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The Transmission Electron Microscope (TEM)

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.

A small routine Transmission Electron Microscope (TEM) used for biological work.

A small routine Transmission Electron Microscope (TEM) used for biological work.


The Scanning Electron Microscope (SEM)

This instrument is quite different from a TEM because electrons are not used to directly image the specimen, but to excite it in such a way that it gives out secondary electrons. These secondary electrons are collected by detectors and used to form the image. Most of the SEM pictures seen in articles and books are secondary electron images. However, the beam not only causes the generation of secondary electrons, but scatters them as well. These backscattered electrons can also be used to form an image. In fact there are many different specimen beam interactions that produce useful information about the specimen. For example, the beam of electrons also causes the specimen to emit X-Rays. These can be collected by a separate detector and used to analyze the elemental components making up the specimen, or even to build up an elemental map of the specimen on a video screen.

In a nutshell -- an electron beam is focused to a small spot by similar lenses to those used in a TEM. This spot, which may be only a few nanometers in diameter, is scanned over the surface of the specimen by electromagnetic scanning coils, in the same way as the beam in a domestic television set. This scanning is digital and performed in fine steps, line by line, until the whole specimen has been covered. In this way the surface of the specimen is divided into a large number of points and the current that flows as a result of the secondary or backscattered electrons at each of these points is used to modulate the beam of a video monitor which is in exact synchronicity with the scanning beam. This results in an image being built up which represents the surface of the specimen. The process is very fast. The scanning rate, the accelerating voltage, the conductivity of the specimen and many other factors all have to be taken into account. For example, in non-conducting specimens such as biological tissue the electrons will collect in the specimen and charge it up so that nothing except bright light will be seen on the monitor. The electrons, after doing their work, need to be conducted away from the specimen and this is done by coating non-conducting specimens with metal or carbon, often both. A very fine layer of gold or platinum is evaporated under vacuum onto the surface. This does not change the structure, or hide detail, because the film is only a few nanometers thick.

A small routine Scanning Electron Microscope (SEM) with an X-Ray Energy Dispersive Spectrometer (EDS). The big tank on the left of the column is the Dewar for liquid Nitrogen used to keep the X-Ray detector cool.

A small routine Scanning Electron Microscope (SEM) with an X-Ray Energy Dispersive Spectrometer (EDS). The big tank on the left of the column is the Dewar for liquid Nitrogen used to keep the X-Ray detector cool.


The Scanning Transmission Electron Microscope

This instrument is usually a normal TEM which has an added scanning system. Instead of a single focused beam of electrons, a small spot is scanned across the specimen and the image is collected on a detector beneath the specimen. This instrument is very useful for the X-Ray microanalysis (EDS) of very small parts of thin specimens.

A Large research Scanning Transmission Electron Microscope used for high magnification imaging and Microanalysis.

A Large research Scanning Transmission Electron Microscope used for high magnification imaging and Microanalysis.


X-Ray microanalysis in the Electron Microscope

All specimens give off X-Rays when they are irradiated by an electron beam. These X-Rays can be collected by detectors and used to identify and quantify the different elements present in the specimen. This can be done in two ways. By detecting and measuring X-Rays of a particular wavelength, this is Wavelength Dispersive Spectroscopy (WDS) or by collecting X-Rays of different energy in Energy Dispersive Spectroscopy (EDS). The spectrometers fitted to a microscope to carry out WDS have special crystals in them to deflect the X-Ray beam into the detectors. These are exceedingly expensive and complicated instruments to operate. They are generally referred to as Microprobes. Microprobes can give very accurate quantitative information.

On the other hand an Energy Dispersive X-Ray Spectrometer (EDS) can be fitted to any reasonably good electron microscope and will give decent qualitative results. However, biological specimens are usually stained with heavy metals such as Uranium (U), Osmium(Os) and Lead (Pb). X-rays emitted from these heavy metals may interfere with the characteristic X-Ray spectrum of other elements and so it is necessary to use unstained material when EDS is to be used. An Energy Dispersive X-Ray detector is kept at the temperature of Liquid Nitrogen to reduce the effect of electronic noise.