The method involves ultra-rapid cooling of small tissue or cell samples to the temperature of liquid
nitrogen (−196 °C) or below, stopping all motion and metabolic activity and preserving the internal structure by freezing all fluid
phases solid. Typically, a sample is plunged into liquid nitrogen or into liquid
ethane or liquid
propane in a container cooled by liquid nitrogen. The ultimate objective is to freeze the specimen so rapidly (at 104 to 106 K per second) that
ice crystals are unable to form, or are prevented from growing big enough to cause damage to the specimen's
ultrastructure. The formation of samples containing specimens in
amorphous ice is the "
holy grail" of biological cryomicroscopy. In practice, it is very difficult to achieve high enough cooling rates to produce
amorphous ice in specimens more than a few
micrometres in thickness. For this purpose, plunging a specimen into liquid nitrogen at its boiling point (−196 °C) does not always freeze the specimen fast enough, for several reasons. First, the liquid nitrogen boils rapidly around the specimen forming a film of insulating gas that slows heat transfer to the cryogenic liquid, known as the
Leidenfrost effect. Cooling rates can be improved by pumping the liquid nitrogen with a
rotary vane vacuum pump for a few tens of seconds before plunging the specimen into it. This lowers the temperature of the liquid nitrogen below its boiling point, so that when the specimen is plunged into it, it envelops the specimen closely for a brief period of time and extracts heat from it more efficiently. Even faster cooling can be obtained by plunging specimens into liquid
propane or
ethane (ethane has been found to be more efficient) cooled very close to their
melting points using liquid nitrogen or by slamming the specimen against highly polished liquid nitrogen-cooled metal surfaces made of
copper or
silver. Secondly, two properties of water itself prevent rapid cryofixation in large specimens. The
thermal conductivity of ice is very low compared with that of
metals, and water releases of
latent heat of fusion as it freezes, defeating rapid cooldown in specimens more than a few micrometres thick. ==High-pressure freezing==