Freeze-fracture autoradiography: the in-vacuo coating technique

Freeze-fracture autoradiography (FFA) was introduced in 1976 as a new method for electron microscopic autoradiography of diffusible compounds (Fisher and Branton, Rix et al.). With the original technique, the film monolayer was applied to the cold specimen in a cryostat at atmospheric pressure. Coating under these conditions did not exclude the risk of artifacts, mainly due to uncontrolled ice contamination of the cold specimen surface. A new method has been developed for coating the frozen specimen, immediately after replication, in the maintained vacuum of the freeze-fracture unit. Two main components of the new technique are described in detail, a specially designed coating device, and the use of spreading substances, promoting adhesion of the film in vacuo. Using this technique artifacts so far inherent in the FFA method can be eliminated.