(i) Source of illumination is a beam of electrons (not visible light)
(ii) There are a series (usually three sets) of electromagnets for focusing the beam of electrons. These are called magnetic lenses (not made out of glass)
The general construction pattern of TEM is same as that explained above. TEM is for studying the structural details of the various components present in a cell or microbe.
The specimens form a contrasting image with all the structural details. Depending on their refractive index, the components of a specimen bring about varying degrees of scattering of the electron rays.
Generally however biological specimens do not produce a great degree of scattering of electrons, hence the contrast is low. Staining is resorted to improve the contrast-But instead of dyes used for staining in light microscopy, electron dense heavy metal salts are used for staining in electron microscopy.
Some problems are encountered while viewing a specimen through TEM. Many a time artifacts appear which can be easily mistaken to be a component of a cell.
(An artifact is the appearance of something in an image due to causes within the optical system or due to the preparation of a specimen and does not represent any component of the specimen under view).
Proper preparation of the specimen, correct adjustment of the electron beams, accurate acumination will help in reducing the chances of artifact appearance.
Preparation of specimen for TEM:
The biological specimens to be examined have to be specially prepared in order to obtain proper structural details with high magnification, but at the same time avoiding the appearance of artifacts. The following steps are necessary before the specimens are ready for observation under TEM.
Dehydration and fixation:
Under light microscopy specimens are kept in water or oilier liquids for observation. But in an electron microscope, the specimens have to be absolutely dry.
Any water present, the specimen would boil (as it is in a vacuum) resulting in disintegrating the structural organization of the specimen.
Additionally the specimen also has to be fixed in its proper orientation. Fixation and dehydration have to be carried out in several stages in order to prevent distortion.
From the point of view of magnification obtained in an electron microscope, microbes are too thick for viewing.
Hence normally they are cut into thin sections using an ultra microtome. The ultra microtome has a device which advances the fixed specimen on a diamond or glass knife surface at a known thickness so that sections of uniform thickness are cut. The microbes are usually embedded in a plastic resin to facilitate sectioning.
In order to enhance the contrast, the specimens are stained with heavy metal containing compounds such as phosphotungstic acid.
The staining can be negative or positive. In negative staining, the sectioned specimen in dipped in heavy metal solution which stains the background and not the specimen. The specimens are then visualized in relief against a dark background.
This is a special technique employed mainly to reveal the details of structure in microbes. In freeze etching, various biochemically defined layers are made visible, including the organelle details. In this technique (instead of using fixatives to preserve the cell structure) the specimens are rapidly struck with a knife blade.
At this temperature (freezing) biological specimens are hard to cut, but they crack along the lines of their natural weakness i.e. they are fractured. The fractured specimen is then etched i.e. the water (ice) is allowed to evaporate from the surface.
This evaporation raises the surface layers of the specimen. A replica then is made of the freeze etched specimen, by exposing it to vapors of heavy metal (platinum) at 45° angle to produce a shadow effect.
The specimen then is rotated at 90, and exposed to vaporized carbon. This produces a replica of the surface of the specimen.
After removing the remnants of biological material, the carbon replica is viewed under electron microscope. Surface details (internal as well external) of organelles can be clearly visualized by the freeze etching technique.
2. Scanning electron microscope (SEM):
SEM is primarily used for visualizing the surface architecture of the specimen (pollen grains, hairs, membranes, etc.) rather than the internal details. In a SEM, an electron beam is scanned across the surface and a three dimensional image is produced.
The construction plan and working principles of a SEM is different from that of Tem. In SEM, an accelerated beam of electrons is produced from the electron gun and is focused on the specimen by the condenser lens.
The magnetic lenses of a SEM are so constructed as to produce an extremely thin beam of electrons. Electrons are then transmitted from the collector to a detector which has a substance that emits light when struck by electrons. The light so emitted is converted to an electrical current which is used to control the brightness of an image on a CRT (Cathode Ray Tube) screen.
The secondary electrons deflected out of the specimen will be a replica of the refractive index of the surface and thus produce an image on the CRT screen revealing all the topographical details.
Image contrast mainly depends on surface topography which determines the number of secondary electrons reaching the detector. The image on the CRT screen will be three dimensional.
Magnification of the image of the specimen in SEM is not achieved through the lenses as in a light microscope or TEM. It is dependent upon the ratio of the length of the scan across the specimen surface to the length of the scan of CRT.
For instance if the electron beam scans 100 nm across of specimen and the image on CRT is 100 nm, the magnification is 100000 times. Thus one can decide the magnification (depending on the requirement) by adjusting the scan distance across the specimen.
SEM also has a resolution equal to that of TEM. A resolution from 1 -10 nm is possible with a corresponding magnification from 10000-100000.
Preparation of specimen for SEM
As SEM also operates on a vacuum like TEM, total dehydration is necessary, but as surface topography is essential, dehydration should be done in such a way as not to disturb the surface configuration.
This is achieved by critical point drying which minimizes artifact formation. In critical point drying at a particular temperature and pressure, the liquid changes to gas without any surface tension damage to the specimen.
The specimen is first immersed in ethanol or acetone to remove water and then in pressurized-liquid of C02 simultaneously raising the temperature above 32°C, the critical point of C02. At this temperature range, the liquid vaporizes without surface tension leaving the specimen perfectly dry.
2. Shadow casting:
In this technique, the specimen is coated with an extremely thin layer of gold, gold-palladium or platinum at an oblique angle, so that the specimen produces a shadow on the uncoated side.
The shadow technique results in the production of a three dimensional topographic image of the specimen. Coating is done with a device called a sputter coater. In some cases double shadowing also can be done by coating the materials from two different angles.
3. Surface replica:
In this technique, widely used for the study of architectural pattern of the wall surface of spores, pollen etc, a thin layer of a coherent material is coated on to the specimen who is then floated on to a water surface, from where it is transferred on to a strong acid or alkali.
This dissolves the specimen without damaging the replica. The replica is then dried and kept on the metal grid for viewing.