Cryogenic electron microscopy (cryo-EM) is atransmission electron microscopy technique applied to samples cooled tocryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment ofvitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquidethane or a mixture of liquid ethane andpropane.[1] 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.[2] This has attracted wide attention to the approach as an alternative toX-ray crystallography orNMR spectroscopy in thestructural biology field.[3]
In the 1960s, the use oftransmission electron microscopy for structure determination of biological samples 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.[6] Bothliquid helium (−269 °C or 4 K or −452.2 °F) andliquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens,[7] however high stability was never achieved. In 1980,Erwin Knapek andJacques 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.[8]
However, these results were not reproducible and just two years later amendments were published,[9] along with a commentary inNature,[10] indicating 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.[10] While cryo-EM samples are routinely collected at liquid nitrogen temperatures,[11] work has continued to understand sample behavior and collect at liquid helium temperatures.[12][13][11]
The energy of the electrons used for imaging (80–300 kV) can lead to the breaking ofcovalent bonds in organic and biological samples.[16] When imaging such specimens it is necessary to limit the electron exposure used to acquire the image. These low exposures require that the images of thousands or even millions of identical frozen molecules be selected, aligned, and averaged to obtain high-resolution maps, using specialized software. A significant improvement in structural features was achieved in 2012 by the introduction ofdirect electron detectors and better computational algorithms.[17]
Advances inelectron detector technology, particularly Direct Electron Detectors, as well as more powerful software imaging algorithms have allowed for the determination of macromolecular structures at near-atomic resolution.[18] Imaged macromolecules includeviruses,ribosomes,mitochondria,ion channels, andenzyme complexes. Starting in 2018, cryo-EM could be applied to structures as small ashemoglobin (64kDa)[19] and with resolutions up to 1.8Å.[20] In 2019, cryo-EM structures represented 2.5% of structures deposited in theProtein Data Bank,[21] and this number continues to grow.[22] An application of cryo-EM iscryo-electron tomography (cryo-ET), where a 3D reconstruction of the sample is created from tilted 2D images.
The 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made todirect electron detectors have led to a "resolution revolution"[23] pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.[24]
More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of sample orientation bias and size limit. Though the minimum size for Cryo-EM remains undetermined,proteins smaller than ~50kDa generally have too low asignal-to-noise ratio (SNR) to be able to resolve protein particles in the image, making 3D reconstruction difficult or impossible.[25][26] Multiple techniques have been reported to improve SNR when determining the structures of small proteins.[27][28] Based on high-affinityDARPins,nanobodies,antibody fragments,[29] these methods rigidly bind the target protein and thereby increase the effective particle size and introduce symmetry to improve SNR for Cryo-EM map reconstruction.
In recognition of the impact cryo-EM has had on biochemistry, three scientists,Jacques Dubochet,Joachim Frank andRichard Henderson, were awarded theNobel Prize in Chemistry "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."[4]
Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.[30] 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.[31] 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.[31]
The resolution of X-ray crystallography is limited by crystal homogeneity,[32] 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.[33] 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,[34][35] 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.[36]
According toProteopedia, the median resolution achieved by X-ray crystallography (as of May 19, 2019) on theProtein Data Bank is 2.05Å,[32] and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å.[37] As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å.[38] However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å,[35] making it a competitor in resolution in some cases.
The biological material is spread on an electron microscopy grid and is preserved in afrozen-hydrated state by rapid freezing, usually in liquidethane nearliquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of theelectron microscope column. Most biological specimens are extremelyradiosensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of transmission electron cryomicroscopy provides an additional protective factor against radiation damage).
Consequently, the images are extremelynoisy. For some biological systems it is possible to average images to increase the signal-to-noise ratio and retrieve high-resolution information about the specimen using the technique known assingle particle analysis. This approach in general requires that the things being averaged are identical, although some limited conformational heterogeneity can now be studied (e.g.ribosome). Three-dimensional reconstructions from CryoTEM images of protein complexes andviruses have been solved to sub-nanometer or near-atomic resolution, allowing new insights into the structure and biology of these large assemblies.
Analysis of ordered arrays of protein, such as 2-Dcrystals oftransmembrane proteins orhelical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is calledelectron crystallography.
