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

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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.