Freeze-Substitution and Freeze-Drying

Abstract

Freeze-substitution (FS) and freeze-drying (FD) are dehydration techniques by which the water is gently removed from a frozen specimen. Both techniques can serve as a link between cryofixation and conventional thin sectioning at room temperature (Fig. 1). They are, therefore, hybrid techniques combining the advantages of the low temperature and the room temperature specimen preparation. With respect to the danger of artefacts, these procedures are much more obscure than “pure” cryotechniques, such as freeze-etching or cryosectioning. Both, FS and FD, are known from light microscopy and have been used in electron microscopy since its early days (for refs. of the older literature see Bullivant 1970; Rebhun 1972; Robards and Sleytr 1985), but only during the last dozen years a breakthrough can be noticed, which is mainly due to improved cryofixation. As for any other cryotechnique in biological electron microscopy, for successful FS and FD the main prerequisite is also good cryofixation with as little freezing damage as possible (see Bachmann and Mayer, Chap. 1; Sitte et al., Chap. 4; Dubochet et al., Chap. 5; Moor, Chap. 8; this Vol.).