Electron microscopy of unstained,freeze-dried macromolecular assemblies

A rapid cooling/cryotransfer system was designed to achieve a high reproducibility in vitrifying thin water films containing biological specimens. In order to improve the contrast these unstained specimens were deliberately freeze-dried in situ in the electron microscope. The preservation of the structure obtained by freeze-drying at 180 K and subsequent microscopy at 90 K is very encouraging as has been shown with two types of test specimen. Tubular photosynthetic membranes were used to examine the effects of a variety of freeze-drying conditions on structural preservation, as judged by flattening of the tubes. A two-dimensional protein crystal was used to evaluate problems of low-dose microscopy of unstained, freeze-dried proteins, e.g. optical density of films, motif detection by cross-correlation and transferring the accurate molecular positions from medium-dose to corresponding low-dose micrographs. The radiation sensitivity of the unstained, freeze-dried protein crystal was investigated by a dose series covering a wide range.