Aspects of cryofixation and cryosectioning for the observation of bulk biological samples in the hydrated state by cryoelectron microscopy.

Abstract

Cryoelectron microscopy allows the observation of hydrated samples at high spatial resolution, and it would be of great interest in biology to apply this method to cells and tissues. However, because of technical problems, the cryo-observation of frozen hydrated ultrathin sections of bulk material has not become an established method. The major limitations are due to the difficulty of achieving the vitrification of such material, and the structural deformation caused by ultrathin sectioning: 1. The vitrification of cells in a physiological environment requires high-pressure freezing. However, new results suggest that the pressure may alter the ultrastructure of the sample. 2. Cryosectioning compresses structures in the cutting direction about 40%. This deformation does not necessarily destroy the character of macromolecular assemblies, but since it depends on the properties of the material, internal standards cannot be used to correct for the deformation of all the structures in a cell.