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.
[Illustration: 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.
[Illustration: 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 15a), 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.
[Illustration: 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.