neither the old DNA nor the RNA would make use of the thymine. We could in this way mark cells preparing to divide. (Actually, thymine itself is not taken up in mammalian cells, but its nucleoside is. A nucleoside is the base plus the sugar, or, in other words, the nucleotide minus the phosphoric acid.) The nucleoside of thymine is called thymidine, and we say that thymidine is a specific component of DNA and can be used, both in laboratory studies and in living organisms, for labeling DNA.

Thymidine labeled with radioactive compounds is available as ¹⁴C-thymidine (thymidine with a stable carbon atom replaced by a radioactive carbon atom) and as ³H-thymidine (thymidine in which a stable hydrogen atom has been replaced by tritium). Thus, when cells actively making DNA are exposed to radioactive thymidine, they incorporate it, and the DNA becomes radioactive.

We have thus found a way to complete the first part of the task, the labeling of new DNA. We still must find out how to distinguish labeled DNA among the many components of the cell. We might do it with a system based on measuring the amount of radioactivity incorporated into the DNA of cells exposed to radioactive thymidine, as an approximation of the frequency of cell division in the group of cells. However, a better method for studying cells synthesizing DNA, and thus preparing to divide, is the use of high-resolution autoradiography.

Detecting DNA with Autoradiography

Autoradiography is based on the same principle as photography. Just as photons of light impinging on a photographic emulsion produce an image, so do beta particles (or alpha particles) emitted by decomposing radioactive atoms. A photographic emulsion is a suspension of crystals of a silver halide (usually silver bromide) embedded in gelatin. When crystals of silver bromide are struck by beta particles, the silver atoms are ionized and form a latent image, so called because it is invisible to our eyes. After the emulsion is developed and fixed, each little aggregate of reduced silver atoms becomes a visible black speck on the emulsion. The distribution and combination of the specks make up the photographic image (see Figure 14). In ordinary photography such an image is a negative, which has to be converted into the positive photograph by printing. In autoradiography we are satisfied to look at the negative image since the clusters of developed silver atoms, appearing under a light microscope as black dots, supply all the information we need.

Figure 14 Schematic diagram of a radioautograph.

The distinction of having made the first autoradiograph belongs to the French physicist, Antoine Henri Becquerel; and to another Frenchman, A. Lacassagne, goes the credit for having introduced this technique into biological studies. Lacassagne used autoradiography to study distribution of radioactive polonium in animal organs. After World War II, when radioactive isotopes were first available in appreciable quantities, autoradiography was further perfected through the efforts of such scientists as C. P. Leblond in Canada, S. R. Pelc in England, and P. R. Fitzgerald in the United States.

Today autoradiography is sufficiently precise to locate radioactively labeled substances in individual cells and even in chromosomes and other structures within the cell. Two conditions must be met to achieve this high resolution: (1) The radiation from the radioactive element in the cells must be of very short range. (2) The cells must remain in close contact with the photographic emulsion throughout the various experimental manipulations. When these conditions are met, the black dots will appear in the emulsion directly above the cell or cell part from which the radiation came (see Figure 14).

Shortness of range is satisfied by use of tritium, since its beta particles travel only about 1 micron (one thousandth of a millimeter) and the diameters of mammalian cells range from 15 to 40 or more microns. A mammalian-cell nucleus is at least 7 to 8 microns in diameter.