EM is a very bulky tool that provides higher resolution and magnification than light microscope.
The EM is best used for studying biological ultra structure and the image obtained is called electron micrograph.
There are two general types of EM:
Transmission electron microscope and scanning electron microscope. The other improved relatives of EM are scanning probe microscope, scanning tunneling microscope and atomic force microscope.
General Principle of EM:
The fundamental principle of EM is similar to those of LM. In EM, a high velocity beam of electrons (instead of light) is used to travel in a vacuum tube. The beam of electrons is focused by a series of electromagnetic lenses analogous to the condenser, objective and eye piece lenses of the light microscope.
The object is placed between the condenser and objective. The magnified image of the object is formed on the fluorescent screen or on photographic film rather than being observed through eye piece. Since the image produced by electrons does not have the colour, the electron micrograph always has shades of black, grey and white.
The objects under examination must be extremely thin and are treated with chemicals or dyes to enhance the contrast as such the live objects cannot be studied. Techniques like negative staining, shadow casting and tracers are commonly used to increase the contrast.
Theoretically, the maximum resolution of the EM is 0.005 nm which is less than the diameter of a single atom, or 40,000 times the resolution of the light microscope and 2 million times that of the naked eye. However, the practical resolution of modern EM is of 0.1 nm (1 A).
(1) Transmission electron microscope (TEM):
The TEM was designed by Knoll and Ruska (1932) of German. The TEM has a magnification of 100,000-300,000 times with resolution of 2-5 A. The resolution of TEM depends upon wave length (A) of the electron beam. The wave length of electron beam is inversely proportional to the square root of the accelerating voltage (V) i.e. λ /V.
Electron beam produced by an electric current of 50,000 Volts (V) has a wavelength of a electron is 0.5 A. It is less than the diameter of smallest atom (Hydrogen atom = 1.06(A). A wavelength of 0.5 A shall produce a resolution power of about 0.25 A.
The same is not achieved due to limitations of:
(i) Electromagnetic lenses,
(ii) Drying up of specimens,
(iii) Sectioning of specimens,
(iv) Creation of perfect vacuum.
Structural parts of a TEM- The structural parts of a TEM are as follows (Fig. 1.6):
(a) Electron gun:
It consists of a tungsten filament or cathode that emits electrons on receiving high voltage electric current (50,000-100,000 volts). Near the top of the tube is an anode which attracts electrons.
(b) Ray tube (Microscope Column):
It is a high vacuum metal tube (2mt. high) through which electrons travel.
(c) Condense lens:
It is the electromagnetic coil which focuses the electron beam in the plane of the specimen.
(d) Objective lens:
It is the electromagnetic coil which produces the first magnified image formed by the objective lens and produces the final image.
(e) Projector lens:
It is also an electromagnetic coil which further magnifies the first image formed by the objective lens and produces the final image. Each electromagnetic coil has a coil of wire encased by a soft iron casing.
(f) Fluorescent Screen or Photographic Film:
Since unaided human eye cannot observe electrons, therefore, a fluorescent screen is used for viewing the final image of the specimen. The final image can be captured on photographic film and die photograph obtained is called an electron micrograph.
Uses:
1. EM has a high magnification and resolving power.
2. Without the aid of EM, biologists would have never known submicroscopic cell organelles (e.g., ribosomes, micro-bodies, centrioles, microtubules, endoplasmic reticulum) and internal structure of microscopic organelles (e.g., chioroplasts, mitochondria).
3. Study of microorganisms, viruses and viroids have been possible only with the aid of EM.
4. It can discern even macromolecules.
This has helped scientists to know the arrangement of molecular aggregates and even their components, e.g. nucleosomes.
Draw backs:
(i) It is complicated and costly.
(ii) There is risk of radiation leak,
(iii) 11 require very high voltage electric current.
(iv) A cooling system is required,
(v) The specimen or object has to be given special treatment including complete dehydration.
Preparation of material for TEM:
The material to be studied under electron microscope must be well preserved, fixed, completely dehydrated, ultrathin and impregnated with heavy metals that sharpen the difference among various organelles. The material is preserved by fixation with glutaraldehyde and then with osmium tetroxide. The fixed material is dehydrated and then embedded in plastic (epoxy resin) and sectioned with the help of diamond or glass razor of ultra-microtome.
The sections are ultrathin about 50-100 nm thick. These sections are placed on a copper grid and exposed to electron dense materials like lead acetate, uranylacetate, palladium vapours, phosphotungstate etc. Now the sections can be viewed in the TEM. The coating with electron dense material enables the specimen to withstand electric bombardment.
(2) Scanning Electron Microscope (SEM):
It is the second type of EM, first built by Knoll (1935) but it was commercially developed by Cambridge Instruments (1965). It is used to study the three dimensional images of the surfaces of cells, tissues or particles. The SEM allows viewing the surfaces of specimens without sectioning. The specimen is first fixed in liquid propane at-180° C and then dehydrated in alcohol at-70°C.
The dried specimen is then coated with a thin film of heavy metal, such as platinum or gold, by evaporation in a vacuum provides a reflecting surface of electrons. The surface of the specimen when scanned by electron beam release secondary electrons that from a three-dimensional image of the specimen on a television screen. Holes and fissures appear dark, and knobs and ridges appear light. Complete scanning from top to bottom usually takes only a few second.