Preparation of thin, fine-grained, tantalum metal replicas for freeze-fracture electron microscopy

Two critical factors in the preparation of metal films on biological specimens are the type of metal used and the potentially damaging effects of radiant energy from the hot metal source. The excessive heating of surfaces is a major limitation to the replication of heat-sensitive aqueous specimens with refractory metals such as tungsten and tantalum, although these metals are known to form smaller grains and thinner films than the more commonly used platinum/carbon deposited under similar conditions. We describe here an electron gun designed for the evaporation of pure tantalum; surface heating is reduced through intermittent deposition controlled by varying the open/closed intervals of a fast shutter that operates in ultra-high vacuum. The effectiveness of the shutter was evaluated with a thin thermocouple in place of the specimen. The composition of the replicas was determined by x-ray microanalysis and by direct observation of the initial melting and subsequent evaporation of the tantalum bead supported on a tungsten rod that remained unchanged during the evaporation. The quality of the tantalum replicas was demonstrated with freeze-fracture replicas of reconstituted proteoliposomes and native membrane vesicles. With shutter intervals of 0.5 sec open and 1.0 sec closed, the surface heating was reduced enough to prevent unintentional etching and to preserve small pits complementary to protein particles in hydrophobic membrane surfaces and in ice.