Cryofixation

Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy. Typical specimens for cryofixation include small samples of plant or animal tissue, cell suspensions of microorganisms or cultured cells, suspensions of viruses or virus capsids and samples of purified macromolecules, especially proteins.^[1][2]

The method involves ultra-rapid cooling of small samples to liquid nitrogen temperature (-196 ^oC) or below, thus stopping all motion and metabolic activity, and preserving the internal structure by freezing all fluid phases solid. 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 sufficiently large to cause damage to the specimen's ultrastructure. The formation of samples containing specimens in amorphous ice is the holy grail of biological cryomicroscopy.

However, 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 of a specimen into liquid nitrogen at its boiling point (-196 ^oC)^[3] does not always produce sufficiently rapid freezing, for several reasons. Firstly, the liquid nitrogen boils rapidly around the specimen forming a film of insulating N₂ 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 reduces the temperature of the liquid nitrogen below its boiling point, so that when the specimen is plunged into it, for a brief period of time it envelops the specimen closely, and extracts heat from it efficiently. Even more rapid cooling can be obtained by plunging specimens into liquid propane or ethane (ethane has been found to be more efficient ^[4]) cooled very close to their melting points using liquid nitrogen^[5] or by slamming the specimen against highly-polished liquid nitrogen-cooled metal surfaces made of copper or silver.^[6] Secondly, two properties of water itself militate against rapid cryofixation in large specimens.^[7] The thermal conductivity of ice is very poor compared with that of metals, and water produces an exothermic release of latent heat of fusion as it freezes, defeating rapid cooldown in specimens more than a few micrometres thick.

Self pressurized rapid freezing (SPRF) which can utilize many different cryogens has recently been touted as an attractive and low cost alternative to high pressure freezing (HPF).^[8]

References

1. ^ Echlin P (1992) Low Temperature Microscopy and Analysis. Plenum Publishing Corporation, New York
2. ^ Dubochet J, Adrian M, Chang J-J, Homo J-C, Lepault J, McDowall AW, Schulz P (1988) Cryo-electron microscopy of vitrified specimens, Quarterly Review of Biophysics 21, 129-228
3. ^ Battersby BJ, Sharp JCW, Webb RI, Barnes GT (1994) Vitrification of aqueous suspensions from a controlled environment for electron microsocopy: an improved plunge-cooling device. Journal of Microscopy 176, 110-120
4. ^ Ryan, Keith P. (1992). "Cryofixation of tissues for electron microscopy: a review of plunge cooling methods.". Scan. Microsc. 6 (3): 715–743. http://www.csa.com/partners/viewrecord.php?requester=gs&collection=TRD&recid=0032394EA. 
5. ^ Bald WB (1984) The relative efficiency of cryogenic fluids used in the rapid quench-cooling of cryogenic samples. Journal of Microscopy 134, 261-270
6. ^ Allison DP, Daw CS and Rorvik MC (1987) The construction and operation of a simple and inexpensive slam freezing device for electron microscopy. Journal of Microscopy 147, 103-108
7. ^ Bald WB (1987) Quantitative Cryofixation. Adam Hilger, Bristol and Philadelphia
8. ^ Leunissen Jan L.M. abd Yi H. (2009). "Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy". J. Microsc. 235 (1): 25–35. doi:10.1111/j.1365-2818.2009.03178.x. PMID 19566624. http://www3.interscience.wiley.com/journal/122464006/abstract.