Structural changes in samples cryofixed by contact with a cold metal block.

Abstract

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.