Importance of Autoradiography in Cell Biology

Any discussion of the uses of radioisotopes as tracers in cell biology would be incomplete without at least a brief description of autoradiography.

The materials and equipment used in this technique differ signifi­cantly from those employed in the methods previously described.

In autoradiography, the biological sample containing the radioisotope is placed in close contact with a sheet or film of photographic emulsion.

Rays emitted by the radioisotope enter the photographic emulsion and expose it in a manner similar to visible light.

After some period of time (usually several days to several weeks), the film is developed and the loca­tion of the radioisotope in the original sample is deter­mined from the exposure spots on the film.

Autoradiography is gener­ally not employed as a quantitative technique (al­though it can be under certain conditions); instead, it is used to determine the specific region of localization of a radioactive tracer.

The method is most often used with histological sections to determine the precise lo­cation of a labeled compound in a tissue or in a cell and may be applied at either the light microscope or elec­tron microscope level.  Autoradiography has also been successfully employed in conjunction with electropho­resis, chromatography (i.e., thin-layer chromatogra­phy, paper chromatography, etc.), and other molecular fractionation methods for identifying zones containing labeled compounds (Fig. 14-9).

Because the main goal of autoradiography is to de­termine the precise location of the tracer, the degree of resolution obtained in the autoradiograph is of pri­mary importance.

Resolution depends on:

(1) The type of photographic emulsion used,

(2) The distance be­tween the radioactive sample and the emulsion, and

(3) The nature of the emitted radiation.

A variety of photographic emulsions are available, varying in sen­sitivity and resolution according to the size and con­centration of their silver halide grains, with the least sensitive films generally offering the highest degree of resolution. Emulsions can also be obtained that are particularly sensitive to specific types of radiation.

Because radiation is emitted in all directions from the radioactive source, the greater the distance between the film and the source, the more diffuse is the result­ing image. It is therefore important to use very thin samples and to place them in very close contact with the film. The highest degree of resolution is obtained with radioisotopes that emit rays of short path length and that have a high specific ionization.

Alpha par­ticles provide high resolution, but radioisotopes emitting this type of radiation are rarely useful in biological experiments. Autoradiographs become in­creasingly diffuse as the E_max of a beta emitter in­creases, but ³H, ¹⁴C, ³⁵S, and ⁴⁵Ca do yield good reso­lution. Gamma rays are inefficient as a result of their extremely high ranges and low specific ionizations.

Autoradiography may be employed with large but thin slices of tissues or organs (gross autoradiogra­phy) or with smaller pieces sectioned and prepared for light or electron microscopy. For example, the deposi­tion of calcium in bone has been studied using ⁴⁶Ca by cutting thin, flat, longitudinal slices through bone and placing them against large sheets of photographic film.

When used in conjunction with light microscopy, the paraffin-embedded tissue sections are first mounted on slides, which are then coated with photo­graphic emulsion and stored in a light-tight, usually lead-lined box (to minimize background exposure that results from the effects of cosmic radiation and other sources of radiation).

Several days or weeks later, af­ter photochemical development, the sections are con­ventionally stained to better visualize the biological material and the distribution of radioisotope is deter­mined microscopically from the location of dark expo­sure spots (called “grains”) on the section (Fig. 14- 10).

Autoradiography may also be used with thin sec­tions of tissues prepared for electron microscopy (Fig. 14-11). This procedure involves preliminary examination and photography of the thin section followed by coating the grid with a very thin layer of photographic emulsion containing silver halide crystals of particu­larly small size.

After several days, the grid is devel­oped and again examined and photographed with the electron microscope to identify those regions of the original section that contain clusters of metallic silver grains and therefore the radioactive tracer.