Relationships between ice crystal size, water content and proton NMR relaxation times in cells

Biological specimens were frozen under controlled conditions. We questioned how the size of ice crystals, as measured in cryosectioned and cryoadsorbed sections of these biological specimens, relates to the water content and to the proton NMR relaxation times (T1 and T2) of the unfrozen specimens. The results permit the following conclusions: After rapid freezing in liquid propane cooled in a liquid nitrogen bath, the average size of ice crystals at distances of 150 microns or more from the surface of a particular tissue was always the same. Thus, the average size of the ice crystals was found to be characteristic of the type of biological tissue studied. Linear regression analysis showed average ice crystal size to have a significant correlation coefficient to T1 relaxation time and to water content. Specifically ice crystal size increased with T1 relaxation time and with water content. Multiple regression and path analysis demonstrated a positive correlation between the T1 relaxation time and the ice crystal size variation. Path analysis showed that both water content and T2 relaxation time were less directly correlated with ice crystal size. The findings from the path analysis and other observations show that the average size of ice crystals in subcellular compartments is best predicted by the proton T1 relaxation time. A working model is put forth to explain differences in ice crystal size observed between specimens enriched in globular or in parallel filamentous proteins.