STEREOMICROSCOPE

It was first designed by GREENOUGH in 1897.

It is also called as BINOCULAR DISSECTING MICROSCOPE.

When one looks at any object , the image recorded by the optical system , in
conjunction with the co-ordinating facility of the brain, has the THREE
DIMENSIONAL EFFECT.

This is the basic principle of the stereomicroscope.

It is designed specially for LOW POWER works due to long working distance.

TYPES OF STEREOMICROSCOPE

GREENOUGH TYPE STEREOMICROSCOPE designed by

GREENOUGH and introduced by CARL ZEISS in 1897.

COMMON- MAIN- OBJECTIVE MICROSCOPE originated by CARL

ZEISS in 1946.

PARTS AND PRINCIPLE OF STEREOMICROSCOPE

GREENOUGH TYPE:
It is essentially two separate compound microscope tubes with matched
objectives and eyepieces mounted side by side at an angle.
In this type of microscope , each eye is provided with its own complete
microscope.
The paired objectives are mounted together.
The approximate angle of binocular vision is 15 degrees.
Each tube contains an erecting prism above the objective to give the 3-D effect.
The rays from the objective pass at an angle through a pair of prisms, which
produces an image that is not inverted and viewed at an angle.
The prisms are similar to that used in field binoculars.
Eyepiece separation is normally achieved by slight rotation of the prism boxes.

IMAGE FORMATION

 Magnification may be altered ranging from 3X to 100X by

exchanging pairs of objectives and eyepieces, or by incorporating a
magnification changer.
Magnification changers are nothing but a series of interchangable
pairs of lenses inserted into the light path.
Alternatively , movable lenses may be incorporated in the light paths
to provide a ZOOM system.
The objectives may be incorporated into a sliding nosepiece.
All these modifications have provided for a great depth of focus , long
working distance , large flat fields , and simplicity of operation.
These instruments are often built with an ability to interchange
between various bases and arms to allow them to be carried over quite
large specimens to work on a particular part.

DISADVANTAGE
Because of the inclination of the tubes , there is a
tendency for the image to be slightly out of focus
towards the edge on each side of the centre line of the
field of view.

COMMON-MAIN-OBJECTIVE TYPE: The most widely used nowadays.

It has a single large-diameter objective with two further separate lens
systems mounted behind it , each carrying an erecting prism below the
eyepieces which are mounted in parallel tubes.
They have their primary image plane parallel to the plane of the object, and
the images are simultaneously in focus across a wider field of view when
compared to greenough type where image is viewed at an angle.
Most of the light forming the image passes at oblique angles through the
periphery of the objective and becomes parallel to each other and these
parallel rays are deviated into two different optical paths by using four
prisms.
The image is upright and laterally correct.
Their numerical apertures are commonly lower than might be supposed, as a
double beam path has to be accommodated within the objective.
Many modern instruments have a zoom magnification changer built in, and
are extremely easy and convenient in use.

IMAGE FORMATION

SUPPORTING THE INSTRUMENT

Stereomicroscopes intended for transmitted light use are mounted on a

base which has an integral low wattage bulb and a ground glass or
clear plate glass insert to support the slide. But the intensity of light is
uneven.
There are bases which can provide dark ground illumination as well.
Stereomicroscopes intended for reflected work are used with stands
ranging from ordinary clamp stands to expensive high-precision
roller-bearing stands.
There are also motor driven and microprocessor controlled stands
supporting a surgical operating microscope.

LIGHTING THE SPECIMEN

For transmitted light photomicrography integral low wattage bulbs are used.
For transmitted light direct visual observation a large source like an OPAL
MAINS LAMP , below a pair of photographic enlarger condensers are sufficient.
For reflected light works ordinary FOCUSABLE TUNGSTEN LAMPS are
sufficient.
The lamps are adjusted at varied angles and distances so as not to impede access to
the specimen.
FIBRE-OPTIC LAMPS are most versatile for lowest magnifications it is easy to
adjust and conveys little heat to specimens.
Filters of different colors can be used with fibre-optic lamps to get a 3-D detail.
Magnification is 5- 50 times as compared to compound microscopes with 50- 1200
times magnification.

