LAMELLAR SYSTEMS 117 



crystal structure and growth has already been undertaken ( Fernan- 

 dez-Moran, 1959d). Further developments along these lines may 

 eventually permit direct electron-optical studies of thin biological 

 specimens in which the native hydrated state has been partially 

 preserved through vitrification at low temperatures. 



Low-Temperature Preparation Techniques 

 for Electron Microscopy 



Standard Freeze-Drying and Freeze-Substitution Techniques. 

 The classical freeze-drying techniques (Gersh and Stephenson, 

 1954) of established value in morphological and histochemical 

 studies of tissues by light microscopy have generally proved to be 

 inadequate for electron microscope preparations. Even when freeze- 

 drying is performed under favorable conditions (Sjostrand and 

 Baker, 1958), the resulting thin sections show extensive vacuoliza- 

 tion due to ice-crystal formation, and generallv deficient preserva- 

 tion of tissue structure. This ice-crystal artifact plays a major role 

 in all low-temperature procedures, and can be largely accounted for 

 in terms of the characteristic phase transformations which water 

 undergoes at different temperatures. If a sufficiently small tissue 

 sample is cooled rapidly enough (in less than -/looo second) below 

 — 100° C with isopentane-liquid nitrogen (Stephenson, 1956), the 

 water in the tissue will solidify in a metastable "vitreous" or glassy 

 state ( Gersh and Stephenson, 1954 ) . In the case of pure water, this 

 vitreous ice turns abruptly into crystalline ice above the critical 

 "glassy transformation temperature," around — 130°C (Meryman, 

 1956). The phase transformations of water in tissues are different 

 from those in pure water (Luyet, 1957; Stephenson, 1956), but even 

 in tissues a transition temperature of about —100° C has been as- 

 sumed by Stephenson (1956). Meryman (1956) has shown that a 

 pure ice crystal can develop in 30 seconds from the glassy state to 

 a length of l/>t at —70° C. Since the subhmation of ice in freeze- 

 drying is usually carried out at temperatures not below —80° G, the 

 rapid growth of ice crystals within the tissue matrix and the ensuing 

 damage occurring during the relatively long periods of vacuum de- 

 hydration are readily understandable. 



In the freeze-substitution process introduced by Simpson (1941), 

 the ice formed within the tissue is slowly dissolved or "substituted" 

 in a fluid solvent at temperatures of about — 70° G (Patten and 



