Ice crystal damage in freeze-dried articular cartilage studied by scanning electron microscopy

The aim of preparing specimens at very low temperatures is to maintain biological tissue in its natural state. The problems which arise are: 1) obtaining the highest possible cooling rate when freezing the tissue; 2) preventing melting with recrystallization during the drying process; and 3) preventing the dried specimens from water vapor contamination. Biological tissue is prepared in a cooling chain by freezing the samples in a supercooled nitrogen bath, breaking the specimens in the frozen state, sputtering the fracture surfaces in a vacuum on the cooling stage of a special device and then transferring the specimens under vacuum to the SEM where they are studied on a cooling stage. They have smooth surfaces, with the liquid and solid phases present simultaneously, and no artefacts. Any shifts or deformations which may occur within the structure cannot yet be determined. Freeze-drying quenched specimens at various temperatures (-140 degrees to -60 degrees C) taking the dynamics of vapor pressure into consideration shows that sublimation, the melting processes with recrystallization and the resulting damage to the structures occur simultaneously during the drying process. The destruction of the tissue is inversely proportional to the ratio of sublimation to melting.