Aim:- To Study Compound microscope

Microscope is a device that magnifies the image of an object that is not visible to
the naked eye to an extent where it can be seen clearly. It was invented by Antony
Leeuwenhoek.

Types of Microscopes:

1. Monocular microscope:- It has only one eyepiece

2. Binocular microscope:- It is a compound microscope having two eyepiece.
Both eyes are used simultaneously that prevents eyestrain.
3. Dissection microscope:- It is a binocular microscope used for micro-
dissection under magnification
4. Dark Field microscope:- It employs a special condenser that causes light
waves to cross on the material under study rather than passing through it.
The field of view appears dark against which the object appears bright. It is
used in microbiology to study spirochetes.
5. Phase Contrast microscope:- A special phase plate is inserted into the
condenser which can retard the speed of some light waves. Since the tissue
cells and organisms have different refractive indices, this microscope uses
these differences to produce an image with good contrast of light and shade.
6. Interference – contrast microscope:- A special prism that can split a beam of
light is added to the condenser. The two split beams are then polarized but
only one resultant beam passes through the specimen while the other does
not. The two beams are then recombined to produce a 3-D image.
7. Polarizing microscope:- It has a polarizer (filter) which is usually placed
between the light source and the specimen, and an analyzer which is located
between the objective and the eyepiece.Used to study tissue with the
property of birefringence (eg. Muscle fiber).
 8. Fluorescence microscope:- A fluorescent dye is used to stain tissue which
are then studied under this microscope.
9. Transmission Electron microscope:- It uses strong beam of electrons instead
of light and electromagnetic fields in place of glass lenses. The magnified
image can be recorded on photographic film. Total magnification obtained
can vary from one to several hundred thousand times.
10.Scanning electron microscope:- With this, a resolution of about 30Å can be
achieved. It has been developed for 3D study of surface topography of cells
and object.

Parts of microscope

A. The Support system:- Functions as a frame work to which various functional

units are attached.
1. Base:- It is heavy metallic ‘U” or horseshoe shaped base or foot which
supports the microscope on the worktable to provide maximum stability.
2. Pillars:- Two upright pillars project up from the base and are attached to the
C-shaped handle. The hinge joint allows the microscope to be tilted at a
suitable angle for comfortable viewing.
3. Handle:- The curved handle that projects from the hinge joint supports the
focusing and magnifying system.
4. Body tube; The past through which the light passes to the eyepiece and thus
conducts the image to the observes eye. It is 16- 17 cm in length and can be
raised or lowered by the focusing system.
5. The stage:- It has two components:-
i) The fixed Stage:- It is a square platform with an aperture in its center,
and fitted to the limb below the objective lens. The slide is placed on
it and centered over the aperture for viewing.
ii) Mechanical Stage:- It is calibrated metallic frame fitted on the right
edge of the fixed stage. There is spring mounted chip to hold the slide
or counting chamber in position while the two screw heads move it
from side to side and forward and backward. The vernier scale on the
frame indicates the degree of movement.
A. The focusing system:- It is employed for raising or lowering the optical
system with reference to the slide under study till it comes into focus. It
consists of two fine & coarse adjustment screws. If one fine or coarse
adjustment is moved, second on the other side rotates simultaneously.
1. Coarse adjustment:- it moves the optical system up or down rapidly
through a large distance via rack and pinion arrangement.
 2. Fine adjustment:- It works in the same way but several rotations of screw
head are required to move the tube through a small distance. One rotation
moves the tube by 0.1mm or less.
B. The optical system:- It can be raised or lowered as desired. It consist of:-
1. The body tube:-
a) Tube length_ Distance between upper end of objective and eyepiece
(16-17 cm)
b) Optical tube length – Distance between the upper focal point of eye
piece and lower focal point of objective (25cm).
2. The eyepiece:- Located/fits at the top of body tube. Most microscopes are
provided with 5X, 8X, 10X eyepieces. Each eyepiece has 2 lenses – one
mounted on top called as eye lens and other at bottom called as field lens.
The field lens collects the divergent rays of primary image and passes to
eye lens which further magnifies the image.
3. Nose piece:- Fitted at lower end of body tube. It has two parts:-
a) Fixed nosepiece
b) Revolving nosepiece:- Carries interchangeable objective lenses that
can be rotated into position being indicated by a ‘Click’.
4. Objective lens:- These are
a) Low power objective:- For initial focus and viewing large area
b) High power objective:- More detailed study of material.
c) Oil immersion objective- Employed for detailed study of morphology
of blood cells and tissues.
d) Scanning objective:- used for scanning or viewing much larger area
on slide.

Objective Magnification Position of Focal Iris Numerical

condenser length diaphragm aperture
Low 10x Low 16mm Partly open 0.25
High 40x Midway 4mm Half open 0.65
Oil 100x High 1.6-2 mm Fully open 1.30
Scanning 3x 40mm 0.10

Racking of microscope:- The cells and their constituents are 3D structures and lie
at different levels. Therefore, it is important to continuously rack the microscope
by using fine adjustment after the specimen has been brought under focus under
any magnification.

Oil Immersion:- A drop of cedar wood oil is first placed on slide and oil immersion
lens dips into oil. Oil is used to increase the numerical aperture and thus the
resolving power of the objective

Refractive index

Air = 1.00

Water = 1.33

Glass = 1.55

Without the oil, there is layer of air trapped between the lens and slide. The
refractive index of air is different from that of glass so there is refraction of light
away from the lens which diminishes the brightness and clarity of image. The oil
used (cedar wood oil) has same refractive index as that of glass so that light passes
directly into lens and image is much more clear. Other medium like glycerin and
paraffin (R.I = 1.35 – 1.40) can also be used. Cedar wood oil though costly gives
better result.
Parfocal System:- When one objective lens (e.g LP) is in focus, the other are more
or less in focus. Thus switching from one lens to another requires only a little turn
of fine adjustment to bring the image into sharp focus.

