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

electron microscopy An electron microscope is a microscope that uses a beam of electrons as a source of illumination. It uses electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing i ...

and

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

. Typical specimens for cryofixation include small samples of

plant Plants are the eukaryotes that form the Kingdom (biology), kingdom Plantae; they are predominantly Photosynthesis, photosynthetic. This means that they obtain their energy from sunlight, using chloroplasts derived from endosymbiosis with c ...

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

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. Viruses are found in almo ...

or virus capsids and samples of purified

macromolecules A macromolecule is a "molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass." Polymers are physi ...

, 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, re ...

. Types of cryo-fixation are freezing-drying, freezing-substitution and freezing-etching.

Plunge freezing

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

nitrogen Nitrogen is a chemical element; it has Symbol (chemistry), symbol N and atomic number 7. Nitrogen is a Nonmetal (chemistry), nonmetal and the lightest member of pnictogen, group 15 of the periodic table, often called the Pnictogen, pnictogens. ...

(−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 a naturally occurring Organic compound, organic chemical compound with chemical formula . At standard temperature and pressure, ethane is a colorless, odorless gas. Like many hydrocarbons, ethane is List of purification methods ...

or liquid

propane Propane () is a three-carbon chain alkane with the molecular formula . It is a gas at standard temperature and pressure, but becomes liquid when compressed for transportation and storage. A by-product of natural gas processing and petroleum ref ...

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 water (known as ice) in crystal structure, symmetrical shapes including hexagonal crystal family, hexagonal columns, hexagonal plates, and dendrite (crystal), dendritic crystals. Ice crystals are responsible for various at ...

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 c ...

. The formation of samples containing specimens in

amorphous ice Variations in pressure and temperature give rise to different phases of ice, which have varying properties and molecular geometries. Currently, twenty-one phases, including both crystalline and amorphous ices have been observed. In modern histor ...

is the "

holy grail The Holy Grail (, , , ) 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 miraculous healing powers, sometimes providing eternal youth or sustenanc ...

" 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 (English in the Commonwealth of Nations, Commonwealth English as used by the International Bureau of Weights and Measures; SI symbol: μm) or micrometer (American English), also commonly known by the non-SI term micron, is a uni ...

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 or film boiling is a physical phenomenon in which a liquid, close to a solid surface of another body that is significantly hotter than the liquid's boiling point, produces an insulating vapor layer that keeps the liquid fr ...

. 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

(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 Copper is a chemical element; it has symbol Cu (from Latin ) 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 pinkish-orang ...

silver Silver is a chemical element; it has Symbol (chemistry), symbol Ag () and atomic number 47. A soft, whitish-gray, lustrous transition metal, it exhibits the highest electrical conductivity, thermal conductivity, and reflectivity of any metal. ...

. 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 heat conduction, conduct heat. It is commonly denoted by k, \lambda, or \kappa and is measured in W·m−1·K−1. Heat transfer occurs at a lower rate in materials of low ...

of ice is very low compared with that of

metals A metal () is a material that, when polished or fractured, shows a lustrous appearance, and conducts electricity and heat relatively well. These properties are all associated with having electrons available at the Fermi level, as against no ...

, 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 Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product and lowering pressure, thereby removing the ice by sublimation. This is in contrast to dehydration by ...

References

{{reflist Microscopy Scientific techniques

Plunge freezing

High-pressure freezing

Freeze-drying

See also

References