USES:

Generally used for examination of objects by reflected light.

Stereomicroscopes with transmitted light base are used for examination of slides
and transparent objects. If base is not fitted with integral lamps object can be
supported over a light box for transmitted light viewing.
Very useful for initial scanning of slides at low magnifications.
Microdissection
For viewing of solid objects gemology, coins.
Industrial purpose.
In dentistry -- for bond strength.
Working distance is much longer than with typical compound microscope allowing
work to be done on the specimen dissecting purpose.

MACROSCOPE

Superficially similar to stereomicroscopes.

Designed principally for photomacrography.
They have only a single imaging system, with an objective of large diameter, often
apochromatic.
Objective aperture is higher than the stereomicroscopes.
Macroscopes are usually fitted with a binocular head and a camera port for
providing an image display.
Macroscopes {LEICA} are specially intended for low power comparison of two
very similar objects, for example in forensic studies, and so has two matched lens
systems linked by a comparison bridge to one binocular eyepiece which presents a
split field to the observer.

It has a large stage and long working distance for the objectives.
It has a zoom lens sometimes fitted, to allow a magnification of range 4X to 80X
{MAKROZOOM LEITZ LENS}
A macroscope should allow the use of both oblique and coaxial incident light and
the base should have an aperture to allow transmitted light to be used.

Fluorescence Microscopy

INTRODUCTION
A fluorescence microscope (epifluorescent
microscope) is a light microscope used to
study properties of organic or inorganic
substances using the phenomena of
fluorescence and phosphorescence instead
of, or in addition to, reflection and
absorption.

 Fluorescence is characterized by the ability

to convert energy of shorter wavelength that
is invisible into a light of longer wavelength
which is visible.
If this light is emitted only during the time of
exposure or for a short time afterwards
(about 9-10 seconds), it is called
FLUORESCENCE.

 If the emission persists after the exciting light is

cut off, it is called PHOSPHORESCENCE.
In fluorescence microscopy, the exciting radiation
is usually in the ultraviolet wavelength (360 nm)
or blue region (400 nm), although longer
wavelength can be used.

Primary or Autofluorescence
It is the ability of some naturally occuring
compounds to fluoresce.
A substance which possesses a fluorophore
will fluoresce naturally. This is known as
primary fluorescence or autofluorescence.
Ultraviolet excitation is required for
optimum results with substances such as
vitamin A, porphyrins and chlorophyll.

Secondary Fluorescence
Demonstration of tissue components stained by
fluorescent dyes.
Dyes, chemicals and antibiotics added to tissues
produce secondary fluorescence, of structures and
are called fluorochromes.
This is the most common use of fluorescence
microscopy and the majority of fluorochromes
require only blue light excitation.

Induced Fluorescence:
Is the term applied to substances such as
catecholamines, which after treatment with
formaldehyde vapor are converted to
fluorescent quinoline compounds.
Pretreatment of tissues causes a reaction
with component under investigation,
producing a reaction which is fluorescent.

Principle of Fluorescence
When a quantum of
light is absorbed by
an atom or
molecule, an
electron is boosted
to a higher energy
level.
When this displaced
electron returns to
its original ground
state, it may emit a
quantum of light.

Principle of Fluorescence
1.

2.
3.

Energy is absorbed by
the atom which becomes
excited.
The electron jumps to a
higher energy level.
Soon, the electron drops
back to the ground state,
emitting a photon (or a
packet of light) - the
atom is fluorescing.

If this light is emitted only during the time of

exposure, or for a very short time afterwards, it is
called FLUORESCENCE.
If this emission persists after the exciting light is
cut off, it is called PHOSPHORESCENCE.
Fluorescence is the property of some substances
which, when illuminated by a light of shorter
wavelength will remit the light at a longer
wavelength.

 Since a certain amount of energy is lost as heat

before the electron returns to its ground state, the
fluorescent or phosphorescent light is at a longer
wavelength (lower energy) then the original
exciting light.

In fluorescence microscopy ultraviolet light (which

is not visible to the human eye) is used as the
exciting light with the resulting fluorescence (of
longer wavelength) being in the visible range.

 Thus an object is
illuminated with a
black light and,
when fluorescent,
appears as a bright
object against a
dark background.

