118 MACROMOLECULAR COMPLEXES 



Brown, 1958). Although addition of chemical fixatives to the sub- 

 stituting fluids (Feder and Sidman, 1958; Fernandez-Moran, 1957) 

 improves the quality of tissue preservation, the artifacts associated 

 with the formation of ice crystals during prolonged immersion at 

 these temperatures— well above the critical transition at — 100°C— 

 cannot be avoided. In both freeze-drying and freeze-substitution, 

 embedding of the tissues produces further extraction and rearrange- 

 ments which, though indiscernible under the light microscope, 

 nevertheless obliterate important structural detail at the submicro- 

 scopic level. The partially undenatured, freeze-substituted tissue 

 components are particularly vulnerable to the lipid-extracting action 

 of the usual transfer and embedding media. All of these artifact 

 sources severely limit the usefulness of standard freeze-substitution 

 techniques for electron microscopy. 



In order to adapt freeze-substitution for the study of tissue fine- 

 structure, it would be necessary to dissolve the ice matrix at tem- 

 peratures preferably below the critical glassy transition in tissues, 

 tentatively assumed to be at —100° C. The entire process of im- 

 pregnation and embedding would also have to be carried out at 

 temperatures of — 100°C to approximately — 50° C, at which the 

 solubility of the labile lipoprotein membrane complexes would be 

 greatly reduced, and the stiffness of the frozen tissue matrix would 

 prevent gross structural rearrangements from occurring. Muller 

 ( 1957), who first successfully applied photopolymerization of meth- 

 acrylate at —10° C for embedding of frozen-dried preparations, 

 noted marked improvements in the preservation of chloroplast fine- 

 structure, and considerably reduced extraction of lipoprotein com- 

 plexes. Freeze-substitution procedures are well suited for infiltra- 

 tion with a monomer and photopolymerization at low temperatures, 

 since, as Feder and Sidman ( 1958 ) have pointed out, the transitions 

 do not involve the disruptive effects of a liquid-gas interface sweep- 

 ing through the specimen, as is the case when frozen-dried tissues 

 are immersed in the embedding fluids. As shown by previous studies 

 (Fernandez-Moran, 1950, 1957), treatment of the tissues with glyc- 

 erol or other protective agents prior to freezing is essential to pre- 

 vent destructive ice-crystal formation in the more commonly en- 

 countered larger tissue specimens. 



Cryofixation and Related Low-Temperature Techniques. 

 Guided by these considerations, and ])ased on the results of earlier 

 work (Fernandez-Moran, 1950, 1957) and the recent technical 



