Autoradiography of Tissue Sections

Autoradiography is the study of biological events in tissue sections using radioactivity. Autoradiography permits the localization of radioactive substances in tissues by means of emitted radiation effects on photographic emulsions. Silver bromide crystals present in the emulsion act as microdetectors of radioactivity in the same way that they respond to light in common photography. The first step of autoradiography is to deliver a radioactive compound to the cells. A variety of molecules, including radioactive amino acids, radioactive nucleotides, and radioactive sugars, can be used, depending on the purpose of the study. These molecules are called precursors, because they may be used by the cells to synthesize larger molecules, such as proteins, nucleic acids, or polysaccharides and glycoproteins. The tissue sections are prepared and are covered with photographic emulsion. The slides are kept in light-proof boxes; after an adequate exposure time they are developed photographically and examined. When the silver bromide crystals present in the photographic emulsion are hit by radiation they are transformed into small black granules of metallic silver, thus revealing the existence of radioactivity in the tissue. The structures that contain radioactive molecules become covered by these granules. This procedure can be used in both light and electron microscopy (Figure 1–11).

Figure 1–11

Autoradiographs from the submandibular gland of a mouse injected with [³H] fucose 8 h before being killed. A: With a light microscope it is possible to observe black silver grains indicating radioactive regions in the cells. Most radioactivity is in the granules of the cells of the granular ducts of the gland. High magnification. B: The same tissue prepared for electron microscope autoradiography. The silver grains in this enlargement appear as coiled structures localized mainly over the granules (G) and in the gland lumen (L). High magnification. (Courtesy of TG Lima and A Haddad.)

Much information becomes available by localizing radioactivity in tissue components. Thus, if a radioactive amino acid is used, it is possible to know which cells in a tissue produce more protein and which cells produce less, because the number of silver granules formed over the cells is proportional to the intensity of protein synthesis. If a radioactive precursor of DNA (such as radioactive thymidine) is used, it is possible to know which cells in a tissue (and how many) are preparing to divide. Dynamic events may also be analyzed. For instance, to determine where in the cell a protein is produced, if it is secreted, and which path it follows in the cell before being secreted, several animals are injected with a radioactive amino acid and are killed at different times after the injection. Autoradiographs of the sections, taken at various times throughout the experiment, will show the migration of the radioactive proteins. To determine where new cells are produced in an organ and where they migrate, several animals are injected with radioactive thymidine and are killed at different times after the injection. Autoradiographs of the sections will show where the cells divide and where (or if) they migrate (Figure 1–12).

Figure 1–12

Autoradiographs of tissue sections from a mouse that was injected with [³H] thymidine 1 h before the organs were collected. Because the autoradiographs were exposed for a very long time, the radioactive nuclei became heavily labeled and appear covered by clouds of dark granules. A: Many cells were dividing at the base of the intestinal glands (arrowheads), but no cells were dividing along the villi (arrows). Low magnification. B: A section of a lymph node shows that cell division occurs mostly at the germinal centers of this structure (arrow). Low magnification. (Courtesy of TMT Zorn, M Soto-Suazo, CMR Pellegrini, and WE Stumpf.)

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