Immunoelectron microscopy can be defined as any technique that uses antibodies, or molecules that interact with antibodies (for example, protein A or protein G), in conjunction with electron microscopy to localize ultrastructurally antigens or antibodies in cells and tissues. In addition, a number of other biological macromolecules whose specific ligand-bind-ing properties are known can be used (for example, lectins). Immunoelectron microscopy was a term that was originally confined to studies that made use of the transmission electron microscope but now include those that use the scanning electron microscope.
In the transmission electron microscope, electrons serve as a pseudo-light source. High-energy electrons emitted from a filament are accelerated and forced by electromagnets into a very fine coherent beam in the center of the microscope column. Because electrons are used, the microscope column and specimen area are maintained in a vacuum (10~6-10~s Torr) to prevent the deflection of electrons by atmospheric gases. The electron beam is focused on to the sample. Here, the more electron-dense molecules in the sample under observation adsorb and scatter electrons, preventing their passage through the sample. Those electrons with enough energy to penetrate are transmitted, further focused, and projected on to a phosphor screen. The image is resolved on the phosphor screen due to the emission of photons in the area struck by the transmitted electrons. The image can be permanently recorded directly on photographic film sensitive to electrons or visualized with photographic cameras interfaced with the electron microscope viewing chamber and a computer to rapidly generate high-resolution computer processed images.
In preparation for transmission electron microscopy, samples are usually fixed with aldehydes (glutaraldehyde, formaldehyde or a combination of the two), post-aldehyde fixed in osmic acid, en bloc stained with a uranyl acetate solution, dehydrated with alcohols and embedded in a plastic resin. The embedded samples are thin sectioned with a glass or diamond knife and subsequently counterstained with heavy metals (uranium and lead salts) to further enhance the electron density of positively or negatively charged molecules in the sample. Specimens thus prepared have good contrast and can be used to observe and record the molecular and subcellular detail of cells and tissues. Alternatively, molecules may be adsorbed to a thin plastic film followed by embedding in an electron-dense stain or angular shadowing with evaporated metals.
The mechanical principles of operation for the scanning electron microscope are virtually the same as for transmission electron microscopes. The major difference is in the formation of the image. In scanning electron microscopes, the focused electrons are scanned over the surface of a sample, as implied by the name. Instead of transmitting electrons, secondary electrons emitted from the interaction of electron beam and sample are collected, electronically deciphered and multiplied, and projected on to a high-resolution cathode-ray tube. These images give a quasi three-dimensional impression of the structure under observation because of the great depth of field of the scanning electron microscope. The image is recorded on film using an ordinary camera focused on the cathode-ray tube. Samples for scanning electron microscopy must be fixed and dehydrated, as in transmission electron microscopy, but instead of embedding the sample in a plastic resin, it is covered with a conductive coating of a fine metal, such as gold, by evaporation; no further processing is necessary.
As some examples of the utility of these techniques in ultrastructural research, transmission electron microscopy . an reveal the structure of macro-molecules adsorbed to a film, the surface detail of samples through surface replicas, or the internal structure of cells or infectious agents through thin sections. Scanning electron microscopes are primarily used for observing the surface detail of cells and tissues. The incorporation of immunoconjugates or other ligands into electron microscopic studies permits the high-resolution study of the antigenic composition of cell organelles and surfaces in concert with ultrastructural analyses. Singularly, or in combination, these two types of microscopic analysis have proved invaluable in the correlative study of morphologic structures and their antigenic composition.
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