Autoradiography is the localization of radioactive atoms within a solid specimen. These atoms are localized by the ability of their emissions to expose a photographic emulsion placed in close proximity to the specimen. For our purposes this specimen can be a thin or thick microtome section, a cryostat or paraffin section, a cell suspension or an electrophoretic gel. A thin film of this "nuclear" halide crystals and are similar to those placed on plastic sheets to produce photographic film. Autoradiography emulsions however have silver halide grain sizes that are smaller and more homogeneous. In microscopy, the usual goal of this technique is to localize a biochemical process or its products. A sample is labeled with a radioactive precursor to this process or product and its position is located within the sample by exposure and subsequent development of the overlaid emulsion. Therefore, to choose the proper precursor it is imperative that the investigator be thoroughly familiar with the biochemical pathway of the chosen system.

RADIOISOTOPES

Due to their biological relevance, the most common radioactive elements ("radioisotopes" or "radionuclides") used in autoradiography are 3H (tritium) and 14C (carbon-14). Hydrogen and carbon can easily be incorporated into nearly any organic molecule. Of equal importance are the "half-life" of the radioisotope and the energy and type of particle emitted. Half-life is a measurement of the probability that a certain isotope will spontaneously decay and emit particles. For the purposes of autoradiography, we want our radioisoptope's half-life to be long enough so that sufficient radioactivity remains after sample processing but not so long that we must wait years fro enough exposure. Tritium has a half-life of 12.26 years and carbon-14 a half-life of 5760 years (which is near the limit of usability. Phosphorous -32 is at the other end of the scale with a half-life of 14.3 days.) There are three main types of emissions from a decaying nuclei; alpha and beta particles and gamma rays. Alpha particles (helium nuclei) and gamma rays (high energy photons) are generally too energetic for use in autoradiography. These emissions can expose silver halide grains at a great distance from their source, thereby giving misleading results. Beta particles (electrons) are much less energetic and better suited for autoradiography.

RADIOCHEMICALS

In autoradiography, a radiochemical is a biologically relevant molecule with tritium or carbon-14 incorporated into it, usually at a specific position. This again points out the importance of a good knowledge of the target pathway. You would not want to use glucose labeled with tritium at the #3 position if the #3 carbon gets cleaved off somewhere in the pathway under observation. Precursors are frequently chosen such that they will be incorporated into macromolecules within the cells being studied. Some specific examples are: Labeled Precursor Macromolecule 3H-thymidine DNA 3H-uridine RNA 3H-cholesterol steroid hormones monosaccharides polysaccharides amino acids proteins Most radiochemicals should be stored at 4°C and will last up to six months (depending on half-life.)

INCORPORATION INTO SAMPLE

Radiochemicals are generally introduced into animal samples via intravenous injection. Due to the typical expense of these precursors, this technique is usually limited to small animals (rats, mice etc.) Incorporation can also be achieved by interperitoneal injection or by inclusion into the animals diet or environment (air or water). Labeling can also be carried out in vitro with cell cultures, tissues or microorganisms. These techniques are known as "continuous incorporation" and can be modified by "pulse-chasing" labeling. In this powerful technique a short initial "pulse" of radiochemical is followed by a much larger treatment of unlabeled precursor, the "chase". By fixing and processing the specimens at differing times after the chase it is possible to follow a discrete pulse of radioactivity through the sample. There are many considerations involved when determining the proper dose of radiochemical for a specimen. Therefore, when designing an autoradiography experiment it is highly advisable to consult relevant texts and published data. Due to the very thin samples required for electron microscopy, a high concentration of radioactive precursor is needed. 10-40 microcuries of radiochemical per gram body weight is typical for injections and 10-200 microcuries/ml is usual for in vitro purposes.

TISSUE AND CELL PREPARATION

The primary concern here is that the radiochemical used and any subsequent modifications of it are preserved and remain at their proper locations within the cell. Tradition glutaraldehyde and/or paraformaldehyde and/or osmium tetroxide fixations generally work well. If, for instance, lipids are of interest, special considerations must be made. Neutral fats tend to be lost during conventional processing. Incomplete dehydration or the addition of digitonin to the fixation helps retrain these fats. The retention of phospholipids can be improved with the addition of calcium salts. The quality of morphological preservation is secondary, however it obviously must be high enough to resolve the structures of interest. EMULSIONS The emulsions used in autoradiography are basically the same as those used in photography but without the paper or plastic base. They usually consist of silver bromide crystals in the gelatin binder. The grain sizes are much more consistent than normal photographic emulsions and for EM level are much smaller (down to 90nm). The relationship between a grain size and its sensitivity and resolution is the same as for photography; small grains have lower sensitivity but better resolution and vice versa. The most commonly used emulsions are Kodak NTB2 for light level and Ilford 14 for EM level.