 It should be remembered that, while an enormous

number of compounds are fluorescent to some
degree, only relatively few give sufficiently
brilliant fluorescence that they may be detected in
small quantities by their autofluorescence, or used
as fluorescent dyes.
Some compounds and dyes, while brilliantly
fluorescent as pure compounds, may lose their
power to fluoresce when bound to other structures.
This is known as QUENCHING of fluorescence.

COMPONENTS
Illuminating system
Filter system
Exciter filter
Contrast or barrier filter
Dichromatic filter attachment
Condenser
Objective

TYPES
TRANSMITTED LIGHT
FLUORESCENCE.
INCIDENT LIGHT FLUORESCENCE.

DIFFERENCES
TRANSMITTED
There is no use of
dichromatic mirror.

INCIDENT
There is use of
dichromatic mirror.

It was being used

earlier.

Most commonly used

these days.

The fluorescence is
produced towards the
light source, and
viewed on the opposite
side.

Fluorescence is
produced on the
observers side, and
therefore more brilliant.

DIFFERENCES
TRANSMITTED

INCIDENT

Two different lenses

are used for, the
objective and
condenser.

The objective in this also

acts as the condenser,
therefore illumination and
objective numerical
apertures are one and the
same, and so optically
correct, and at their most
efficient condition.

Transmitted light fluorescent

microscope.

Incident light fluorescent microscope

APPLICATIONS:
INDUCED FLUORESCENCE: pretreatment of
tissues causes a reaction with component under
investigation, producing a reaction which is
fluorescent.
Eg: 5-hydroxytryptamine fluorescence induced by
formalin fixed tissue.
SECONDARY FLUORESCENCE: demonstration
of tissue components stained by fluorescent dyes.

CONFOCAL
MICROSCOPY

 The principle of confocal imaging was

patented by Marvin Minsky in 1957.
Confocal microscopy is an optical imaging
technique used to increase micrograph contrast
and/or to reconstruct three-dimensional images
by using a spatial pinhole to eliminate out-offocus light in specimens that are thicker than
the focal plane.

 Confocal microscopy aims to overcome some limitations

of traditional wide-field fluorescence microscopes.
In a conventional (i.e., wide-field)
fluorescence microscope, the entire specimen is flooded in
light from a light source.
All parts of the specimen in the optical path are excited and
the resulting fluorescence is detected by the microscope
photodetector or camera as background signal.
In contrast, a confocal microscope uses point illumination
and a pinhole in an optically conjugate plane in front of the
detector to eliminate out-of-focus information - the name
"confocal" stems from this configuration.

PRINCIPLE OF CONFOCAL
MICROSCOPY

TYPES
Three types of confocal microscopes are
commercially available:
Confocal laser scanning microscopes
Spinning-disk (Nipkow disk) confocal
microscopes
Programmable Array Microscopes (PAM)

DISADVANTAGE
However as much of the light from sample
fluorescence is blocked at the pinhole this
increased resolution is at the cost of
decreased signal intensity so long
exposures are often required.

USES
This technique has gained popularity in the
scientific and industrial communities.
Its typical applications are in life sciences
and semiconductor inspection.

ELECTRON MICROSCOPE

INTRODUCTION
The EM has gained its fundamental superiority
over light microscope because of its high
resolving power, and thereby producing extreme
fine details.
In light microscope the highest resolution
possible theoretically is half the wavelength of
light. (thus the limit of resolution of light
microscope is 0.2 microns = 2000A0)

 With the usage of ultraviolet rays, this can be

improved to 0.1 microns = 1000A0).
But intracellular components, certain bacteria and
most of the viruses are smaller than this and cannot
be visualized.
So attempts were made to use other types of
radiation.
This lead to the new type of an instrument, which
has been designed for much higher resolving power,
than the light microscope ELECTRON
MICROSCOPE.
By using an electron beam instead of light rays, the
EM gives much better resolution.

HISTORY
o The first electron microscope prototype was built
in 1931 by Max knoll and Ernst Ruska.
o This initial instrument was capable of magnifying
objects by only four hundred times, although it
demonstrated the principles of an electron
microscope.
o Two years later, Ruska constructed an electron
microscope that exceeded the resolution possible
with an optical microscope.

