cryofixation

Cryofixation is a sample preparation technique that preserves biological structures by freezing specimens rapidly enough to prevent the formation of ice crystals that would disrupt cellular architecture. The goal is to immobilize biological processes and retain a near-native ultrastructure for subsequent analysis, most notably by electron microscopy.

Two primary approaches are used. Plunge freezing involves rapidly immersing a small, thin sample on a carrier into a cryogen such as liquid ethane cooled by liquid nitrogen, producing vitrified, glass-like ice. High-pressure freezing enlarges the allowable sample thickness by applying high pressure during cooling, which helps suppress ice crystal formation. Cryoprotectants like glycerol or dextran may be used to improve vitrification, but they can introduce artifacts if not carefully controlled.

In modern workflows, cryo-electron microscopy (cryo-EM) and cryo-electron tomography rely on samples kept in vitreous ice at cryogenic temperatures to image macromolecular complexes and cellular components at high resolution. Freeze substitution is a related technique in which water is gradually replaced with organic solvents at low temperature after cryofixation, enabling embedding for conventional EM while attempting to retain structural details.

Advantages of cryofixation include excellent preservation of ultrastructure with minimal chemical modification and compatibility with high-resolution imaging methods. Limitations involve the need for specialized equipment, sensitivity to devitrification and ice crystal artifacts, difficulties with thick specimens, and potential artifacts from cryoprotectants or subsequent processing steps. The technique has become central to structural biology and cell biology since its development in the mid-20th century, culminating in the routine use of cryo-EM for near-native structural analysis.