cryo-Transmission Electron Microscopy (cryoTEM)

cryoTEM (or simply cryo-electron microscopy - cryoEM) is a buzzword that encompases the field of structural biology where the principle experimental technique is TEM followed by extensive image processing. bacterio rhodopsinThe "cryo" designation in cryoTEM arises from the observation made in the 1980's that the extensive radiation damage to unstained biological materials due to the electron beam can be lowered to managable levels by holding the specimen near liquid N2 temperatures GroELand using "minimal dose imaging techniques" during the image recording process. These innovations have allowed structural biology using the transmission electron microscope to begin to fullfill the promise of near atomic resolution structures of biological macromolecules, starting with thin protein crystals such as bacterio-rhodopsin (PDB ID 1BRD) and more recently extending to such macro-molecular assemblies as ribosomes, GroEL (PDB ID 3CAU) and some icosahedral viruses that have been imaged under near native conditions using rapid vitrification in biological buffers.

cryoTEM is often a synonym for the electron microscopy of frozen, hydrated specimens and several things have happened in the decades since the initial observations about low temperature effects that have expanded this field from the domain of a select number of specialized labs to a more general and widely practiced area:

At the same time that it has become easier and easier to work with vitrified specimens at low temperatures, the term cryoTEM has been expanded to include most structural biology using electron microscopy whether or not vitrified specimens or even low temperatures are involved. A more formal description of this general field of structural biology might be "electron crystallography of biological macromolecules" which is the title of a recent book that gives an excellent over-view of the entire field. However, cryoTEM (or cryoEM) seems to be the more commonly used designation and is certainly easier to remember and say.

The experimental details of an investigation using cryoTEM are dictated by the macromolecular order and the symmetry of the system being examined and can be fundamentally divided into the following sub-areas:

The first two areas involve the application of diffraction theory to TEM images and are more related to each other than to the third area. However, crystallographic diffraction and helical diffraction are rather different and it is useful to divide the diffraction-based approaches into distinct sub-areas.

The third sub-area (also referred to as "single particle analysis") can be further sub-divided based on the three-dimensional point group symmetry present in the macromolecule: specific techniques have been developed for the analysis of icosahedral complexes (532 point group symmetry) such as virus particles and certain enzyme complexes that are rather different from the techniques used for lower or absent point group symmetry. There are also several new developments in the analysis of helical protein cystovirus imagefibers that more properly belong to the sub-area of single particle analysis than to the diffraction-based methods mentioned above.

A very recent development in this general area is that the field of biological electron tomography (both with room temperature samples embedded in plastic resins and frozen samples that have been vitrified in different ways) has come to be included under the general umbrella of cryoTEM. This development has been triggered mainly by the wide availability of robust and dependable tomographic data collection software for all recently purchased TEMs, and while not everyone who works in electron tomography also works in the general area of cryoTEM (electron crystallography of biological macromolecules!), it does seem that almost every lab that is devoted to cryoTEM has recently become involved in electron tomography to some extent.

It is also very important to note that while the term "cryoTEM" equates to "structural biology using the electron microscope," the actual work done with the microscope includes conventional TEM, EFTEM and electron diffraction and is likely to expand to include areas such as STEM, EELS and EDS that are usually reserved for the materials science community.