On estimating freezing times during tissue rapid freezing

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 micron depths in specimens. The calculations indicate that cooling rates in excess of 4 X 10(4) K s-1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 micron, the times obtained are 0.1-0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about +/- 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 microseconds, which can be taken as an upper limit for the resolution time for rapid freezing.