C. The illumination System:- It should provide uniform and bright illumination

of entire field of view. It Consist of
1. Source of light:- It may be
a) External Source:- Diffuse natural daylight reflected and scattered by the
atmosphere. Artificial source – A fluorescent tube or an electric lamp
housed in a lamp box with frosted glass window – can also be used.
b) Internal light source- Frosted tungsten lamp to provide uniform white
light.
2. The mirror – A double sided mirror, one plane and other concave fitted back
to back in metal frame is located below the condenser that can be rotated in
all directions.
a) Plane mirror – Used with distant source of light.
b) Concave mirror – When source of light is near the microscope.
3. The condenser:- It is system of lenses fitted in short cylinder mounted below
the stage. It can be raised or lowered by rack and pinion and focuses the
light rays into solid cone of light on to the material under study. Raising or
lowering the condenser can vary its NA.
4. The iris diaphragm:- Fitted within the condenser with small lever on its side
that can adjust the size of the aperture Narrowing the aperture (reducing the
size of field of view) decreases NA of the condenser.
5. Filter:- A metallic ring can accommodate a blue or green filter since
monochromatic light is ideal for microscopy.

Physical basis of microscopy:

 1. Resolving power (resolution):- Ability to show closely located structures as
separate and distinct fro each other.
R.P of unaided human eye = 0.15-0.25mm
0.61×𝜆
R.P = ( 𝑊𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡 𝑢𝑠𝑒𝑑)
𝑁.𝐴

2. Magnification:- It is mentioned on the eyepiece and objective lens used.

Total magnification (M) = M1 X M2

M1 = Magnification of eye piece (10X)

M2 = Magnification of objective
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑖𝑚𝑎𝑔𝑒 𝑓𝑟𝑜𝑚 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒
M2 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑜𝑏𝑗𝑒𝑐𝑡 𝑓𝑟𝑜𝑚 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒
𝐵𝑜𝑑𝑦 𝑡𝑢𝑏𝑒 𝑙𝑒𝑛𝑔𝑡ℎ
=
𝐹𝑜𝑐𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ

(i) Low power

160
M2 = = 10
16

M = 10 x 10 = 100X

(ii) High power

160
M2 = = 40
4

M = 10 x 40 = 400X
(iii) Oil immersion
160
M2 = = 100
1.6

M = 10 x 100 = 1000X

3. Numerical Aperture – It is a power of resolution of lens, is the ratio of its

diameter to its focal length.
As N.A (numerical aperture) increases, resolving power of lens increases.

N.A is also an index of light gathering power of a lens i.e the amount of light
passing through the lens.

N.A – n sine Alpha

Where n = Refractive index

Alpha = Angle subtended by the lens at the objective.

Image formation:- It is the objective that starts the process of magnification. It

forms a real, inverted and enlarged image in the upper part of body tube. The field
lens of the eye piece collects the divergent rays of the light of primary image and
passes these through the eye lens. The image seen by the eye is virtual, inverted
and magnified appearing to be about 25cm in front of the eye.
Procedure for use of microscope:-

Principle:- A focused beam of light passes through the material under study into
the microscope. Parts of the specimen that are optically dense and having a high
refractive index or coloured with stain cast a potential shadow which is magnified
in 2 main stages as it passes into the observes eye.

Procedure/Steps:

1. Place the microscope on your work table in an upright position and raise the
body tube 7 – 8 cm above the stage. Put the slide on the stage and using the
mechanical stage, bring the specimen over the central aperture.
2. Select and adjust the mirror so that light shines on the specimen.
3. Always start with the low power objective. Rack the condenser well down
(low position) and partly close the diaphragm to cut down excess light.
4. Looking from the side and using the coarse adjustment, bring the body tube
down so that the LP lens is about 1 cm above the slide.
5. Now look into the eyepiece and gently raise the tube till the specimen comes
into focus. When the image comes into focus, scan the entire field, racking
the fine adjustment all the time.
6. For focusing under high power, rotate the nosepiece so that HP lens clicks
into position. Raise the condenser to mid-position and open the diaphragm to
admit enough light. Use fine adjustment as required.
7. To use oil immersion lens, shift the objective lens from HP lens to OI lens.
Place a drop of cedar wood oil on the slide, and looking from side, slowly
bring the objective down till it just enters the oil drop. The oil will spread
out in the capillary space between the slide and lens. While looking into the
eyepiece, slowly and very carefully raise the objective with coarse
adjustment till the cells comes into view. Use fine adjustment for fine
tuning.

Precautions:-

1. Select a stool or chair of suitable height so that your eyes are at a level
slightly above the eyepiece. This will ensure comfortable working for long
period.
2. Ensure that all the lenses are clean and free from dust and smudges. Donot
touch them with your fingers, nor blow them to remove dust.
3. Check the position of the objective, condenser and diaphragm, to ensure
optical illumination.
4. Never lower any objective from any height while looking into the
microscope.
5. Once the specimen has been focused continuously rack the microscope.
6. Clean the microscope. Never leave cedar wood oil on the OI lens because it
may seep into the body of objective and damage the lens permanently. Dried
 oil is difficult to remove. Remove oil with lens paper, then xylene to clean
the lens.
7. Cover the microscope with plastic cover after use.