Preparation of cultured cells using high-pressure freezing and freeze substitution for subsequent 2D or 3D visualization in the transmission electron microscope

Abstract

Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples and it is particularly useful when determining virus–host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast, and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the material. Here we describe a method which aims to minimize preparation artifacts by freezing the samples at high pressure to instantaneously preserve ultrastructural detail, then rapidly substituting the ice and infi ltrating with resin to provide a fi rm matrix which can be cut into thin sections for imaging. Thicker sections of this material can also be imaged and reconstructed into 3D volumes using electron tomography.