Electron Microscopy: Meaning, Types and Techniques!
Meaning of Electron Microscopy:
Virus diagnosis by electron microscopy relies on the detection and identification of virus on the basis of their characteristic morphology. Earlier attempts to see viruses with even the most powerful optical microscopes of the day was largely unsuccessful.
This was so because visible light radiations with an average wavelength of about 5500 A°. (A°-Angstrom unit, equivalent to 10-8 centimeter) were unable to lighten the finer and detailed aspects of virus particles, which are comparatively smaller in size.
The light wavelengths are relatively long. Therefore, particles having smaller size cannot be properly resolved i.e. distinctly and separately identified. This problem was solved with the development of electron microscope by Knoll and Ruska in 1931.
These instruments do not use electromagnetic radiations with longer wavelengths. Instead, strong electron beams are projected from a suitable source to resolve the object under observation. The wavelengths of such electron beams were very small, often less than 1A°. On the other hand, the distance between different atoms in a molecule is more than that.
Therefore, it is theoretically possible to obtain resolutions at the atomic level with the help of these. Once resolution at such a fine degree is obtained, it is possible to have enlarged and magnified images to the desired extent. We can achieve a magnification of up to 2,000,000X where-as from ordinary light microscope it is up to 2000X. Using binoculars the image can be amplified to a maximal magnification of 2,000,000X with the JEM 100 S.
The electron microscope consists of an electron gun, central column, electro-magnetic lenses and a fluorescent screen (Fig. 3). Electron gun is located at the top of the microscopic body which serves as source of electrons. The gun consists of tungsten filament at 30 KV to 150 KV potential. It is surrounded by a negative shield with an aperture through which an electron beam is drawn off.
The central column is an evacuated metal tube which is present on the top of the gun. Electromagnetic lenses or coils are similar to the condenser, objectives lenses and ocular lens of the light microscope and are called condenser, objective and projector coils respectively (Fig. 4).
Each coil has coils of electric wire winded on a hollow metal cylinder. The electric current while passing through these magnetic coils produces an axially symmetric field in the centre of the lens. The magnetic field forces the electrons to spiral around the central axis and functions as magnifying lens. Focusing can be achieved by adjusting the voltage.
Most electron microscopes operate in the voltage rang of 25-200 KV. A few operate at 1000 KV or more. The image formed is observed on a fluorescent screen and not directly, since electrons are harmful to eyes. To obtain a permanent image record, photographic material has been incorporated into the microscope for direct exposition of the electron beams.
Types of Electron Microscope:
There are two basic types of electron microscopes:
(a) Transmission Electron Microscope (TEM):
This electron microscope can be compared with a light microscope (LM). It uses transmitted electrons that can penetrate the thin sample.
(b) Scanning Electron Microscope (SEM):
This can be compared with a dissection or stereoscopic microscope. SEM uses scattered electrons from the sample surface, either secondary or back scattered, thus rendering a tridimensional image.
Technique:
The first plant virus to be observed under the electron microscope was Tobacco Mosaic (Williams and Wykoff, 1943. Since then electron microscopy technology has improved considerably. Many techniques related to electron microscopy have brought out reasonably clear pictures of (table 1) a large number of viruses.
These techniques are:
Ultrathin Sectioning:
This technique is helpful in studying the particle within the host cell. It is also useful for studying the crystal structure. Ultrathin sections (25-90 nm) are produced from fixed, dehydrated and embedded biological materials, using special microtomes (thermal or mechanical advance microtomes) and glass or diamond knives with very sharp and hard cutting edges.
The optimal characteristics of an ultra-tin sections are:
(i) Thickness of 30 to 60 nm.
(ii) Enough consistency to support the electron beam.
(iii) Susceptible to good contrast through its affinity with the tinctures.
Glass knives are made by cutting a special glass to obtain a sharp cutting edge. The first model was designed by Sorvall in 1953 and is known as Sorvall MT1. A swedish company manufactures commercial ultra-microtomes under the “ultratome” brand name.
Negative Staining:
Negative staining is an easy qualitative method for examining the structure of isolated organelles, individual macromolecules and viruses at electron microscopy level. It is a very useful technique because of its ease and rapidity, and also because it requires no specialized equipment other than that found in a regular electron microscopy laboratory.
Hall (1955) was the first to accidentally demonstrate the negative staining effect in a study in which panicles were being positively stained with phosphotungstic acid. Imperfectly washed particles were surrounded and embedded in the dried reagent, and instead of appearing dark on a light background, they were seen light on a dark background (Fig. 5).
Huxley (1955) independently noticed the same effect with Tobacco Mosaic Virus. Brenner et. al. (1959) also observed the same phenomenon and named it is negative staining. In this technique the stain metal compounds are deposited around virus particles in such a way that they delineate the surrounding virus and make it electro-dense. By contrast, the entire virus particles area remains trans lucid to the electrons.
Some principal negative stains with their normal pH for use are:
Sodium or Phosphotungstate (PTA) – 5 to 8
Uranyl acetate – 4.2 to 4.5.
