high pressure freezing

High pressure freezing, one of rapid freeze fixation techniques, is to fix biological specimens such as biological tissues, biological cells and bacteria.  Rapid freezing under an about 2000 atm suppresses formation of ice crystals, which give rise to the destruction of the tissues, by decreasing the melting point of water by about -20 °C and increasing its viscosity.  This technique provides a uniform freezing under amorphous ice with a depth more than one order of magnitude larger (about 200 μm) compared with the freezing depth at the atmospheric pressure.  Specimen preparation for TEM observation of the frozen specimen is carried out by one of the following three procedures.  (1) Applying freeze sectioning to the specimen, (2) Applying resin embedding and ultrathin sectioning to the specimen after the specimen is subject to freeze substitution and is returned to room temperature, and (3) Making a replica of the specimen by freeze fracturing.

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