Long back, the biologists could learn about living things, only from what they could see with the naked eye. But progress in science, is closely linked with the advent of new tools and techniques. This has particularly been true for biology. In the course of history of biology, various new tools and techniques have developed, like microscopy, paper chromatography, etc. In this lesson you will learn about some of these.

After completing this lesson you will be able to :

• trace the development of microscopes and their working.
• list the parts of a compound microscope.
• compare the working principle of compound microscope with that of an electron microscope.
• explain the basic functioning of other microscopes like the phase contrast microscope, scanning microscope.
• describe the basic aspects of some other techniques like cytochemistry, autoradiography, paper chromatography, centrifugation and tissue culture.

The first microscope-was constructed by Antonym Van Leeuwenhock (1632-1723). This microscope consisted of a single biconvex lens filled in a small window of a "board" and the object was viewed through ii. This was a simple microscope Next stage was a very primitive compound microscope in which two lenses were used (Fig. 38.1) cImprovements continued and newer and newer microscopes were designed and are still being improved.

f'ig. 38.1 Crude microscope used by Robert Hook 38.4

There are different types of microscopes which are used in studying the various structures and activities taking place inside a ceil. Some of these are as follows :

1. Simple microscope.

2. Compound microscope

3. Phase-Contrast microscope

4. Electron microscope

5. Scanning electron microscope (SEM)

Magnification : It is the ratio of the size of image to the object.

1. Simple Microscopes : These are of two types

(i) Hand Lens : It consists of a biconvex lens, mounted on a handle. The lens is of, different sizes and different magnifying powers. is commonly used to magnify an entire object.

(ii) Dissecting Microscope : It consists of a biconvex lens which is moved up and down by an adjustment screw, to bring the object in sharp focus. The light is focussed with the help of a concave mirror fitted below. A full object can be seen under it.

2. Compound Microscope : It comprises of two lenses : an objective lens and an eyepiece. placed al either end of the body tube.

It also has a condenser, having a simple mirror on one side and concave on the other. The object is placed first below the objective lens over the stage. The objective lens forms an image of the object. This image is further magnified by the eye piece.

1. Basically one biconvex lens is used

2. It can magnify upto 20 times

3. The whole object can be seen

Basically two lenses are used It can magnify upto 1500 times

Only a part of the object or a thin section can be seen.

It uses light which is transmitted through the object

1. It is operated in open condition.
2. The objective lens is simply a glass lens.
3. The source of illumination is light.
4. The final image of an object is observed through an eye-piece.
5. It magnifies the object upto 1500 times.
6. Resolution power is upto 2500A.
7. It can be used to see a living ceil.

It is operand only in vacuum condition.

The objective lens is electromagnetic lens.

The source of illumination is an electron beam .

The final image of an object is projected on a fiourescent screen.

It magnifies the object upto 200,000 times.

Resolution power is upto 2.5A.

It can be used to see only dead cells.

3. Phase-Contrast microscope : It has an annular diaphragm located below the condenser an objective having a phase plate. When light is transmitted through lenses, some of its rays pass directly while others are diffracted laterally The diffracted light rays are thus separated from the direct light and an image of strong contrast is produced. Mainly it is used to

1. examine living cells
2. oserve the nuclear and cytoplasmic changes taking place during mitosis.

By this type of microscope, three dimensional images are developed. It gives more detailed and clear structure of cells. It is particularly used for the study of the surface of an object.

There are other types of tools and techniques that have been developed. Some of them are given below :

1. Cytochemical Methods : These methods are used to locate specific chemical constituents within the cells. It is done either by the use of certain dyes or by using the substrates of enzymes e.g. Schiff's reagent is used to localize the presence of DNA in a cell.
2. Autoradiography : This technique is used for study the synthesis of molecules and to trace metabolic events in the cells. The radio-labelled compounds are injected into the organism. Then various tissues are investigated to find out where the radio-activity is located. This is done by using photo-sensitive film.

3. Paper Chromatography : In this method the chemical substances are separated from a mixture. A spot of the mixture is put on the distal end of a long strip of the Whatman filter paper. The filter paper is hanged in a manner that the distal spotted end dips into the solvent mixture kept in the tray/jar. As the liquid is drawn up on the paper, different substances in the mixture begin to separate according to their molecular weight & size and rise up to different heights on the paper. It is then analysed by using certain chemicals for further investigation.

4. Centrifugation : By this method different organelles of cells such as nucleus, mitochondria, ribosomes etc. having different particles size & weights are separated.

The Cells are homogenised by a special method. It is then put into tubes and tubes are placed in a centrifuge. The centrifuge is rotated at a high speed. By doing so under the influence of centrifugal force orgnells separate according to their particle density and sizes, lighter particle at the top and the heaviest particle settle at the bottom. The layers are then studied separately and the structure in detail is known.

5. Tissue Culture : This technique involves growing of living cells outside the -organism by providing ail necessary conditions for their survival and growth. The cells from an organism are grown in laboratory on a nutritive medium at a suitable temperature. Using this technique it has been possible to develop a whole organism from a single cell. Some new fully grown plants have been developed in this way.

Fungal tissue placed on PDA Medium

Mother spawn

WHAT YOU HAVE LEARNT

• Biologists depend heavily on a number of tools and techniques for studying organisms.
• Microscopes, such as the simple (direction) microscope, compound microscope and the electron microscope are used to study organisms.
• Compound microscope uses light and can give magnification up to about 1500 times whereas the electron microscope uses electron beam and magnifies the image upto 2,00,000 times.
• Phase contrast microscope is chiefly used for observing activities inside the living cells.
• Scanning electron microscope is used for introducing three-dimensional images chiefly of the surfaces.
• Cytochemical methods, autoradiography, centrifugation are helpful in studying cell chemistry synthesis of substances inside the living organism and isolation of cell organelles respectively
• Paper chromatography is used for separating chemical substances in a mixture.
• Tissue culture involves growing of cells tissues outside the body of the organism.

1. Name the scientist who constructed the first microscope.

2. Mention differences between a compound microscope and an electron microscope.

3. Define the term ultra-centrifugation

4. Name the microscopes under which we study a living cell and a dead cell.

5. List the main points under the technique of autoradiography.

6. Give the uses of cytochemical methods and centrifugation.

7. Mention the importance of tissue culture.

1. Biconvex lens

2. upto 1500 times

3. Any two points given in the table 38.1

4. Electrons

1. Radio labelled

2. Cytochemical methods

3. Centrifugation