Autoradiography

The process of localizing radioactive materials onto a cell is known as autoradiography. ₃H (tritium) is used in cell analysis because it is a relatively weak beta emitter (thus making it safer to handle) and, more significantly, can be localized within cell organelles. ₁₄C and ₃₂P are also used, but are more radioactive, require significantly more precautions in handling, and are inherently less capable of resolving intracellular details. They are used at the tissue or organ level of analysis.

Radioactive isotopes can be incorporated into cellular molecules. After the cell is labeled with radioactive molecules, it can be placed in contact with photographic film. Ionizing radiations are emitted during radioactive decay and silver ions in the photographic emulsion become reduced to metallic silver grains. The silver grains not only serve as a means of detecting radioactivity but, because of their number and distribution, provide information regarding the amount and cellular distribution of the radioactive label.

The process of producing this picture is called autoradiography and the picture is called an autoradiogram. The number of silver grains produced depends on the type of photographic emulsion and the kind of ionizing particles emitted from the cell. Alpha particles produce straight, dense tracks a few micrometers in length. Gamma rays produce long random tracks of grains and are useless for autoradiograms. Beta particles or electrons produce single grains or tracks of grains. High-energy beta particles (such as those produced by ₃₂P) may travel more than a millimeter before producing a grain. Low-energy beta particles (₃H at 14°C) produce silver grains within a few micrometers of the radioactive disintegration site, and so provide very satisfactory resolution for autoradiography.

The site of synthesis of cellular molecules may be detected by feeding cells a radioactive precursor for a short period and then fixing the cells. During this pulse labeling, radioactivity is incorporated at the site of synthesis but does not have time to move from this site. The site of utilization of a particular molecule may be detected by chase labeling. Cells are exposed to a radioactive precursor, radioactivity is then washed or diluted away, and the cells allowed to grow for a period of time. In this case, radioactivity is incorporated at the site of synthesis, but then has time to move to a site of utilization in the cell.

₃H-thymidine can be used to locate sites of synthesis and utilization of DNA. Thymidine, the deoxyribose nucleoside of thymine, can be purchased with the tritium label attached to the methyl group of thymine. Thymidine is specifically incorporated into DNA in Tetrahymena. Some organisms can remove the methyl group from thymine, and incorporate the uracil product into RNA. Even in this case, RNA would not be labeled because the tritium label would be removed with the methyl group. Methyl-labeled thymidine, therefore, serves as a very specific label for DNA.

This is known as pulse labeling, after which the cells are washed free of the radioactive media. All remaining radioactivity would be due to the incorporation of the thymidine into the macromolecular structure of DNA. The cells will be fixed, covered with a photographic emulsion, and allowed to develop.

During this time, the activity emanating from the ₃H will expose the photographic emulsion, causing the presence of reduced silver grains immediately above the location of the radioactive source (DNA). Thus, it will be possible to localize the newly synthesized DNA, or that which was in the S phase of mitosis during the time period of the pulse labeling.

Rules for Safe Handling of Radioactive Isotopes

• All work with radioactive material must be done in a tray lined with absorbant paper.
• All glassware and equipment contacting radioactive material must be appropriately labeled and kept inside the tray. The only exception is that microscope slides of labeled cells may be removed from the tray after the drop of labeled cells has been applied to the slide and allowed to dry.
• Plastic gloves should be worn when handling radioactive material.
• All waste solutions containing radioisotopes, all contaminated gloves, paper, etc., must be placed in appropriate liquid or dry radioactive waste containers.