Freeze-substitution for thin-section study of biological specimens.

Freeze-substitution as a preparation technique for frozen specimens is briefly reviewed. Recent advance in cryotechniques, especially quick freezing followed by freeze-substitution, has made it possible to preserve the ultrastructural features of cells and tissues to reflect more closely their living state and capture transient physiological processes as evanescent as fusion and fission of biological membranes. The technique is also advantageous to retain soluble substances of the cellular components. Thus, histochemical, autoradiographic and/or immunocytochemical analyses of quick-frozen, substitution-fixed samples allow us to obtain more precise localization of chemical components in cells than in the case of conventional chemical fixation. Several examples indicating the superiority of the preparatory method are presented.