 Siemens produced the first commercial

Transmission Electron Microscope (TEM) in 1939,
but the first practical electron microscope had been
built at the University of Toronto in 1938, by
Eli Franklin Burton and students Cecil Hall,
James Hillier, and Albert Prebus.
In the same decade Manfred von Ardenne pioneered
the scanning electron microscope.
Although modern electron microscopes can magnify
objects up to two million times, they are still based
upon Ruska's prototype

PRINCIPLE
An electron microscope is a type of
microscope that uses a particle beam of
electrons to illuminate a specimen and
create a highly-magnified image.
The electron microscope is constructed
based on the same optical principle as
the light microscope.
Convergence of a light beam by a
convex glass lens has its counterpart
in the convergence of an electron beam
as it passes through the core of a
circular magnetic field.

 Beams of electrons are

used to produce images.
Wavelength of electron
beam is much shorter
than light, thus resulting
in much higher
resolution.
The physical basis for this
benefit lies in the
formula:
R = 0.61 lambda
NA

R = 0.61 lambda
NA
Where: R, the RESOLUTION = represents the capacity
of the optical system to produce separate images of
objects very close together.
Lambda is the wavelength of the incident illumination.
NA is the numerical aperture of the lens.
Thus for any given lens, resolution is directly related to
the wavelength of the source radiation.

The optical theory states that, the resolution of any

optical system is limited by the wavelength of light
employed.
Both electron and light microscopes have resolution
limitations, imposed by the wavelength of the
radiation they use.
Therefore by using electron beam instead of light
rays, EM gives much better resolution.

 The wavelength of moving electrons depends on

their velocity.
At an acceleration of 50,000 volts they have
wavelength of 0.01 A0. It is this extremely short
wavelength that gives the EM its fundamental
superiority over light microscope. (therefore
resolves images of this order).
But due to lens defects which can be corrected in
the light microscope, but have not been corrected in
EM, the resolution is limited to 4 A0.

 Electron microscopes have much greater

resolving power than light microscopes , and can
obtain much higher magnifications of up to 2
million times, while the best light microscopes are
limited to magnifications of 2000 times.

Parts of an EM
The electron beam is obtained from
a heated tungsten filament(cathode)
which is surrounded by a metal
cylinder known as the whenalt cap.
This cap serves to shape the
electron beam.
Just beyond the whenalt cap is the
anode which has an aperture
through which the electron beam
passes.
A large voltage is passed between
the cathode and anode, which gives
the electron their high velocity.
They pass through the rest of the
microscope without any
acceleration.

 ELECTRON GUN:
the device responsible
for generating the
beam of electron is
the electron gun.
The most important
components of the
gun are tungsten
filament, wehnelt
sheild and anode.

1. FILAMENT: electrons are generated by thermionic

emission from a v shaped length of tungsten wire.
however, significant improvement in electron yield
occurs if lanthanum hexaboride filaments are used.
2. WHENELT SHEILD: The filament is covered by
an apertured electrode known as whenelt sheild.
The whenelt cap is given a voltage slightly lower
than the filament, and this voltage is usually
variable so that the flow of electrons from the
cathode can be controlled. This is known as the
BIAS voltage.

 The like charge 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, forcing the electrons into a circle (or
spot) of decreasing size but increasing brilliance.
Optimum 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.

3. ANODE: the anode, an apertured disk is

placed a short distance from the shield.
As the anode is kept at zero potential,
electrons are attracted away from 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 known as the ACCELERATING
VOLTAGE.
In most microscopes this can be varied in
stages from 20 kv to 120 kv during
operation.

 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 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 decrease in electron scatter as the beam passes
through the specimen, giving lower contrast in the final image.
Therefore, the general view is that the microscope should be
operated at highest accelerating voltage that allows structures in the
specimen to be clearly discriminated.
For most situations this will be 80 120 kv.

