Freeze Substitution and Low-Temperature Embedding

Abstract

Although freeze substitution and low-temperature embedding are two distinct processes, they are considered together because they complement each other and are frequently used sequentially during sample preparation. Freeze substitution is a chemical dehydration process in which ice in frozen-hydrated specimens is removed and replaced by an organic solvent. This procedure is in sharp contrast to the process of freeze-drying discussed in the previous chapter, where the water is removed by a purely physical process and is not replaced. Low-temperature embedding seeks to replace either the spaces once occupied by water in a freeze-dried sample or the organic fluid that has been used during freeze substitution, by infiltrating the sample at low temperatures with resins, which are, in turn, polymerized at low temperatures. The temperatures at which these processes are carried out are critical. If it is too high, the rate of ice recrystallization will damage the sample. If it is too low, the organic solutions become so viscous that they will not adequately penetrate the specimen. The compromise is to use lower temperature (193–173 K) for substitution and somewhat higher temperatures (253–238 K) for embedding.*