The potential of cryofixation and freeze substitution: observations and theoretical considerations

SUMMARY The theoretical and experimental evidence in favour of cryofixation and freeze‐substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.