Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for

electron microscopy An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...

and

cryo-electron microscopy Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample sol ...

. Typical specimens for cryofixation include small samples of

plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...

animal Animals are multicellular, eukaryotic organisms in the Kingdom (biology), biological kingdom Animalia. With few exceptions, animals Heterotroph, consume organic material, Cellular respiration#Aerobic respiration, breathe oxygen, are Motilit ...

tissue, cell suspensions of microorganisms or cultured cells, suspensions of

viruses A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...

or virus capsids and samples of purified

macromolecules A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...

, especially

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

. Cryo fixation Types 1.Freezing-drying 2.Freezing-substitution 3.freezing-etching

Plunge freezing

The method involves ultra-rapid cooling of small tissue or cell samples to the temperature of liquid

nitrogen Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...

(−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 Ethane ( , ) is an organic chemical compound with chemical formula . At standard temperature and pressure, ethane is a colorless, odorless gas. Like many hydrocarbons, ethane is isolated on an industrial scale from natural gas and as a petr ...

or liquid

propane Propane () is a three-carbon alkane with the molecular formula . It is a gas at standard temperature and pressure, but compressible to a transportable liquid. A by-product of natural gas processing and petroleum refining, it is commonly used a ...

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 Ice crystals are solid ice exhibiting atomic ordering on various length scales and include hexagonal columns, hexagonal plates, dendritic crystals, and diamond dust. Formation The hugely symmetric shapes are due to depositional growth, na ...

are unable to form, or are prevented from growing big enough to cause damage to the specimen's

ultrastructure Ultrastructure (or ultra-structure) is the architecture of cells and biomaterials that is visible at higher magnifications than found on a standard optical light microscope. This traditionally meant the resolution and magnification range of a co ...

. The formation of samples containing specimens in

amorphous ice Amorphous ice (non-crystalline or "vitreous" ice) is an amorphous solid form of water. Common ice is a crystalline material wherein the molecules are regularly arranged in a hexagonal lattice, whereas amorphous ice has a lack of long-range order ...

is the "

holy grail The Holy Grail (french: Saint Graal, br, Graal Santel, cy, Greal Sanctaidd, kw, Gral) is a treasure that serves as an important motif in Arthurian literature. Various traditions describe the Holy Grail as a cup, dish, or stone with miracul ...

" of biological cryomicroscopy. In practice, it is very difficult to achieve high enough cooling rates to produce

in specimens more than a few

micrometres The micrometre (American and British English spelling differences#-re, -er, international spelling as used by the International Bureau of Weights and Measures; SI symbol: μm) or micrometer (American and British English spelling differences# ...

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 The Leidenfrost effect is a physical phenomenon in which a liquid, close to a surface that is significantly hotter than the liquid's boiling point, produces an insulating vapor layer that keeps the liquid from boiling rapidly. Because of this re ...

. Cooling rates can be improved by pumping the liquid nitrogen with a

rotary vane vacuum pump A rotary vane pump is a positive-displacement pump that consists of vanes mounted to a rotor that rotates inside a cavity. In some cases these vanes can have variable length and/or be tensioned to maintain contact with the walls as the pump rotates ...

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

(ethane has been found to be more efficient) cooled very close to their

melting points The melting point (or, rarely, liquefaction point) of a substance is the temperature at which it changes state from solid to liquid. At the melting point the solid and liquid phase exist in equilibrium. The melting point of a substance depend ...

using liquid nitrogen or by slamming the specimen against highly polished liquid nitrogen-cooled metal surfaces made of

copper Copper is a chemical element with the symbol Cu (from la, cuprum) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pinkis ...

silver Silver is a chemical element with the Symbol (chemistry), symbol Ag (from the Latin ', derived from the Proto-Indo-European wikt:Reconstruction:Proto-Indo-European/h₂erǵ-, ''h₂erǵ'': "shiny" or "white") and atomic number 47. A soft, whi ...

. Secondly, two properties of water itself prevent rapid cryofixation in large specimens. The

thermal conductivity The thermal conductivity of a material is a measure of its ability to conduct heat. It is commonly denoted by k, \lambda, or \kappa. Heat transfer occurs at a lower rate in materials of low thermal conductivity than in materials of high thermal ...

of ice is very low compared with that of

metals A metal (from Greek μέταλλον ''métallon'', "mine, quarry, metal") is a material that, when freshly prepared, polished, or fractured, shows a lustrous appearance, and conducts electricity and heat relatively well. Metals are typicall ...

, and water releases of

latent heat of fusion In thermodynamics, the enthalpy of fusion of a substance, also known as (latent) heat of fusion, is the change in its enthalpy resulting from providing energy, typically heat, to a specific quantity of the substance to change its state from a s ...

as it freezes, defeating rapid cooldown in specimens more than a few micrometres thick.

High-pressure freezing

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). 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.

Freeze-drying

Drying times are reduced by up to 30% with proper freeze drying.

References

{{reflist Microscopy Scientific techniques

Plunge freezing

High-pressure freezing

Freeze-drying

See also

References