Sunday, December 28, 2008

Transmission Electron microscope

This instrument employs a beam of electrons to image the specimen and no light is involved. An image of the specimen is viewed on a phosphor coated screen and the final pictures, called electron micrographs, are made on photographic plates or film. The source of the electrons is a heated tungsten filament, a single Lanthanum Hexaboride crystal, or a very sharp metal tip. The electron beam is accelerated down a vertical column (under vacuum) by high voltage and focused by electro-magnetic lenses. Electrons cannot be focused by glass lenses. In fact, even very thin glass will stop an electron beam. The voltages used to accelerate the beam can be from a few thousand up to a million volts in some research instruments. The usual potential is somewhere between 10 000V and 150 000V in routine instruments. Specimens for examination by TEM need to be very thin, of the order of 50 nanometers or less.

A common method for the preparation of biological specimens is sectioning. Fixed biological material is infiltrated with a plastic, often Methacrylate or an epoxide such as Araldite, which hardens to a solid block. Very thin slices, or sections, are cut from this block with an Ultra-Microtome using a glass or diamond knife. The sections are mounted on copper grids for examination in the TEM. Metals and other conducting material can be thinned by chemical means, mechanical polishing, or by electrolysis.

Specimens such as virus particles, proteins or DNA molecules are placed on very thin carbon films on a copper grid made by the evaporation of carbon in high vacuum. Specimens are usually stained or shadowed with heavy metal to increase the contrast. It has taken decades to develop suitable methods for EM preparation and it is still the most difficult part of the process.

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