Diagram of a typical light microscope. This drawing shows a cross-sectional view of the microscope, its operating components, and light path. imaging illuminating (Courtesy of Carl Zeiss, Inc., Thorn-

backed by extracellular space is called the E-face; the face backed by the protoplasm (cytoplasm) is called the P-face. The specimen is then coated, typically with evaporated platinum, to create a replica of the fracture surface. The tissue is then dissolved, and the surface replica, not the tissue itself, is picked up on grids to be examined with the TEM. Such a replica displays details at the macromolecu-lar level (see Fig. 2.5, page 23).

In scanning electron microscopy, the electron beam does not pass through the specimen but is scanned across its surface

In many ways, the SEM more closely resembles a television tube than the TEM. For examination of most tissues, the sample is fixed, dehydrated by critical point drying, coated with an evaporated gold-carbon film, mounted on an aluminum stub, and placed in the specimen chamber of the SEM. For mineralized tissues, it is possible to remove all the soft tissues with a bleach and then examine the structural features of the mineral.

Scanning is accomplished by the same type of raster that scans the electron beam across the face of a television tube. Electrons reflected from the surface (backscattered electrons) and electrons forced out of the surface (secondary electrons) are collected by one or more detectors and reprocessed to form a three-dimensional-like image on a high-resolution CRT.

Photographs may then be taken of the CRT to record data, or the image may be recorded on videotape. Other detectors can be used to measure x-rays emitted from the surface, cathodoluminescence of molecules in the tissue below the surface, and Auger electrons emitted at the surface.

The scanning-transmission electron microscope combines features of the TEM and SEM to allow electron probe x-ray microanalysis

The SEM configuration can be used to produce a transmission image by inserting a grid holder at the specimen level, collecting the transmitted electrons with a detector, and reconstructing the image on a CRT. This latter configuration of a SEM or scanning-transmission electron microscope (STEM) facilitates the use of the instrument for electron probe x-ray microanalysis.

Detectors can be fitted to the microscope to collect the x-rays emitted as the beam bombards the section, and with appropriate analyzers, a map can be constructed that shows the distribution in the sections of elements with an atomic number above 12 and a concentration sufficient to produce enough x-rays to analyze. Semiquantitative data can also be derived for elements in sufficient concentration. Thus, both the TEM and the SEM can be converted into sophisticated analytical tools in addition to being used as "optical" instruments.

focusing control eyepiece source

field diaphragm objective auxiliary condenser exit pupil (eyepoint)

real intermediate image exit pupil of objective specimen condenser diaphragm light source (jj exit pupil (eyepoint)

real intermediate image exit pupil of objective specimen condenser diaphragm field diaphragm light source (jj focusing control source objective eyepiece auxiliary condenser stage condenser diaphragm condenser stage control field diaphragm

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