An illustration of the high-pressure freezing-freeze substitution method.
figure by Neta Varsano

Vitrification provides the best-known means for the preservation of aqueous samples in their native state, since the only perturbation that occurs during vitrification is the lowering of kinetic energy and possibly a slight change of volume. However, the processing of frozen samples for electron microscopy (EM) is technically demanding and the visualization of the sample in the microscope is limited by damage caused by electron-beam irradiation.  In contrast, it is relatively easy to apply chemical fixation procedures in order to prepare samples for EM observation and the processed samples are resilient to the electron beam. However, these advantages come with a price – the structure of chemically fixed samples may deviate from the native structure due to preparation artifacts (shrinking, swelling, washing out of material, collapse etc.).
Freeze substitution combines many of the advantages of the two methods (freezing and chemical fixation) while minimizing their disadvantages. The sample is usually frozen by HPF, which allows for good structural preservation. Once frozen, the sample is incubated in an organic solvent kept at a temperature close to its melting point (e.g., acetone at -90°C), which replaces water molecules in the sample and dehydrates it over a few hours or days. Aldehydes and heavy metal complexes can be added to the solvent in order to chemically fix the sample and provide contrast in the microscope. This dehydration in the cold is very gentle and it involves much less artifacts than dehydration applied during chemical fixation. Once dehydrated, the temperature of the sample can be safely raised to higher temperatures without the risk of crystallization. From this stage the sample can be processed as in chemical fixation by embedding in plastic resin, sectioning, contrasting with additional heavy metal complexes and observation in the electron microscope.