Freeze‐substitution and isothermal freeze‐fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (‐21°C)

Abstract

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethylsulphoxide, 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 configuration 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.