4. ELECTRON LENS:
The lenses used to refract electrons are electromagnetic
coils or solenoids.
When energized these generate a magnetic field that
forces electrons into a spiral trajectory and towards a focal
point on the opposite side of the lens.
All lenses of TEM operate in the same way the
focussing effect of each lens can be changed simply by
varying the power and hence the strength of the magnetic
field.
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 focussed 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 first place.
Apertures made from molybdenum or platinum need to be cleaned
regularly unlike those made from gold foil which are self cleansing.
CHROMATIC ABERRATION: is due to electrons of different speed
(and thus wavelength) being focused at different planes.
It is primarily a function of electrons losing speed as they pass
through the section itself and it gives rise to blurred image.
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 also reflects flaws in lens construction.
It is also exacerbated by contamination of 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.

 CONDENSER:
The electron beam first passes through the condenser
lens as in light microscope.
This lens serves to focus the beam on the object, and so
provide illumination.
The magnetic lenses of an EM can have different
powers, depending on the amount of current flowing in
the electric coils. (In light microscope, the lens power is
fixed, but the lens can be moved so that the image can be
focused.
In EM all lenses are fixed rigidly but their focal points
are variable by adjusting the lens current.
Thus the illumination of the object is achieved by
varying the current to the condenser lens.

 IMAGING SYSTEM:
The imaging system consists of three
lenses;
* The objective
* the intermediate
* the projector lenses
This gives three stages of
magnification and makes it possible
to achieve high magnification in a
reasonable amount of space.
The objective lens is placed with its
focal point close to the object.
Intermediate images are formed
between each lens.
The projector lens throws its image
on to a fluorescent screen, which may
be substituted by a photographic plate
to make a permanent record

 The entire illuminating and imaging system is usually referred to

as MICROSCOPE COLUMN, and its constructed upside down
compared to the light microscope; that is the electron gun and
the condenser lens are placed above and the image is formed
below.
The column is very rigidly constructed and is maintained in high
vacuum, since air molecules will deflect the electron beam.
Because the specimen has to be placed inside the vacuum, it is
not possible to examine living material in EM.
Electron optics are essentially similar to light optics, one
important difference is that the formation of an image is due to
the scattering of electrons by molecules of the specimen and this
scattering depends solely on mass densities.

 Elements of high atomic weight such as the lead or uranium cause

marked electron scatter and appear very dense in the electron image.
The lighter elements such as carbon, oxygen or nitrogen cause little
electron scatter and have poor contrast
In light microscope the image is due to absorption of light which
depends more on molecular structure than atomic weights.
Histological stains depend on absorption of certain wavelengths of
light due to their molecular structure and are composed mainly of
carbon, hydrogen and nitrogen atoms.
Since these are all of low atomic weight they have little electron
scattering power and are not generally useful as stains for EM.
Unstained tissues have very poor contrast in EM, but may be stained
by a variety of heavy metal salts.
Most such electron stains are relatively non specific.

Types of EM

Transmission electron microscope

Scanning electron microscope
Reflecting electron microscope
Scanning transmission electron microscope.

Transmission EM
The original form of electron
microscope, the
transmission electron microscope (TEM)
uses a high voltage electron beam to
create an image.
When the electron beam emerges from
the specimen, the electron beam
carries information about the structure
of the specimen that is magnified by
the objective lens system of the

 The spatial variation in this information (the

"image") is viewed by projecting the magnified
electron image onto a fluorescent viewing
screen coated with a phosphor or scintillator
material such as zinc sulfide.
The image can be photographically recorded by
exposing a photographic film or plate directly
to the electron beam, or a high-resolution
phosphor may be coupled by means of a lens
optical system or a fibre optic light-guide to the
sensor of a CCD (charge-coupled device)
camera. The image detected by the CCD may
be displayed on a monitor or computer.

 In the TEM, the

focused,
monochromatic
electron beam
interacts with and is
transmitted through
the sample, focused
into an image and
projected onto a
phosphor coated
screen which emits
visible light.

 The brighter areas of the image

represent areas where more electrons
have passed through the sample.
The darker areas represent areas where
fewer electrons have passed through as
a result of higher specimen density.
A TEM can magnify up to about
500,000x.

Scanning EM
Unlike the TEM, where electrons of the high
voltage beam carry the image of the specimen,
the electron beam of the
Scanning Electron Microscope does not at any
time carry a complete image of the specimen.
The SEM produces images by probing the
specimen with a focused electron beam that is
scanned across a rectangular area of the specimen
(raster scanning).

