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.
Figure 15 Cells being prepared for autoradiography. (a) Cells being coated with a photographic emulsion. (b) Coated cells being exposed to produce a latent image.
The condition of close contact between cells and emulsion is achieved by the technique of dip-coating autoradiography. In this process the glass slide on which the cells are carried is dipped into a melted photographic emulsion (see [Figure 15]a), a thin film of which clings to the slide. After it has been dried, the slide is placed in a lighttight box and kept in a refrigerator for the desired period of exposure, usually several days or weeks. During this period disintegrating radioactive atoms within the cells continue to emit beta particles, which, in turn, produce a latent image in the overlying emulsion. After the exposure is complete, the slide is developed and fixed like a photographic plate, and a stain is applied which penetrates the emulsion so that the outlines of the cells and their internal structures can be seen. The fixing process removes all silver bromide that has not been ionized so that the emulsion is reduced to a thin, transparent film of gelatin covering the stained cells and containing only the clusters of silver grains that were struck by the beta particles.
Figure 16 Radioautographs of tumor cells. Above, tumor cells and blood cells. Below, magnification of tumor cells.
When the finished autoradiograph is examined under the microscope, it will look like the radioautographs of tumor cells in [Figure 16]. In the upper micrograph the tumor cells are the larger ones and the smaller ones are blood cells. The dense structures in the center of the tumor cells are nuclei. The cells were exposed to tritium-labeled thymidine, and those synthesizing DNA at the time of exposure took up the thymidine and became radioactive. They can be identified by the black dots overlying the nuclei; the dots are the aggregates of silver grains struck by the beta particles.
Notice that only the nuclei contain radioactivity; the reason for this is that radioactive thymidine is incorporated only into DNA localized in the nuclei of cells. This picture identifies not only the cells that were making DNA at the time the label was administered but also the cells that were destined to divide in the immediate future, since cells synthesize DNA in preparation for cell division.
If we want to compare two populations of cells to find out which is proliferating (dividing) more actively, counting the fraction of cells labeled will give the number of cells synthesizing DNA in preparation for cell division. Of course, a rough approximation of the proliferating activity can be obtained by simply counting the number of cells actually dividing. But with tritiated thymidine we can obtain not only much more accurate measurements but also considerable information that cannot be obtained by simply counting the number of cells in mitosis. We shall discuss the cell cycle later on, but for the moment we should emphasize that much of our knowledge of the cell cycle stems from the use of high-resolution autoradiography.
It is clear that autoradiography enables us to find out which cells are dividing in a cell population and how many of them do so. For instance, in a given tissue or organ, not all cells are capable of dividing into two daughter cells. In the epidermis, which is the thin outer layer of the skin, only cells in the deepest portion can divide. The other cells, although originating from cells in the deep layer, have lost the capacity to divide, and eventually die without further division. If we take a bit of skin, expose it to tritiated thymidine, and determine the amount of radioactivity incorporated into the skin cells’ DNA, we obtain a fair measurement of the amount of DNA being synthesized. However, this purely biochemical investigation cannot possibly give any information on which specific cells are synthesizing DNA. For this, autoradiography provides the information we need.