Freeze-fracture, deep-etch, and freeze-substitution studies of olfactory epithelia, with special emphasis on immunocytochemical variables.

Freeze-fracturing and deep-etching are a well-suited set of methods to study membrane and cytoplasmic features. Various approaches are available. Possible variables include tissue preparation, fracturing only or fracturing followed by etching, modes and materials of replication, and various ways of combining freeze-fracturing and/or deep-etching with (immuno)cytochemistry. Freeze-substitution, in particular combined with embedding in methacrylate resins such as the Lowicryls, is becoming rather widely accepted for purposes of ultrastructural (immuno)cytochemistry. Most investigators active in this field agree that this combination yields superior results compared to (immuno)cytochemistry combined with more conventional means of thin section transmission electron microscopy. Yet relatively little information is available on the variations that can occur with different approaches of freeze-substitution immunocytochemistry. This review deals with some of the variations in freeze-fracturing, freeze-etching, and freeze-substitution as applied to olfactory epithelial structures and with the effectiveness of observations obtained by application of the above sets of methods in relating the special morphology of olfactory epithelial cellular structures with those obtained by other approaches. Indeed, the data obtained continue to provide an integral image in which that morphology can be related to the special biochemistry, cell and molecular biology, and electrophysiology of olfactory epithelial structures.