Functional Considerations: Development of Electron Microscopy
The electronic principles of both the TEM and the SEM are similar to those of a cathode ray tube (CRT), such as those used in television sets. In fact, the first EMs, built in the early 1930s, were developed independently in several countries by scientists and engineers working on the development of television. Although some viruses and other dried paracrystalline materials were studied with the EM in the 1930s, it was not until adequate fixation, embedding, and sectioning methods were developed in the 1950s that the TEM could be applied as a routine tool in biologic research.
tude smaller or thinner than those used for light microscopy. The TEM, whose electron beam has a wavelength of approximately 0.1 nm, has a theoretical resolution of 0.05 nm.
Because of the exceptional resolution of the TEM, the quality of fixation, i.e., the degree of preservation of subcellular structure, must be the best achievable.
Routine preparation of specimens for transmission electron microscopy begins with glutaraldehyde fixation followed by a buffer rinse and fixation with osmium tetroxide
Glutaraldehyde, a dialdehyde, preserves protein constituents by cross-linking them; the osmium tetroxide reacts with lipids, particularly phospholipids. The osmium also imparts electron density to cell and tissue structures because it is a heavy metal, thus enhancing subsequent image formation in the TEM.
Ideally, tissues should be perfused with buffered glutaraldehyde before excision from the animal. More commonly, tissue pieces no more than 1 mm5 are fixed for the TEM (compared with light microscope specimens, which may be measured in centimeters). The dehydration process is identical to that used in light microscopy, and the tissue is infiltrated with a monomeric resin, usually an epoxy resin, which is subsequently polymerized.
The plastic-embedded tissue is sectioned on specially designed microtomes using diamond knives
Because of the limited penetrating power of electrons, sections for routine transmission electron microscopy range from 50 nm to no more than 150 nm. Because abrasives used to sharpen steel knives leave unacceptable scratches on sections viewed in the TEM, diamond knives with nearly perfect sharpness are used. Sections cut by the diamond knife are much too thin to handle; they are floated away from the knife edge on the surface of a fluid-filled trough and picked up from the surface onto plastic-coated copper mesh grids. The grids have 50 to 400 holes/inch or special slots for viewing serial sections. The beam passes through the holes in the copper grid, then through the specimen, and the image is then focused on the viewing screen or photographic film.
Routine staining of transmission electron microscopy sections is necessary to increase the inherent contrast so that the details of cell structure are readily visible and photographable
In general, transmission electron microscopy sections are stained by adding materials of great density, such as ions of heavy metals, to the specimen. Heavy-metal ions may be bound to the tissues during fixation or dehydration or by soaking the sections in solutions of such ions after sectioning. Osmium tetroxide, routinely used in the fixative, binds to the phospholipid components of membranes, imparting additional density to the membranes.
Uranyl nitrate is often added to the alcohol solutions used in dehydration to increase the density of components of cell junctions and other sites. Sequential soaking in solutions of uranyl acetate and lead citrate is routinely used to stain sections before viewing with the TEM to provide high-resolution, high-contrast electron micrographs.
Freeze fracture is a special method of sample preparation for transmission electron microscopy, especially important in the study of membranes
The tissue to be examined may be fixed or unfixed; if it has been fixed, the fixative is washed out of the tissue before proceeding. A cryoprotectant, such as glycerol, is allowed to infiltrate the tissue, and the tissue is then rapidly frozen to about -160°C. Ice crystal formation is prevented by the use of cryoprotectants, rapid freezing, and extremely small tissue samples. The frozen tissue is then placed in a vacuum in the freeze fracture apparatus and struck with a knife edge or razor blade.
The fracture plane passes preferentially through the hydrophobic portion of the plasma membrane, exposing the interior of the plasma membrane
The resulting fracture of the plasma membrane produces two new surfaces. The surface of the membrane that is
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