 At each point on the specimen the incident electron

beam loses some energy, and that lost energy is
converted into other forms, such as heat, emission of
low-energy secondary electrons, light emission (
cathodoluminescence) or x-ray emission.
The secondary electrons reflected from the surface
of the specimen are collected by a secondary
electron detector, passed through a photomultiplier
tube and onto a cathode ray tube where image is
displayed.

 Back scattered electrons are also reflected from the

surface of the specimen, these are electrons with less
energy loss than secondary electrons.
Biological specimens are largely non conductive, so
it is necessary to impart conductivity.
Gold, platinum, and carbon are some of the
substances used for this purpose.
It is used in research and development, quality
control and failure analysis.
Range:- 20,000 30,000 times.
Voltage employed is:- 5 40 kv.

 The condenser lens is used to produce a narrow

pencil like beam of electrons, this passes through a
scanning coil that moves it back and forth over the
surface of the specimen in a rapid scanning motion
corresponding to the scanning pattern on a
television screen.
At each place that the scanning beam strikes the
specimen, secondary electrons are emitted from its
surface coating.

 In the SEM, a set of scan

coils moves the electron
beam across the specimen
in a 2 dimensional grid
fashion.
When the electron beam
scans across the
specimens, different
interactions take place.
These interactions are
decoded with various
detectors situated in the
chamber above the
specimen.

DIFFERENCES BETWEEN TEM AND SEM

TEM
Electron beam passes
through thin sample
Specially prepared thin
samples or particulate
material are supported on
TEM grids.

SEM
Electron beam scans over
surface of sample.
Sample can be of any
thickness and is mounted
on an aluminum stub.

Specimen stage halfway

down column.

Specimen stage in the

chamber at the bottom of
the column.
Image shown on TV
monitor.
Image is the surface of the
sample.

Image shown on
fluorescent screen.
Image is a two
dimensional projection of
the sample.

RESOLUTION OF MICROSCOPES
MICROSCOPE

RESOLUTION

MAGNIFICATIO
N
1000X

OPTICAL

200nm

TEM

0.2nm

500 000X

SEM

2nm

200 000X

REFLECTING EM
In the Reflection Electron Microscope (REM) as in
the TEM, an electron beam is incident on a surface,
but instead of using the transmission (TEM) or
secondary electrons (SEM), the reflected beam of
elastically scattered electrons is detected.

Scanning transmission EM
o The STEM rasters a focused incident probe across a specimen
that (as with the TEM) has been thinned to facilitate detection of
electrons scattered through the specimen.
o The high resolution of the TEM is thus possible in STEM. The
focusing action (and aberrations) occur before the electrons hit
the specimen in the STEM, but afterward in the TEM.
o The STEM's use of SEM-like beam rastering simplifies
annular dark-field imaging, and other analytical techniques, but
also means that image data is acquired in serial rather than in
parallel fashion.

USES
o Transmission electron microscopy has been widely used in routine
histopathology in recent years particularly in the field of renal
disease, tumor pathology and viral infections.
o The electron microscope is an essential item of equipment in many
laboratories.
o Researchers use them to examine biological materials (such as
microorganisms and cells), a variety of large molecules, medical
biopsy samples, metals and crystalline structures and the
characteristics of various surfaces.
o The electron microscope is also used extensively for inspection,
quality assurance and failure analysis applications in industry,
including, in particular, semiconductor device fabrication .

ELECTRON
MICROSCOPE

PRINCIPLE

Formation of an image is
due to scattering of
electrons by molecules of
the specimen and
scattering depends
solely on mass densities.
Elements of high
molecular weight such as
lead and uranium cause
marked electron scatterused for back ground
staining

Depends on wavelength
RESOLUTIO of moving electrons ;
N
resolution of 2 A0 can be
obtained.
VOLTAGE

KV

SOURCE

ELECTRONS

LIGHT MICROSCOPE
Image formation is due to
absorption of light which
depends more on
molecular structure than
on atomic weights C, n2
and H atoms- low
molecular wt .
Histological stains
depends more on certain
wavelenght of light, due to
molecular structure.
Limited to about 200 nm

LIGHT

Microsc
ope
image
formatio
n

Convergence of electron
beam is by passing
through circular
magnetic field - EM
lenses.
Beam is obtained from
heated tungsten
filament.
Lenses used are
condenser, intermediate
and projector lenses.