Ammonium molybdate – 5 to 7
Methylamine tungstate – 5 to 7
Phosphotungstate (PTA) is one of the most commonly used negative statin. The virus preparation were stained with 2% aqueous solution of PTA, adjusted to pH 7.0 with NaOH or KOH. Floating the grids with adsorbed virus particles, with a drop of 0.1% glutaraldehyde for 5 minutes before staining with PTA will reduce the damage to the virus particles.
Ammonium molybdate at pH 6 to 7 can be used for staining soya-bean dwarf virus strains (SbDV). The main significance of negative staining is to surround or embed the biological object in a suitable electron dense material which provides high contrast and good preservation.
Positive Staining:
In this technique heavy metal salts attach to the various organelle or macromolecules within the section to increase their electron density and they appear dark against a lighter background. Some positive stains are: Uranyl acetate (UA), Reynoldi’s lead citrate. Uranyl ions react strongly with phosphate and amino groups, so that the nucleic acids and certain proteins are highly stained. A 25% solution of UA is prepared in absolute methanol. This saturated solution is clarified by filtering through a syringe filter (2 µm pore size) just before to use.
In Reynoldi’s lead citrate stain lead ions bind to negatively charged component and osmium-reacted areas i.e. membranes. This stain can be prepared by adding 1.33 gm. lead nitrate and 1.76 gm. sodium citrate in 30 ml. CO2 free distilled water. It is boiled for 10 minutes. To this milky suspension 5 to 7 ml/N NaOH is added. It clears the suspension. This stain is stored in 50 ml volumetric flask.
Comparison between Negative Staining and Positive Staining:
Negative staining is a technique used in preparing specimens for electron microscopic examination. The specimen is mixed with an electron dense material that penetrates the interstices of the specimen but not the material of the specimen itself. The specimen then appears transparent against an opaque background.
The positive stain-stick with specimen and gives its colour where-as negative stain does not mix with the specimen but settle around its outer boundary and forming a silhouette (outline). The negative stain produces a dark background around the cell (Fig. 5).
Metal Shadowing:
Metal shadowing is a technique that makes surface details of very small particles visible in the electron microscope. In this technique a thin layer of evaporated metal such as gold or platinum is laid at an angle in a biological sample. An acid bath dissolves the biological
material, leaving a metal replica of its surface, which can then be examined in the transmission electron microscope (TEM).
Variation in the angle and thickness of the deposited metal allow an image to be formed because some incident electrons will be scattered in various directions rather than pass through the preparation. If the metal is deposited mainly on side of the sample, the image seems to have “shadows” where the metal appears dark and the shadows appear light.
Positive staining is also used in preparing specimen for electron microscopic examination but in this technique charged biological macromolecules or structures are rendered electron dense by allowing heavy metal ions of opposite charge to bind together.
Method:
The sample is spread on a mica sheet and then dried in a vacuum evaporator. A filament of heavy metal, such as platinum or gold is heated electrically so that the metal evaporates and some of it falls over the sample grid in a very thin film. In order to stabilize the replica, the specimen is then coated with a carbon film evaporated from a over-head electrode. The biological material is then dissolved by acid to observe only the metal replica of the sample. In electron micrographs of such preparation, the image is usually reversed Carbon coated areas appear light and platinum shaded areas appear dark.
Freeze Drying:
To remove the moisture (e.g., from food) by first freezing and then subjecting to a high vacuum used as a wild method for drying foods and chemicals while causing little decomposition is called freezing drying or a method drying food or blood plasma or pharmaceuticals or tissues without destroying their physical structure, material is frozen and then warmed in a vacuum so that ice sublimes (for biochemical the term lyophilized is often used).
Freeze drying technique helps in getting a correct idea about the shape of the particles. The morphology of the virus has been studied with a variety of electron microscopic methods. However, most of these techniques require a pre-treatment which may introduce structural changes in the object. The metal shadowing and negative staining which require no fixation may cause shrinkage during the drying process.
In addition they may alter or disrupt the object as has been shown for whole virus particles. In order to overcome harmful effect of drying on biological structure Wychoff tried to prevent the virus by freezing the specimen in a cold metal block and evaporating the ice in a vacuum evaporator. Williams developed a better procedure for freeze drying of virus, membranes etc.
Method:
The freezing dying technique has been described in detail by Williams (1952). This technique involves the rapid freezing of the specimen and the thin layer of water covering it on the grid to which it had adsorbed followed by sublimation of the ice around it. A thin layer of heavy metal is then deposited on the dehydrated surface to provide contrast.
Carbon Replica:
Preparation of carbon replica similar to plaster of Paris moulds are prepared in many cases to bring out the surface characteristics of virus particles.
Significance of Electron Microscopy:
Electron microscopy shows morphology and allow:
(i) Measurement of size of virus particles
(ii) Might indicate possible genus
(iii) Useful when nothing is known about the identity of the virus.
(iv) Results within 1-2 hours.