Many small molecular precursors of larger molecules, such as the amino acids that make up proteins and the nucleotides that make up nucleic acids, may be tagged by incorporating a radioactive atom or atoms into their molecular structure. The radioactivity is then traced to localize the larger molecules in cells and tissues. Labeled precursor molecules can be injected into animals or introduced into cell or organ cultures. In this way, synthesis of DNA and subsequent cell division, synthesis and secretion of proteins by cells, and localization of synthetic products within cells and in the extracellular matrix have been studied.
FIGURE 1.8 • Examples of autoradiography used in light and electron microscopy. a. Photomicrograph of a lymph node section from an animal injected with tritiated [3H]thymidine. Some of the cells exhibit aggregates of metallic silver grains, which appear as small black particles (arrows). These cells synthesized DNA in preparation for cell division and have incorporated the [3H]thymidine into newly formed DNA. Over time, the low-energy radioactive particles emitted from the [3H]thymidine strike silver halide crystals in a photographic emulsion covering the specimen (exposure) and create a latent image (much like light striking photographic film in a camera). During photographic development of the slide with its covering emulsion, the latent image, actually the activated silver halide in the emulsion, is reduced to the metallic silver, which then appears as black grains in the microscope. 1,200. (Original slide specimen courtesy of Dr. Ernst Kallenbach.) b. Electron microscopic autoradiograph of the apical region of an intestinal absorptive cell. In this specimen, 125I bound to nerve growth factor (NGF) was injected into the animal, and the tissue was removed 1 hour later. The specimen was prepared in a manner similar to that for light microscopy. The relatively small size of the silver grains aids precise localization of the 125I–NGF complexes. Note that the silver grains are concentrated over apical invaginations (inv) and early endosomal tubular profiles (tub). 32,000. (Electron micrograph courtesy of Dr. Marian R. Neutra.)
Sections of specimens that have incorporated radioactive material are mounted on slides. In the dark, the slide is usually dipped in a melted photographic emulsion, thus producing a thin photographic film on the surface of the slide. After appropriate exposure in a light-tight box, usually for days to weeks, the exposed emulsion on the slide is developed by standard photographic techniques and permanently mounted with a coverslip. The slides may be stained either before or after exposure and development. The silver grains in the emulsion over the radioactively labeled molecules are exposed and developed by this procedure and appear as dark grains overlying the site of the radioactive emission when examined with the light microscope (Fig. 1.8a).
These grains may be used simply to indicate the location of a substance, or they may be counted to provide semiquantitative information about the amount of a given substance in a specific location. For instance, after injection of an animal with tritiated thymidine, cells that have incorporated this nucleotide into their DNA before they divide will have approximately twice as many silver grains overlying their nuclei as will cells that have divided after incorporating the labeled nucleotide.
Autoradiography can also be carried out by using thin plastic sections for examination with the EM. Essentially the same procedures are used, but as with all TEM preparation techniques, the processes are much more delicate and difficult; however, they also yield much greater resolution and more precise localization (Fig. 1.8b).
A microscope, whether simple (one lens) or compound (multiple lenses), is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye. The simplest microscope is a magnifying glass or a pair of reading glasses.