Ice crystal growth in skeletal muscle fibres

Ice crystal growth was studied in rapidly frozen skeletal muscle fibres which were treated with cryo-protective additives (glycerol, DMSO, sucrose) or which were untreated. Freeze cleaving and etching was the basic method, with conventional plastic embedding and cryo-ultramicrotomy arred during freezing in all unprotected fibres. Just below the fibre surface the crystals were numerous but small, while deeper in the fibre they were fewer but larger. The deeper within the specimen a fibre was located, the larger, in general, was the crystal size. The crystal volume density was about 55%, irrespective of crystal size. Ice recrystallization was practically absent at the temperature normally used in cryo-sectioning (-70 degrees C). Anti-freeze treatment eliminated crystal growth. If the anti-freeze agents were used in non-toxic concentrations, however, their effect on crystal growth was very limited. 'Dry'-cut, freeze-dried ultra-thin cryosections of protected and unprotected fibres confirmed these observations, while sections obtained by 'wet' cryo-cutting showed no apparent signs of crystal growth. In plastic sections of frozen and thawed fibres a previous occurrence of crystals was only slightly indicated. In interpreting the ultrastructure in 'wet'-cut cryo-sections of unprotected frozen muscle fibres, the distorting effects of ice crystals through mechanical compression and alterations in sectioning conditions, must be taken into consideration. Crystal growth also strongly limits the possibilities of using 'dry'-cut sections of untreated frozen tissue for analytical electron microscopy; only the most superficial parts of the fibres seem to be suitable for microanalysis.