# Cryogenic 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 solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization.
In 2017, the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution." Nature Methods also named cryo-EM as the "Method of the Year" in 2015.

## History
### Early development

In the 1960s, the use of transmission electron microscopy for structure determination methods was limited because of the radiation damage due to high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage. Both liquid helium (−269 °C or 4 K or −452.2 °F) and liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens. In 1980, Erwin Knapek and Jacques Dubochet published comments on beam damage at cryogenic temperatures sharing observations that:
   Thin crystals mounted on carbon film were found to be from 30 to 300 times more beam-resistant at 4 K than at room temperature... Most of our results can be explained by assuming that cryoprotection in the region of 4 K is strongly dependent on the temperature.
However, these results were not reproducible and amendments were published in Nature just two years later informing that the beam resistance was less significant than initially anticipated. The protection gained at 4 K was closer to "tenfold for standard samples of L-valine", than what was previously stated.
In 1981, Alasdair McDowall and Jacques Dubochet, scientists at the European Molecular Biology Laboratory, reported the first successful implementation of cryo-EM. McDowall and Dubochet vitrified pure water in a thin film by spraying it onto a hydrophilic carbon film that was rapidly plunged into cryogen (liquid propane or liquid ethane cooled to 77 K). The thin layer of amorphous ice was less than 1 μm thick and an electron diffraction pattern confirmed the presence of amorphous/vitreous ice. In 1984, Dubochet's group demonstrated the power of cryo-EM in structural biology with analysis of vitrified adenovirus type 2, T4 bacteriophage, Semliki Forest virus, Bacteriophage CbK, and Vesicular-Stomatitis-Virus.

### Recent advancements

The 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made to direct electron detectors have led to a "resolution revolution" pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.
   Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) formed a consortium with engineers at the Rutherford Appleton Laboratory and scientists at the Max Planck Society to fund and develop a first prototype. The consortium then joined forces with the electron microscope manufacturer FEI to roll out and market the new design. At about the same time, Gatan Inc. of Pleasanton, California came out with a similar detector designed by Peter Denes (Lawrence Berkeley National Laboratory) and David Agard (University of California, San Francisco). A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company (San Diego, California).
More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of sample orientation bias and size limit. Proteins smaller than ~50 kDa generally have too low a signal-to-noise ratio (SNR) to be able to resolve protein particles in the image, making 3D reconstruction difficult or impossible. The SNR of smaller proteins can be improved by binding them to an imaging scaffold. The Yeates group at UCLA was able to create a clearer image of three variants of KRAS (roughly 19 kDa in size) by utilising a rigid imaging scaffold, and using DARPins as modular binding domains between the scaffold and the protein of interest.

### 2017 Nobel Prize in Chemistry

In recognition of the impact cryo-EM has had on biochemistry, three scientists, Jacques Dubochet, Joachim Frank and Richard Henderson, were awarded the Nobel Prize in Chemistry "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."

## Comparisons to X-ray crystallography

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules. However, the aforementioned improvements in cryo-EM have increased its popularity as a tool for examining the details of biological molecules. Since 2010, yearly cryo-EM structure deposits have outpaced X-ray crystallography. Though X-ray crystallography has drastically more total deposits due to a decades-longer history, total deposits of the two methods are projected to eclipse around 2035.
The resolution of X-ray crystallography is limited by crystal homogeneity, and coaxing biological molecules with unknown ideal crystallization conditions into a crystalline state can be very time-consuming, in extreme cases taking months or even years. To contrast, sample preparation in cryo-EM may require several rounds of screening and optimization to overcome issues such as protein aggregation and preferred orientations, but it does not require the sample to form a crystal, rather samples for cryo-EM are flash-frozen and examined in their near-native states.
According to Proteopedia, the median resolution achieved by X-ray crystallography (as of May 19, 2019) on the Protein Data Bank is 2.05 Å, and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å. As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å. However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å, making it a competitor in resolution in some cases.

## Correlative light cryo-TEM and cryo-ET

In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.

## Scanning electron cryomicroscopy

Scanning electron cryomicroscopy (cryoSEM) is a scanning electron microscopy technique with a scanning electron microscope's cold stage in a cryogenic chamber.

## Cryogenic transmission electron microscopy

Cryogenic transmission electron microscopy (cryo-TEM) is a transmission electron microscopy technique that is used in structural biology and materials science. Colloquially, the term "cryogenic electron microscopy" or its shortening "cryo-EM" refers to cryogenic transmission electron microscopy by default, as the vast majority of cryo-EM is done in transmission electron microscopes, rather than scanning electron microscopes.

## Centers

The Federal Institute of Technology, the University of Lausanne and the University of Geneva opened the Dubochet Center For Imaging (DCI) at the end of November 2021, for the purposes of applying and further developing cryo-EM. Less than a month after the first identification of the SARS-CoV-2 Omicron variant, researchers at the DCI were able to define its structure, identify the crucial mutations to circumvent individual vaccines and provide insights for new therapeutic approaches.
The Danish National cryo-EM Facility also known as EMBION was inaugurated on December 1, 2016. EMBION is a cryo-EM consortium between Danish Universities (Aarhus University host and University of Copenhagen co-host).

## Advanced methods

* Cryogenic electron tomography (cryo-ET), a specialized application where many images are taken of individual samples at various tilt angles, resulting in a 3D reconstruction of a single sample.
* Electron crystallography, method to determine the arrangement of atoms in solids using a TEM
* MicroED, method to determine the structure of proteins, peptides, organic molecules, and inorganic compounds using electron diffraction from 3D crystals
* Single particle analysis cryo-EM, an averaging method to determine protein structure from monodisperse samples.
* Cryo-EM image of GroEL suspended in amorphous ice at 50000× magnification
* Structure of alcohol oxidase from Pichia pastoris by Cryo-EM
* Cryo-EM image of an intact ARMAN cell from an Iron Mountain biofilm. Image width is 576 nm.
* Cryo-EM image of the CroV giant marine virus (scale bar represents 200 nm)