LIGHT LEVEL AUTORADIOGRAPHY

All EM autoradiography investigations should begin with a light level run. Depending on the specific project, sections of 0.25µm to 50µm are placed on gelatin - chrome alum coated glass slides. Before the emulsion can be applied it must be liquefied by warming in a water bath at 43° to 45°C. This is carried out under safe lights and in a dark room where the humidity can be raised. The slides are dipped into the emulsion, slowly withdrawn and allowed to dry. The slides are then placed, along with a packet of desiccant, in a light safe box and allowed to expose for 3 to 28 days. It is a good idea to prepare four or five such boxes so that one can be removed at intervals and developed without disturbing others. The slides are developed, fixed and washed, much like a TEM negative, and observed in a light microscope. Additional slides are developed until the desired level of signal is reached. As a rule of thumb, EM level exposures take ten times as long as the corresponding light level exposure.

EM LEVEL AUTORADIOGRAPHY

The first step in this technique is to collect gold interference color thin sections on formvar coated nickel or gilded grids. Cooper grids are to be avoided due to the possibility of interaction with the nuclear emulsion. A large number of grids (30-40) should be collected so that several different exposure boxes can be prepared. As with light level work this allows one box to be developed without disturbing the others. The grids are then post-stained in the usual manner with uranyl acetate and lead citrate. Next the grids are coated with a thin layer of carbon to prepare a smooth surface for the emulsion and to reduce chemographic effects (see next section.) At this point the grids are mounted section side up on a cork using double sided tape with a hole just smaller than the grid punched in it. The grids are then taken to the darkroom for the application of the emulsion. There are several methods for applying the nuclear emulsion to the grids and the most common, the "loop" technique will be discussed here. For maximum resolution we want to coat the sections with a single layer of silver halide crystals. This is accomplished by first liquefying the emulsion, as in the light level method. Instead of dipping the sections in the emulsion, however, we will dip a 3cm platinum loop into the emulsion and withdraw it. If the conditions (temperature, humidity, emulsion dilution) are right the film of emulsion carried on the wire will gel into a monolayer of silver halide crystals. When held up to the safelight, the "loop" should be uniform opacity and should not be flowing. The cork is then passed though the center of the loop, grid side first. The corks thus produced are allowed to fully dry and are packed into light-tight boxes with dessicent and stored for exposure at 4°C. Before applying the emulsion to the samples, several test grids should be made. This is done by laying a loop of emulsion on a formvar-coated grid with no sections on it. The grid is then placed in the TEM and the emulsion is checked for thickness and uniformity. As in light level autoradiography, boxes of grids are removed at regular intervals and developed. This process continues until an acceptable exposure level is reached.

PHOTOGRAPHIC CONSIDERATIONS

Depending in the sample and the size of the silver grains, most EM autoradiographs are viewed at between 10,000x and 30,000x final magnification. It is advisable to record a fair number of micrographs that are representative of the morphology and distribution of the silver particles from several different sections. It is recommended to observe at least 500 silver particles. Before the micrographs can be properly interpreted, control observations should be made. One type of control is to determine the background grain count. This is done by taking a series of random micrographs of the support film a few micrometers from the edge of the section. If the background grain frequency is greater than 5% of the grain frequency over the tissue, the grid should be abandoned. Another concern is positive and negative chemography. This effect is brought about by chemicals within the tissue that may affect the nuclear emulsion. Positive chemography is caused by chemicals that may expose the emulsion by direct chemical action. This can be tested for by preparing identical but unlabelled samples for autoradiography. If large and complex silver grains are observed positive chemography has occurred. In negative chemography, chemicals within the sample inhibit the formation of latent images and may even reverse their formation. To test for this, light-fog a few specimens for each batch of newly coated sections. Any area where the silver grain density is reduced or eliminated may have been caused by negative chemography.