The steps required to prepare biological material for electron microscopy are challenging. How can we be sure that the image of the fixed, dehydrated, resinembedded specimen bears any relation to the delicate, aqueous biological system present in the living cell? The best current approaches to this problem depend on rapid freezing. If an aqueous system is cooled fast enough and to a low enough temperature, the water and other components in it do not have time to rearrange themselves or crystallize into ice. Instead, the water is supercooled into a rigid but noncrystalline state—a “glass”—called vitreous ice. This state can be achieved by slamming the specimen onto a polished copper block cooled by liquid helium, by plunging it into or spraying it with a jet of a coolant such as liquid propane, or by cooling it at high pressure.
Some rapidly frozen specimens can be examined directly in the electron microscope using a special cooled specimen holder. In other cases, the frozen block can be fractured to reveal interior cell surfaces, or the surrounding ice can be sublimed away to expose external surfaces. However, we often want to examine thin sections. A compromise is therefore to rapidfreeze the tissue, replace the water with organic solvents, embed the tissue in plastic resin, and finally cut sections and stain. Although technically still difficult, this approach stabilizes and preserves the tissue in a condition very close to its original living state (Figure 9–44).
Image clarity in an electron micrograph depends upon having a range of contrasting electron densities within the specimen. Electron density in turn depends on the atomic number of the atoms that are present: the higher the atomic number, the more electrons are scattered and the darker that part of the image. Biological tissues are composed mainly of atoms of very low atomic number (primarily carbon, oxygen, nitrogen, and hydrogen). To make them visible, tissues are usually impregnated (before or after sectioning) with the salts of heavy metals such as uranium, lead, and osmium. The degree of impregnation, or “staining,” with these salts will vary for different cell constituents. Lipids, for example, tend to stain darkly after osmium fixation, revealing the location of cell membranes.
We have seen how antibodies can be used in conjunction with fluorescence microscopy to localize specific macromolecules. An analogous method—immunogold copper grid covered with carbon and/or plastic flm
Figure 9–43 The metal grid that supports the thin sections of a specimen in a transmission electron microscope.
Figure 9–44 Thin section of a cell. this thin section is of a yeast cell that has been very rapidly frozen and the vitreous ice replaced by organic solvents and then by plastic resin. the nucleus, mitochondria, cell wall, golgi stacks, and ribosomes can all be readily seen in a state that is presumed to be as lifelike as possible. (Courtesy of andrew staehelin.) 0.5 µm electron microscopy—can be used in the electron microscope. The usual procedure is to incubate a thin section first with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electrondense and can be seen as a black dot in the electron microscope (Figure 9–45). Different antibodies can be conjugated to different sized gold particles so multiple proteins can be localized in a single sample.