Freeze-Substitution & Low-Temperature Embedding

Although freeze-substitution and low-temperature embedding are two distinct processes, they are considered together because they complement each other and are usually 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.

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, polymerised at low temperatures.

Uses
The two techniques serve as a convenient link between the idealistic approach of purely physical cryofixation techniques for biological specimens, and the convenience of thin sectioning at room temperatures. It is, in theory, the best approach to the preservation of ultrastructure and functionality.