LAMELLAR SYSTEMS 119 



contributions by Feder and Sidman (1958) and by Miiller (1957), 

 improved low-temperature preparation methods liave been devel- 

 oped for the study of tissues by high-resolution electron microscopv 

 (Fernandez-Moran, 1959a, 1959c, 1960). The designation "cryo- 

 fixation techniques" (Fernandez-Moran, 1959c) was suggested to 

 distinguish these procedures from the related freeze-sulDstitution 

 methods (Feder and Sidman, 1958; Patten and Brown, 1958; Simp- 

 son, 1941 ) because of the different type of approach, involving freez- 

 ing with liquid helium II and low-temperature polymerization, 

 which will become of increasing operational value in the course of 

 further developments. 



The basic cryofixation techniques comprise sequential application 

 of the following procedures : ( 1 ) rapid or ultrarapid freezing of 

 thin, fresh specimens or of glycerinated tissues with liquid helium II 

 at 1° to 2° K; (2) staining at — 150° C with halogens or organo- 

 metallic compounds dissolved in isopentane; (3) substitution of the 

 ice matrix with solutions of heavy-metal salts in alcohol-acetone and 

 alkyl halide mixtures, or alternatively in certain organometallic com- 

 pounds, at temperatures of —130° to —80° C; (4) infiltration of the 

 specimens at —100° to —80° C with acryhc monomers; and (5) 

 final embedding of the specimens by photopolymerization with ul- 

 traviolet light at temperatures of -80° to -20° C (Fox et al, 1958). 



In an important technical variant used mainly for examination of 

 specimens which have been subjected to a minimum of chemical 

 fixation, all intermediate staining procedures are eliminated, and 

 only freeze-substitution is carried out with acetone-ethyl chloride 

 mixtures at temperatures of about —130° to —110° C, which are still 

 below the critical transition range for tissues. 



A detailed account of the crvofixation and related low-tempera- 

 ture preparation procedures is now being prepared for publication ^ 

 and only representative methodological aspects will be reviewed 

 here. 



Freezing of Biological Specimens With Liquid Helium II. 

 The use of liquid helium as an indispensable refrigerant for attain- 

 ing temperatures close to absolute zero is based on important recent 

 developments in the fields of low-temperature chemistry ( Klein and 

 Scheer, 1958) and free-radical research (Broida, 1957; Minkoff, 

 1959). The temperature domain below the glassy transformation of 



^ H. Fernandez-Moran. Ultrastructure of the Nervous System. Part I, Techniques- 

 (To be published by Academic Press, Inc., New York. ) 



