Quench cooled ice crystal imprint size: a micro-method for study of macromolecular hydration

Quench cooling (rate greater than 4000 degrees C/sec) of biological specimens limits growth of ice crystals by processes different from slow cooling methods. Quench cooling in liquid propane cooled in liquid nitrogen induced ice crystal segregation compartments, as imaged by scanning transmission electron micrographs of freeze-dried cryosections of tissues and protein solutions. The observed imprints of ice crystals were relatively small and roughly spherical. The size of these ice crystal imprints increased with distance from the specimen/quenchant surface. Beyond a depth of 150 microns the size of the imprints was constant but differed among subcellular compartments. The size of the imprints was found to be dependent on: water content, extent of hydration water and the degree of protein aggregation. Determination of extent of hydration water and degree of protein aggregation in protein solutions by measurements on the size of ice crystal imprints yields data in agreement with macroscopic methods. Thus ice crystal imprints give information about the interactions of macromolecules and water at a subcellular level of resolution.