High Pressure Freezing Technique and following-up procedures

Rapid freezing techniques permit time resolution of dynamic biological events in the millisecond range. Freezing techniques at ambient pressure allow adequate freezing of aqueous layers up to 10 micrometer thickness. Freezing at a pressure of 2100 bar results in adequate cryoimmobilisation of biological samples <200 micrometer thick.

CryoTEM of cryosections of high pressure frozen, native, biological material

Cryoimmobilised biological material is, in general, further processed for microscopical analysis by freeze-fracturing or by freeze-substitution; the latter technique is frequently used in combination with low temperature embedding for immunocytochemical studies. Neither technique permits differentiation between vitrified and crystalline cellular water.

The direct observation of cryosections in the CryoTEM is the only technique which allows one simultaneously to characterize the state of the frozen water by electron diffraction and that of the biological structures (McDowall et al., (1983), J. Microsc. 131: 1-9.).

Cryosectioning of tissue cultures and suspensions, as a purely physical technique, must be established in order to quantitate the extent to which the living state can be described by the currently used, routinely handled specimen preparation methods e. g. freeze-substitution.

Freeze-Substitution

Freeze-substitution replaces cellular water at low temperatures under controlled conditions, with an organic solvent, allowing the sample to be further processed for TEM observation. The addition of metal salts, e.g. uranyl acetate or osmium tetroxide, to the organic solvent used for freeze-substitution, stabilizes the structures. After completion of the dehydration process the samples are infiltrated with the embedding resin and polymerized.

With respect to the preservation of the structural integrity this hybrid technique is less reliable than the purely physical follow-up procedures such as cryosectioning or freeze-fracturing, since effects of the organic solvents and the embedding chemistry could not be excluded.

High Pressure Freezing Literature Compilation >>