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Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)
Authors:C. J. Hunt  M. J. Taylor  D. E. Pegg
Abstract:Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min?1 or 2 K min?1 were related to the distribution of the ice phase within the tissue.
Keywords:Freeze-substitution  isothermal freeze-fixation  cryopreservation  smooth muscle  ice
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