Cryogenic fixation, also known as vitrification, is the transformation of water from a liquid to an amorphous solid state without inducing the nucleation of ice crystals. HPF devices synchronise pressurisation and cooling of the sample (to below the glass transition temperature (Tg)) within 20ms, extending the depth of vitrification to as much as 200μm.

Cryo-fixed samples prepared this way require further sample preparation to make them compatible with a transmission electron microscope. They must either be sectioned and imaged below the Tg or converted to a room-temperature stable state by Freeze Substitution (FS) into a resin.

Freeze-substitution is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration. During freeze substitution the "frozen" water is dissolved by an organic solvent, which usually also contains chemical fixatives. Freeze-substitution links instant physical immobilization of the cell constituents (cryo-fixation) and resin embedding. Once substitution is complete, samples are gradually warmed-up and processed further as for conventionally prepared samples.

Successful cryo-fixation followed by FS shows superior preservation of fine structure compared to chemical fixation techniques.

Related equipment

Leica AFS-2 Freeze Substitution (FS)