Cryoelectron Microscopy of Fission Yeast.

Abstract

Fission yeast cells can be prepared for electron microscopy (EM) in the frozen-hydrated state. This eliminates the requirement for dehydration and heavy metal staining when preparing samples for EM. As with room temperature imaging, however, the yeast must be sectioned to make them thin enough for transmission of the electron beam. Cutting sections of vitreous ice with a microtome is challenging. An alternative method that uses a focused ion beam to make a thin sample by milling away much of the sample at liquid nitrogen temperatures is under development but is not yet available for routine use. Imaging frozen-hydrated samples by EM is also a challenge. The technique involves battling low image contrast, high sensitivity to the electron beam, and mechanical distortions produced during the sectioning process. When used successfully, however, the method holds promise of providing excellent molecular detail without the disruption characteristic of dehydration or isolating a structure from its cellular environment. Cryo-EM of tilted views can be used to examine small structures and macromolecular complexes in their native cellular environment. If a structure exists in multiple copies, or has a repeating unit, it can be investigated at higher resolution using subvolume averaging. This protocol focuses on the preparation of cells for cryo-EM.