130 MACROMOLECULAR COMPLEXES 



of ice-crystal formation and growth within ordered multilayered 

 systems during protracted processing at low temperatm^es. When 

 fresh nerve trunks are rapidly frozen and freeze-substituted at 

 —75° C and subjected to low-temperature embedding, the resulting 

 thin sections reveal numerous large internal patches of stRictural 

 obliteration which correspond mainly to regions of extensive ice- 

 crystal growth. The associated extraction, diffusion, and rearrange- 

 ment artifacts are more marked after substitution in methanol than 

 in acetone. Bearing in mind the rapid ice-crystal growth at —70° C 

 observed by Meryman (1956), it is evident that at temperatures 

 above the critical glassy transition point of water, considered to be 

 around — 100° C in tissues (Stephenson, 1956), ice-crystal growth 

 can assume significant proportions during the prolonged freeze-sub- 

 stitution periods of days or weeks in certain specimens. More direct 

 observations can be performed with a special cold stage and a 

 fluorescence microscopy attachment, on model systems consisting 

 of thin gelatin strips containing riboflavin-phosphate-sodium. As 

 demonstrated by Szent-Gyorgyi ( 1957 ) , the characteristic yellow- 

 ish-green fluorescence emitted by a watery riboflavin solution under 

 ultraviolet light is replaced by an orange phosphorescence on the 

 formation of ice during freezing. This phenomenon can be used as 

 a sensitive indicator of ice under certain conditions, and the ribo- 

 flavin enclosed in the ice matrix serves also as a visible marker by 

 which to follow the freeze-substitution process. Moreover, as the 

 riboflavin dye is released from the dissolving ice, its progressive 

 spread throughout the alcohol-solvent column gives an indication 

 of the diffusion processes still operative at temperatures below 

 -75° C. 



Upon performing freeze-substitution at temperatures of —120° 

 to —90° C, using acetone-ethyl chloride mixtures, substantial im- 

 provement in the preservation of the microstructure was noted, due 

 to absence of larger ice-crystal formations and to the diminished 

 extracting effects of the substituting fluids at these lower tempera- 

 tures. However, at temperatures below —90° C, the time factor 

 enters as an important consideration. Only thin specimens can be 

 freeze-substituted at these temperatures within reasonable time 

 periods; for thicker specimens, the complete processing c\cle ma\ 

 require several weeks. 



After elimination of gross ice-crystal artifacts, it is now possible 

 to recognize some of the direct and indirect effects of submicro- 



