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.
RNA SYNTHESIS: HOW TO TRANSLATE ONE LANGUAGE INTO ANOTHER
Mathematicians are like Frenchmen: whatever you say to them they translate into their own language, and forthwith it is something entirely different.
Wolfgang von Goethe
We have mentioned previously that there are two main types of nucleic acids: DNA, the genetic material itself, and RNA, the molecule that translates the genetic message from DNA into terms the cell can use as “instructions” for making protein. Cells differ from each other on the basis of kinds of proteins they contain, and, since differences among cells determine differences among organisms, it follows that differences in the composition of DNA serve to explain the variety in living organisms populating the world. However, if differences between two organisms can be explained by differences in the chemical composition of their respective DNA’s, how can we explain differences between cells of the same organism? How can we explain that cells of the human pancreas secrete insulin, whereas other cells in man produce no insulin? Or how can we explain that certain cells make bone and others make fat? If indeed all cells in the same organism contain the same amount and kind of DNA (since all DNA in an organism derives from the duplication of the DNA of the fertilized egg cell and its descendants), it would seem, at first glance, that DNA is not the molecule responsible for differences among the cells. The clarification of this apparent contradiction is found in the remarkable properties of the other nucleic acid, the translator molecule, RNA.