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AIM: To study the various types of Microscopy
THEORY:
Microscope is the most commonly used piece of apparatus in the laboratory.
It produces greatly enlarged images of minute objects.
LIGHT MICROSCOPE
A Light Microscope can be simple or a compound microscope.
[A] SIMPLE MICROSCOPE This is a simple hand magnifying lens. The
magnification power of hand lens is from 2x to 200x.
[B] COMPOUND MICROSCOPE This has a battery of lenses which are
fitted in a complex instrument. One type of lens remain near the object
(objective lens) and another type of lens near the observer’s eye ( eye piece
lens). The eye piece and objective lenses have different magnification. The
compound microscope can be monocular having single eye piece or,
binocular which has two eye pieces. The usual type of microscope used in
clinical laboratories is called light microscope.
A compound microscope has the following parts:
▪ Stand
▪ Body
▪ Optical system
▪ Light/ Illumination system
1. Stand
This is Horse-shoe shaped in monocular microscope. It gives stability to the
microscope. Binocular microscopes have a variety of ergonomic shapes of
stand.
2. Body
It consists of limb which arises from the joint with which microscope can be
moved in comfortable position. The stand and limb carry the following:
i. Body tubes
ii. Stage
iii. Knobs for coarse and fine adjustment
Body Tubes
There are two tubes: external tube which carries at its lower end a revolving
nose piece having objective lenses of different magnification while internal
tube is draw tube which carries at its upper end eye pieces.
Stage
This is a metallic platform which accommodates glass slide having mounted
object over it to be seen. Stage is attached to the limb just below the level of
objectives. It has an aperture in its centre which permits the light to reach the
object. Slide on the stage can be moved horizontally or vertically by two
knobs attached to slide holder. Just below the stage is substage which
consists of condenser through which light is focused on the object. The
substage can be moved up or down. The substage has an iris diaphragm,
closing and opening of which controls the amount of light reaching the
object.
Knobs for Coarse and Fine Adjustments
For coarse and fine adjustments, knobs are provided on either side of the
body. Coarse adjustment has two bigger knobs, the movement of which
moves the body tubes with its lenses. Fine adjustment has two smaller knobs
either side of the body. The fine focus is graduated and by each division
objective moves by 0.002 mm.
3. Optical System
Optical System is comprised by different lenses which are fitted into a
microscope. It consists of eye piece, objectives and condensers.
Eye piece
In monocular microscope, there is one eye piece while binocular microscope
has two. Eye piece has two plano-convex lenses. Their magnification can be
5x, 10x, or 15x.
Objective
These are made of a battery of lenses with prisms incorporated in them.
Their magnification power is 4x, 10x, 40x and 100x.
Condenser
This is made up of two simple lenses and it condenses light on to the object.
4. Light/ Illumination system
For day light illumination, a mirror is fitted which is plane on one side and
concave on the other side. Plane mirror is used in sunlight while concave in
artificial light. Currently, most of the microscopes have in-built electrical
illumination varying from 20 to 100 watts.
MAGNIFICATION AND RESOLVING POWER OF LIGHT MICROSCOPE
Magnification power of the microscope is the degree of image enlargement.
It depends upon the following:
• Length of optical tube
• Magnifying power of objective
• Magnifying power of eye piece
With a fixed tube length of 160 mm in majority of standard
microscopes, the magnification power of the microscope is obtained by the
following:
Magnifying power of objective x Magnifying power of eye piece .
Resolving power represents the capacity of the optical system to produce
separate images of objects very close to each other.
Resolving power (R) = 0.61 λ
NA
Where λ is wavelength of incidental light; and
NA is Numerical aperture of lens
Resolving power of a standard light microscope is around 200nm.
HOW TO USE A LIGHT MICROSCOPE
1. The microscope must be kept in a comfortable position.
2. Appropriate illumination is obtained by adjusting the mirror or
intensity of light.
3. When examining colorless objects, the condenser should be at the
lowest position and iris diaphragm closed or partially closed.
4. When using oil immersion, 100x objectives should dip in oil.
5. After using oil immersion clean the lens of the objective should be
cleaned with tissue paper or soft cloth.
OTHER TYPES OF MICROSCOPY
DARK GROUND ILLUMINATION (DGI)
This method is used for examination of unstained living micro-organisms
e.g. Treponema pallidum.
Principle
The micro-organisms are illuminated by an oblique ray of light which does
not pass through the micro-organism. The condenser is blackened in the
centre and light passes through its periphery illuminating the living micro-
organism on a glass slide.
POLARISING MICROSCOPE
This method is used for demonstration of birefringence e.g. amyloid, foreign
body, hair etc.
Principle
The light is made plane polarized. Two discs made up of prism are placed in
the path of light, one below the object known as polarizer and another placed
in the body tube which is known as analyzer. Polarizer sieves out ordinary
light rays vibrating in all directions allowing light waves of one orientation
to pass through. The lower disc (polarizer) is rotated to make the light plane
polarized. During rotation, when analyzer comes perpendicular to polarizer,
all light rays are canceled or extinguished. Birefringent objects rotate the
light rays and therefore appear bright in a dark background.
