Electron microscopy of unstained, freeze‐dried macromolecular assemblies

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Abstract
SUMMARY: 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.