13 Autoradiographic Methods for Electron Microscopy

This chapter focuses on the autoradiographic methods used for electron microscopy. The isotope of choice is tritium. It is the most easily adapted isotope because of its low average energy and consequently its high specific ionization. 3H emits a β-particle of 18 keV maximum energy and, therefore, gives excellent resolution and sensitivity. The nuclear emulsions used for autoradiography are composed of crystals of silver halide and of gelatin. The ideal emulsion must be rich in silver bromide, with grains of regular size and small enough to give a good resolution. It must be easy to handle and give perfectly reproducible results. All the methods of fixation, dehydration, and embedding employed with electron microscopy can be used for autoradiographic methods. A fading of the latent image can be produced through chemical interaction between the specimen and the emulsion in the case of a fixation with osmium tetroxide. This effect can be avoided by putting a carbon layer on the specimen before layering the emulsion. Electron autoradiography can also be used on shadow-cast viruses or macromolecules. In this case, however, because of the interaction between metals and emulsion, a carbon layer must be put between the specimen and the emulsion.