Aqueous glasses as matrices in freeze-fracture electron microscopy

Abstract

FREEZING is an important technique in the preservation of the morphology of biological materials for electron microscopic examination. Methods relying on freeze-fracturing, sometimes followed by etching, have been developed and have led to advances in the interpretation of biological ultrastructure¹. Other, related applications of freezing are for the preservation of biological activity² and the preparation of biological specimens for microanalysis of low molecular weight components³. The freezing of cells and tissues can, like all other methods of fixation, give rise to artefacts, although probably to a lesser extent than the more traditional methods. The most important factors governing the quality of biological freezing include the size distribution of the ice crystals, the extent of supercooling and the possibility of osmotic dehydration and shrinkage of cells. Very high cooling rates are considered essential to minimise the deleterious effects of ice crystals, and to overcome excessive osmotic shrinkage of cells by extracellular ice formation, the use of cryoprotective agents is common. It is believed that an effective, penetrating cryoprotectant must diffuse into the cell without, at the same time, interfering with its morphology. Glycerol and dimethylsulphoxide fulfil these conditions and are widely used as cryoprotectants, although it is known that they can interfere with the physiological functions of some cells⁴.