ELECTRON MICROSCOPY

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Electron Microscope

EM was invented by Knoll and Ruska (1932). It works on the principle similar to that of a light microscope except that an electromagnetic field and a beam of electrons act in a way similar to the action of a glass lens and a beam of light . An electron beam when accelerated through an electric field of 100 KV, has a wavelength of only 0.04 nm which is about 10,000 times shorter than the wave-length of visible light.

The resolving power and magnification of an electron microscope is therefore much higher than any light microscope.

In an electron microscope, a beam of electrons is projected from a cathode (electron gun) and is passed through a series of electro-magnetic lenses. The condenser lens collimates the electron beam on the specimen and an enlarged image is produce by a series of magnifying lenses.

The specimens which are focussed can not be directly seen, their image is rendered visible by projection on a phosphorescent screen. Since the penetrating power of the electrons through solid matter is weak, only very thin sections of specimen can be examined. Two types of electron microscopes are in use today.

Prinicple of electron microscope

Transmission Electron Microscope (TEM)

TEM is used to see the fine structure of cells; an object as small as 1 nm may be viewed. Ultra-thin sections of the objects are prepared, by embedding or freezing the specimen and sectioning it with a diamond of glass knife. Sections are floated in water and picked up on a wire grid.

They are stained with a heavy metal (gold or pallidium) to make certain part dense, and inserted in the vacuum chamber of the microscope. A 100,000 volt electron beam is focussed on the section and manipulated by magnetic lenses. A photograph prepared from the image may be enlarged with enough resolution to achieve magnification of 500,000 to 1,000,000 times at I nm resolution.

Scanning Electron Microscope (SEM)

SEM allows surfaces of objects to be seen in their natural state without staining. The specimen put into the vacuum chamber and covered ­with a thin coating of gold to increase electrical conductivity and thus forms a less blurred image. The electron beam then sweeps across the object building an image line by line as in a TV camera.

As electrons strike the object, they knock loose showers of electrons that are captured by a detector to form the image. Magnification with this microscopy is limited to about 10,000-1,000,000 times at 1-10 nm resolution.

Comparison of Various Types of Electron Microscopes

Type of microscope

Maximum magnification

Resolution

Remarks

Transmission electron microscope (TEM)

500,000-1,000,000 ×

1 nm

Ultrastructure of microbes viewed.

Sc S SScanning electron mmimnmicroscope (SEM)

10,000-1,000,000 ×

1-10 nm

Details of surface structures of microbes viewed; produces a three dimensional image.

Acoustic Microscope

In 1949, a Russian Physicist S.Y. Sokolov proposed that the property of sound waves (sound waves travel as longitudinal vibrations whose velocity depends on the elasticity and temperature of the medium) may be used for viewing intricate inside details of a solid body. However, the technology to convert sound signals into light signals did not exist at that time. Subsequently in the 1960s, Professor C. Quate of U.S.A. and E. Ash of England developed this principle and applied it in microscopy; the first practical microscope based on sound waves, namely, acoustic microscope, was commercialized in 1974. The principle on which the acoustic microscope works is based on the fact that the speed of sound in an environment is directly related to physical properties of that environment, such as the density and elasticity.

In an acoustic microscope, the transmitted mode of the impinging sound wave by the specimen is captured, and the vibration in intensity due to various parts of the specimen is recorded. The inner surface of a solid body is not accessible to optical light and only poorly to X-rays. With the use of proper electronics, acoustic waves can do the job of revealing its inner structure easily. Moreover, the specimen need not be stained.

The acoustic lens is a spherical surface ground into a material such as saphire through which sound travels quickly. The surface of the lens is kept immersed in a fluid of relatively low density (generally water). Sound waves derived from optically opaque objects are then converted into light signals.

This type of microscope has a future in microbiology because unlike light and electron microscopy, the biological specimens can be examined without any alteration.

Acoustic microscope has so far been used mainly in biological studies. However, its significant application lies in the material sciences. They are also being used in studying the effect of cardioactive drugs on internal valves, malignant tissue, varying states of fibrosis in lung cancer, etc.