Cell Biology Jntemetional Reports, Vol. 4, No. 7, July 1380 625 MINI-REVIEW QUICK FREEZING.VS. CHEMICAL FIXATION: CAPTURE AND IDENTIFICATION OF MEMBRANE FUSION INTERMEDIATES Pedro Pinto da Silva and Bechara Kachar Laboratory of Pathophysiology, Bldg. 10, Rm. 5B-47 National Cancer Institute, National Institutes of Health Bethesda, Maryland 20205, U.S.A. INTdbDUCTION Conventional application of freeze-fixation methods to the study of cellular anatomy requires tissue impregnation in a cryo- protectant (usually glycerol at 25-30%) and, in consequence, chemical pre-fixation (usually glutaraldehyde or glutaraldehyde-formaldehyde). It is clear that these requirements are not welcome because they leave open the possibility of structural changes before, during (i.e., fixative induced), or after fixation (i.e., those allowed by inherent limitations of chemical fixation). The severity of these limitations depends on the morphological analysis and on the objective of the study. For *this reason, they have been more acutely felt by researchers of highly dynamic membrane phenomena such as the events involved at the synapse during transmitter discharge, and other secretory processes. In order to overcome the need for chemical fixation and cryo- protectant impregnation, a rapid rate of .freezing must be achieved in which there is no significant damage and/or distortion caused by the formation of ice crystals (Meryman, 1957, Stephenson, 1960, Van Venrooij et. al., 1975). Freeze-fixation methods, initially proposed by Altmann (see Gersh, 1932), Eranko (1954) and others (see Erdnkt), 1954) were developed which involved: 1) direct immersion of the specimen into a superconductive liquid coolant (Fernandez-Moran, 1960: Bullivant,l960); 2) freezing in liquid nitrogen of specimens under high hydrostatic pressure (Moor and Riehli, 1968; Moor, 1971); 3) freezing of small droplets of cellular or sub-cellular suspensions (spray-freezing) (Bachmann and Schmitt, 1971; Plattner et. al., 1972); 4) contact of the specimen with the polished surface of a cooled metal block (Van Harreveld and-Crowell, 1964; Kretzschmer and Wilkie, 1969; Chrystenson, 1971; Van Harreveld et. al., 1974; Dempsey and Bullivant, 1976a; Dempsy and Bullivant, 1976b). Recently, the "quick freezing" technique (Heuser et. al., 1976; Heuser et. a1.,1979) has been proposed which is rapidly gaining acceptance (see Shotton, Nature. News and views,. 283: 12-14, 1980) in the preparation of 0209-1651/8Ol07W2~1&‘$02.00/0 0 1980 Acdomii Rosa Inc. (London) Ltd.