Convergence of a light
beam is by a convex
glass system
Light rays - are
obtained by a lamp
source/ natural light.
Lenses - condenser,
objective and eyepeice.

Magnetic lenses have

different powers depending
on amount of current
flowing, but all lenses are
rigidly fixed.

Lens power is fixed, but the

lenses are made movable
with relation to object.

Focal points are variable by

adjusting lens currents and
image.

Focal point is achieved by

moving the lens system by
proper illuminating
apparatus

Image - only one set of

image.
The image is formed and
magnified image is

2 set of images are formed,

inverted, real image forms
by objective lens and is
converted to virtual image,

System

Column is rigidly
constructed and
maintained at high
vacuum since air
molecules would
deflect electron beam.
Specimen must be
placed inside
microscope living
materials cant be
examined.

Since light rays are

employed, vacuum
creating is not
necessary.
Living organisms can
be visualized, as also
stained specimens.

Illuminating condenser
unit are placed below
the object and lens are
Constructed upside
placed above and
down is electron gun
image can be
and condenser lens are visualized.
placed above and
image formed below.
Unstained sections are
visualized and contrast
is increased by
staining with lead /

Tissue
processing
SPECIMEN
HANDLING

Fixatives are
buffered within ph
range of 7.2 7.6
and osmolarity,
ionic composition
is maintained.
Washing after
fixation - tissue is
rinsed in buffer
after post fixation
(immersed in 2%
aqueous
uranylacetate
enbloc staining)

DEHYDRATION

ETHANOL, ACETONE
are used.
Acetone is
inflammable, hence
avoided.
Propylene oxide is
used with ethanol to
ensure complete

To maintain the
morphology of
tissues and for
stabilizing proteins
it is fixed usually in
fixative (10%
formalin routinely
used )
Throughly washed
after fixation in
running water.

Increasing
concentration of any
organic solvent like
ethanol , acetone is
used
Not as sensitive as in
EM.

CLEARING
AND
EMBEDDING

MICROTOMY
KNIVES AND
SECTION
THICKNESS

Resin Embedding
for ultra thin
sections . Rigid
material
necessary,
withstand
vacuum and heat
generated,
preserve tissue
structure.
Epoxy resin or
acrylic resin is
used

ULTRAMICROTOMY
Glass knives or
diamond knives
used.
0.5 micron

Dehydrating agents are

not miscible with paraffin
wax and hence clearing is
necessary( xylene,
chloroform, toluene)
Paraffin wax is used for
embedding cheap, easily
handled , easy sectioning,
wide range of melting
points.
After 3 changes in
increasing concentration of
paraffin wax for 1 hour
each it is embedded (5860o C)
NORMAL MICROTOMY
disposable blades or stainless
steel blades are employed.
Ranges from 3 to 6 micron
thickness.

2 step procedure
Prepare and examining semi
thin sections 1 micro m.
Trough fluid is used for
collection of sections(10 to
15% ethanol or acetone is
used.
Sections are collected onto
grids using an eye lash probe
or fine point forceps.
For drying grids should be
placed on filter paper in lidded
container.
Standard method for staining
sections.
Grids are floated .
Section is placed side down
and drops of staining solution
is added for required time.
It is stained with uranyl
acetate (2% aqueous) or
reynolds lead citrate stain.

1 step procedure
Placed in water
bath, camel brush is
used to fix it on
slightly along with
egg albumin and
thymol crystal
adhesive.
To fix the section it
is placed on slide
warmer and heated
at 600 C for 10 mins
It is then stained
routinely for various
histological stains.
Glass slides are
placed on stage and
viewed.

REFERENCES
BRADBURY and B.BRACEGIRDLEINTRODUCTION TO LIGHT
MICROSCOPY.
BANCROFT,HISTOLOGICAL
TECHNIQUES,5th edition.
ROYAL MICROSCOPICAL SOCIETY.
INTERNET SOURCES

THANK
YOU