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 32
P) may travel more than a millimeter before
producing a grain. Low-energy beta particles (3
H 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
During this time, the activity emanating from the 3
H 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