Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for theelectron microscopy andcryo-electron microscopy.[1] Typical specimens for cryofixation include small samples ofplant oranimaltissue, cell suspensions of microorganisms orcultured cells, suspensions ofviruses orvirus capsids and samples of purifiedmacromolecules, especiallyproteins.[2][3]
Types of cryo-fixation are freezing-drying, freezing-substitution and freezing-etching.[citation needed][clarification needed]
The method involves ultra-rapid cooling of small tissue or cell samples to the temperature of liquidnitrogen (−196 °C) or below, stopping all motion and metabolic activity and preserving the internal structure by freezing all fluidphases solid. Typically, a sample is plunged into liquid nitrogen or into liquidethane or liquidpropane in a container cooled by liquid nitrogen. The ultimate objective is to freeze the specimen so rapidly (at 104 to 106 K per second) thatice crystals are unable to form, or are prevented from growing big enough to cause damage to the specimen'sultrastructure. The formation of samples containing specimens inamorphous ice is the "holy grail" of biological cryomicroscopy.[citation needed]
In practice, it is very difficult to achieve high enough cooling rates to produceamorphous ice in specimens more than a fewmicrometres in thickness. For this purpose, plunging a specimen into liquid nitrogen at its boiling point (−196 °C)[4] does not always freeze the specimen fast enough, for several reasons. First, the liquid nitrogen boils rapidly around the specimen forming a film of insulatingN
2 gas that slows heat transfer to the cryogenic liquid, known as theLeidenfrost effect. Cooling rates can be improved by pumping the liquid nitrogen with arotary 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 liquidpropane orethane (ethane has been found to be more efficient)[5] cooled very close to theirmelting points using liquid nitrogen[6] or by slamming the specimen against highly polished liquid nitrogen-cooled metal surfaces made ofcopper orsilver.[7] Secondly, two properties of water itself prevent rapid cryofixation in large specimens.[8] Thethermal conductivity of ice is very low compared with that ofmetals, and water releases oflatent heat of fusion as it freezes, defeating rapid cooldown in specimens more than a few micrometres thick.
High pressure helps prevent the formation of large ice crystals. Self pressurized rapid freezing (SPRF) can utilize many different cryogens has recently been touted as an attractive and low cost alternative to high pressure freezing (HPF).[9] Cold pressurized nitrogen substitutes ethanol at temperatures roughly 123K. The warm ethanol is then expelled by the freezing LN2 and most likely produces an ethanol-nitrogen mixture that gradually becomes colder and colder.[10]
Drying times are reduced by up to 30% with properfreeze drying.[11]