Plunge freezing for electron cryomicroscopy

Aqueous biological samples must be "preserved" (stabilized) before they can be placed in the high vacuum of an electron microscope. Among the various approaches that have been developed, plunge freezing maintains the sample in the most native state and is therefore the method of choice when possible. Plunge freezing for standard electron cryomicroscopy applications proceeds by spreading the sample into a thin film across an EM grid and then rapidly submerging it in a cryogen (usually liquid ethane), but success depends critically on the properties of the grid and sample, the production of a uniformly thin film, the temperature and nature of the cryogen, and the plunging conditions. This chapter reviews plunge-freezing principles, techniques, instrumentation, common problems, and safety considerations.