Biological freezing and cryofixation

Abstract

Freezing and freeze fixation are commonly used to achieve ultrastructural and biological preservation. Freezing in biological materials is complex because of their heterogeneous nature-water is unevenly distributed and the various domains are separated by semi-permeable membranes. Processes to be considered include: (1) osmotic gradients leading to redistribution of water, (2) nucleation and uncontrolled growth of ice crystals, (3) recrystallization of nucleated aqueous substrate. To avoid ultrastructural deformation in biological specimens cryofixatives are commonly employed. These are water soluble molecules, able to penetrate cell membranes (e.g. glycerol and dimethylsulphoxide). Interacting strongly with water, ions and bipolymers, they give rise to metabolic and physiological changes which render them useless for X-ray microprobe analytical studies. However, they can enable tissues to survive low temperature storage. Some plants and animals develop in vivo mechanisms which enable them to avoid or tolerate freezing. Alternative means of cryofixation have recently been developed. They rely on non-penetrating polymers of high and specific water binding capacity. These polymers enable the extracellular spaces to be vitrified rather than frozen. Such suppression of ice nuclei enables the cell contents to be maximally subcooled, resulting in the formation of nm dimension ice crystals. Since the polymers have a low osmotic activity and do not penetrate membranes, the interior of the cell is substantially undisturbed. Also hydrophilic polymers used as cryofixatives are physiologically less active than conventional cryoprotectants at equivalent weight concentrations, and th eir mechanical properties render them useful as matrices for cryosectioning.