Cryoultramicrotomy

Cryoultramicrotomy is a specimen preparation technique used to produce ultrathin sections of frozen biological material for electron microscopy. By freezing samples, water is immobilized and chemical fixation is minimized, allowing closer preservation of cellular architecture and labile components.

Typically, the specimen is rapidly frozen to form vitreous ice or is prepared by freeze substitution, then mounted on a cryo-ultramicrotome equipped with a cooled stage and knife. Thin sections, often 50 to 100 nanometers thick, are cut at temperatures well below freezing and collected on electron microscopy grids or specialized support films, sometimes with mild staining or immunogold labeling performed under cryogenic conditions. These sections can be imaged in the transmission electron microscope while kept at low temperature, or subjected to additional processing for cryo-electron tomography.

Cryoultramicrotomy is widely used in cell biology, virology, and pathology to study membranes, organelles, and viral particles in a near-native state. Freeze substitution can improve contrast and preservation for conventional EM after sectioning frozen samples. The technique enables visualization of hydrated structures that are difficult to preserve with routine fixation.

Challenges include the need for specialized equipment and expertise, as well as artifacts such as compression, chatter, crevasses, and knife-induced damage. Achieving uniform ultrathin sections and preventing ice contamination require careful technique. Historical development of cryofixation and cryo-EM methods in the mid-to-late 20th century established cryoultramicrotomy as a key tool in structural biology and pathology.