The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing (cryofixation) in ethane (up to tens of μm in thickness) or more commonly byhigh pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in acryoultramicrotome at temperatures lower than −135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called transmission electron cryomicroscopy of vitreous sections (CEMOVIS) or transmission electron cryomicroscopy of frozen-hydrated sections.
In addition to allowing vitrified biological samples to be imaged, CryoTEM can also be used to image material specimens that are too volatile in vacuum to image using standard, room temperature electron microscopy. For example, vitrified sections of liquid-solid interfaces can be extracted for analysis by CryoTEM,[39] and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.[40]
Even though in the majority of approaches in electron microscopy one tries to get the best resolution image of the material, it is not always the case in cryo-TEM. Besides all the benefits of high resolution images, the signal to noise ratio remains the main hurdle that prevents assigning orientation to each particle. For example, in macromolecule complexes, there are several different structures that are being projected from 3D to 2D during imaging and if they are not distinguished the result of image processing will be a blur. That is why the probabilistic approaches become more powerful in this type of investigation.[41] There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998[42] and relatively recently adapted Bayesian approach.[43]
Themaximum likelihood estimation approach comes to this field from the statistics. Here, all the possible orientations of particles are summed up to get the resulting probability distribution. We can compare this to a typicalleast square estimation where particles get exact orientations per image.[44] This way, the particles in the sample get "fuzzy" orientations after calculations, weighted by corresponding probabilities. The whole process is iterative and with each next iteration the model gets better. The good conditions for making the model that closely represent the real structure is when the data does not have too much noise and the particles do not have any preferential direction. The main downside of maximum likelihood approach is that the result depends on the initial guess and model optimization can sometimes get stuck at local minimum.[45]
TheBayesian approach that is now being used in cryo-TEM is empirical by nature. This means that the distribution of particles is based on the original dataset. Similarly, in the usualBayesian method there is a fixedprior probability that is changed after the data is observed. The main difference from the maximum likelihood estimation lies in special reconstruction term that helps smoothing the resulting maps while also decreasing the noise during reconstruction.[44] The smoothing of the maps occurs through assuming prior probability to be a Gaussian distribution and analyzing the data in the Fourier space. Since the connection between the prior knowledge and the dataset is established, there is less chance for human factor errors which potentially increases the objectivity of image reconstruction.[43]
With emerging new methods of cryo-TEM imaging and image reconstruction the new software solutions appear that help to automate the process. After the empirical Bayesian approach have been implemented in the open source computer program RELION (REgularized LIkelihood OptimizatioN) for 3D reconstruction,[46][47] the program became widespread in the cryo-TEM field. It offers a range of corrections that improve the resolution of reconstructed images, allows implementing versatile scripts usingpython language and executes the usual tasks of 2D/3D model classifications or creatingde novo models.[48][49]
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.[59]
^Venables JA (1963). "Liquid helium cooled tilting stage for an electron microscope".Review of Scientific Instruments.34 (5):582–583.
^Knapek E, Dubochet J (August 1980). "Beam damage to organic material is considerably reduced in cryo-electron microscopy".Journal of Molecular Biology.141 (2):147–161.doi:10.1016/0022-2836(80)90382-4.PMID7441748.
^Lepault, J.; Dubochet, J.; Dietrich, I.; Knapek, E.; Zeitler, E. (January 1983). "Amendment to: Electron beam damage to organic specimens at liquid helium temperature".Journal of Molecular Biology.163 (3): 511.doi:10.1016/0022-2836(83)90073-6.
^Dickerson, Joshua L; Russo, Christopher J (25 July 2025). "A Physical Theory For Cryo-EM At Liquid-Helium Temperatures".Microscopy and Microanalysis.31 (Supplement_1).doi:10.1093/mam/ozaf048.514.
^Henderson, Richard (May 1995). "The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules".Quarterly Reviews of Biophysics.28 (2):171–193.doi:10.1017/s003358350000305x.
^Gilman, Morgan S. A.; Skiba, Meredith A. (15 August 2025). "Hidden gems bring proteins into view".Nature Chemical Biology.doi:10.1038/s41589-025-01959-4.
![Cryo-EM image of the CroV giant marine virus (scale bar represents 200 nm)[65]](/mt/?noimg=&dark=on&url=https%3a%2f%2fen.wikipedia.org%2fwiki%2fFile%3aCroV_TEM_(cropped).jpg)
