RDK PARAMEDICAL STUDENT STUDY: MICROSCOPE

MICROSCOPE

Introduction of microscope

The microscope is indispensable for a clinical laboratory technician. It is very important to have a sound knowledge of the microscope which is going to be a constant comparing in his daily work. To obtain desired magnification for visualizing the objects, the scientists did untiring efforts. The world microscope is derived from two Greek words - ''micro'' (small) and ''scope'' (view). Simple microscope discoveries by Anthony von Leeuwenhoek (1632-1723) and subsequently compound microscope by Robert Hook (1635-1703) were made. It is an instrument by which very small things can be seen which are not seen by the necked eye. This is done by producing an enlarged and well defined image of the objects by a system of lens.

Principle of microscope

The light beam, which falls on the object, passes through a series of lenses giving a clear enlarged vision of an object. Such magnifying instruments could be of the following types:

1. Simple magnifying lens

2. simple dissecting microscope

3. Compound microscope

4. Dark field microscope

5. Phase contrast microscope

6. Polarising microscope

7. Fluorescent microscope

8. Electron microscope

1. Simple magnifying lenses: - This is the simplest form of microscope. These are available in various size and having different magnifications image.

2. simple dissecting microscope :- it consists of the base, plane mirror, vertical limb, stage, stage clip, adjustment screws, folding and revolving arm & eye piece.(Note:- This is commonly used in biology labs to carry out dissections.)

3. Compound microscope: - This is the most commonly used type of microscope. It is very important to know about it in greater detail. There are two convex lenses fitted in two different tubes. It is a convex lens of short focal length and small aperture. This is mounted on a narrow tube. The eye piece which is towards the eye consists of two convex lenses separated at a suitable distance. The eye piece is of wider aperture and larger focal length. The distance between the eye piece and the objective can be varied by sliding the narrow tube in the inner tube.

It is consists of the following parts.

The stand / base: - it comprises a heavy foot, often horse shoe shaped to provide stabiles and support to the microscope. The limb which bears the optical system. The limb is attached to the foot by a hinged joint so that the microscope can be set at a comfortable angle for the observer.
Pillar: - having the inclination joint, helps to move microscope in forward and backward direction.
Plane & concave mirror: - helps to direct beam of light towards an object. The light source is a white cloud, however, bulb may also be used for this purpose.
Iris / Diaphragm: - helps to regulate the amount of light and should not be completely closed.
Sub-stage condenser: - condenses the light on the object to be studied by rising and lowering it with the help of condenser screw.
Stage & Stage clips: - On stage we keep slide, and clips help to hold the slide in proper position.
Stage screws: - right side stage screw helps to move slide in backward & forward direction, however left side screw helps to move slide in right or left side direction.
Aim: - used to handle the microscope. Lower side of the arm gets fixed on pillar with inclination joint and upper side of the arm holds body tube.
Coarse adjustment screw: - helps the moving body tube in upward & downward direction to focus the object approximately.
Fine adjustment screw: - helps most accurate focus of the object.
Body tube: - holds eyepiece, lower portion of body tube holds fix portion of nose-piece on which a movable portion of nose-piece holds objective lenses.
Eye piece (Occular lens):- can be changed as per need, for example, 6X, 10X, etc.
Objective lenses: - are commonly three or four objective lenses, fitted on the movable portion of nose piece. The magnifying power is indicated by ‘X’. The objective lenses used are-

10X or 15X - low power,

44X or 45X - high power,

95X or 100X – Oil immersion lens.

(Oil immersion lens is used by placing a drop of immersion oil on the slide).

Magnification

Magnification is always expressed in terms of diameter and not the area. Magnification of 100 diameter is equivalent to 10,000 times. The magnifying power of an objective is worked out by dividing the length of optical tube by the focal length of the objective lens. The total magnification power will be worked out as follows:

Measurement of the microscopic objects

The unit of microscopic measurement of the object is known as, ‘micron’ (µ) which is equal to 0.001 mm or 1/10 nm.

Use of micrometer

The occular micrometer is a glass disc of graduated scale fitted in eye piece of the microscope. The size of the object is determined in terms of the divisions given.
The stage micrometer is also a glass slide with ruled scale divided into hundredth of one mm fitted on the stage of the microscope. The corresponding values of the occular and stage micrometer are worked out in microns, which holds good only for a particular objective lens used. In place of stage micrometer, the counting slide of hemocytometer covered with cover slip can also be used.

Steps using compound microscope

Clean the microscope properly with the help of lens cleaning fluid or xylol & muslin cloth.
Open iris completely and adjust condenser at mid position.
Turn low power objective lens and view through eye piece and adjust mirror at proper angle to get good cloudy beam of light.
Keep the slide on the stage bring low power lens closer to the object on slide.
Now turn coarse adjustment to move objective lens slowly upward until you see good focused object.
Lastly you use fine adjustment screw to focus the object on the slide very clearly.
Once you adjust slide, you may simply turn high power or oil immersion lens for further use.

4. Dark field microscope: - The consists of blackening out the central rays of light and diverting the peripheral rays against the microscopic object from the side. Only those rays which strike the object are reflected upward and pass into the objective. The object hence appears bright in a black background. By this form of illumination very minute organisms can be seen. It is specially useful for the study of the spirochetes.

5. Phase contrast microscope: - In this microscope the light beam that passes through the tissue is combined with a reference beam directed through an optically uniform zone. Combined beams interfere producing dark area where there is destructive interference and light area where there is constructive interference. This microscope can be used to see the object even without staining.

(Note: - The different tissue components in an undtained preparation appears darker or lighter. This is mainly used as a research tool to study unstained preparations.)

6. Polarising microscope: - Two prisms or polarising filters are used – one below the condenser and the other above the objective. When both these are crossed then no light passes through the upper one. If a birefringent object like cholesterol crystal, amyloid etc. are placed at the object plane, then those light rays that pass through the crystals pass through the upper prism as the prisms are no. longer crossed for these rays. The rays that do not pass through these crystals are absorbed by the upper prism. Thus the crystals are seen as bright objects against a black background.

7. Fluorescent microscope: - Fluorescent microscope makes use of UV light. The tissue sample are stained with a dye fluoresces when irradiated with UV light in fluorescent microscope.

8. Electron microscope: - Electron microscope involves electron beams which could be focused by electron and magnetic lenses instesd of glass lenses to form an image. The wave length of electron is shorter than the visible light as a result electron microscope can show details much better than light microscope with magnification up to 250,000in electron microscope and maximum of 1000 in light microscope is of two types – viz.,

a. Transmission electron microscope (TME) &

b. Scanning electron microscope (SEM).

Exercise for Students

Clean the microscope with muslin cloth and study parts in terms of name, purpose and movement of each.
Practice the manipulation necessary to locate particles of dust which appear in the microscopic field.
Study the effect of following manipulations:

a. Opening and closing of the diaphragm.

b. Raising and lowering of condenser.

c. Using oil immersion objective without oil, with oil between slide and objective.

d. Changing the place and direction of mirror.