Preparation of frozen-hydrated specimens for high resolution electron microscopy

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A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at — 120°C have shown that the structure can be preserved to a resolution of 3.5 Å. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.