It consists of an electron gun as the source of “illumination” at the top of a column containing a series of lenses, the specimen, and the imaging system. The layout of the TEM closely resembles that of a compound light microscope, which is also shown alongside for comparison purposes. Both microscopes use a condenser lens to focus the illuminating beam (either electrons or light) onto the sample and an objective lens to produce a focused and magnified image of the illuminated area. One or more projector lenses are then used to project a magnified image of the specimen onto the imaging system. In the case of an optical microscope this is the eye, film, or a charge-coupled device (CCD), while in a TEM images are viewed on a screen that fluoresces when struck by electrons.
Two factors complicate the imaging of samples in a TEM. First, due to the limited penetration of electrons in matter, specimens for TEM must be extremely thin (approximately 0.1 micron). Second, since the specimens must be inserted into the electron microscope column which is under vacuum, they must be completely dry. This latter point requires biological samples to be dehydrated and fixed prior to viewing. There is a range of TEM specimen preparation techniques available depending on the type of sample. Solid, inorganic samples can be thinned using a combination of mechanical, chemical, and ion beam thinning. Thin biological specimens are normally prepared using ultramicrotomy, which involves slicing thin sections of the sample using a sharp glass or diamond knife. Surfaces can be replicated as thin films of carbon and shadowed with heavy metals to produce remarkably realistic images of surface relief. As with most kinds of microscopy, specimen preparation is an important and specialized area of expertise.
Contrast in TEM images can arise in several ways. Variation of mass and/or thickness gives rise to contrast due to greater absorption or scattering of electrons from heavier and/or thicker parts of the specimen. To increase contrast in biological specimens, tissues are often “stained” with heavy metals. Another contrast mechanism is known as phase contrast. Unlike phase contrast in optical microscopy, phase contrast results from the interference between electrons, which have different phases after passing through the specimen. Phase contrast can be used to directly image the crystal structure of a specimen at atomic resolution.
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