Recent Progress In High-Resolution Shadowing For Biological Transmission Electron Microscopy

Abstract

Freeze-drying followed by heavy metal shadowing is a long established and straight forward approach to routinely study the structure of dehydrated macromolecules. Very thin specimens such as isolated membranes or single macromolecules are directly adsorbed on C-coated grids. After rapid freezing the grids are transferred into a suitable vacuum equipment for freeze-drying and heavy metal shadowing.To improve the resolution power of shadowing films we introduced shadowing at very low specimen temperature (−250°C). To routinely do that without the danger of contamination we developed in collaboration with Balzers an UHV (p≤10-9 mbar) machine (BAF500K, Fig.2). It should be mentioned here that at −250°C the specimen surface acts as effective cryopump for practically all impinging residual gas molecules from the residual gas atmosphere.Common high resolution shadowing films (Pt/C, Ta/W) have to be protected from alterations due to air contact by a relatively thick C-backing layer, when transferred via atmospheric conditions into the TEM. Such an additional C-coat contributes disturbingly to the contrast at high resolution.