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This article was updated on 28 January 2020
Cryo-electron microscopy (Cryo-EM) is a scientific technique used for studying the structures of cells, viruses and proteins at the molecular level.
Understanding biology and biochemistry at the molecular level is key to the study of drug development and disease treatment. For example, during a recent outbreak of the Zika virus in Brazil, a group of scientists produced a high-resolution 3D model of the virus' structure that acted as a foundation for identifying locations on the virus that might be targeted by drugs.
Imaging Biomolecules Using Cryo-Electron Microscopy
When it comes to imaging biomolecules, there are several potential options, but many of them have significant drawbacks. X-ray diffraction can provide high-resolution images of biomolecules. However, the sample must be crystallized, and many proteins won’t crystallise without being altered.
Nuclear Magnetic Resonance (NMR) offers high-definition images of biomolecules, but the technique can only be used on fairly small proteins or aspects of proteins.
Transmission electron microscopes (TEMs) can image the structures of biomolecules at the atomic scale by using a beam of electrons, rather than the light source of an optical microscope. Because the resolution of a microscope is directly affected by the wavelength of the radiation source, the resolution achievable with an optical microscope is considerably less than what is possible with a beam of electrons. However, some materials, especially biomolecules, are not able to tolerate the vacuum conditions and powerful electron beam of a TEM.
To address these TEM shortcomings, Cryo-EM applies low-power electron beams to frozen samples. Around 40 years ago, scientists were able to show a relatively low-power electron microscope used at cryogenic temperatures could minimize the damaging impact of radiation. In 2017, Jacques Dubochet, Joachim Frank and Richard Henderson received the Nobel Prize in Chemistry for developing high-resolution Cryo-EM.
The technique has made it possible for researchers to view the ways that biomolecules move and interact as they carry out their various functions. Liquid forms of nitrogen and helium have been successfully used to freeze samples to generate 3D images of molecules nearly at atomic-level resolution. A meticulous comparison of these two cryogens has shown very comparable protective effects against radiation.
The standard cryo-EM method starts with vitrification; a process involving the test sample being cooled in a way that does not allow for water molecules to crystallize. The result is an amorphous solid and intact sample structure.
For years, the vitrification process has been a demanding and intricate process that was performed manually. Automated vitrification systems have recently made the process faster and easier, addressing a major bottleneck in the Cryo-EM process. A system from SPT Labtech called “chameleon” uses an algorithm to calculate the optimal time for plunging a sample into the cooling medium.
The cooled sample is then screened-in for particle orientation, concentration and distribution. Then, a number of two-dimensional (2-D) pictures are taken. In the last step, the visual information is run through processing software that produces a comprehensive, 3D model of complex biological structures at the molecular scale. These models can show molecular interactions that cannot be seen without the technique, which has been crucial to several scientific discoveries.
There are multiple sub-categories of cryo-electron microscopy, including cryo-electron tomography, single-particle cryo-electron microscopy and electron crystallography. All have been used effectively to investigate biological structures in various contexts. Cryo-electron tomography involves the 3-D imaging of macromolecular surfaces by capturing a series of 2-D images at different tilts relative to the incident electron beam. Single-particle cyro-electron microscopy uses a similar process to produce a 3-D image of a single particle. Electron crystallography is used for structural and functional studies of membrane proteins and involves the crystallization of these proteins in 2-D arrays.
Cryo-EM has been applied across a wide spectrum of biological research, including the imaging of tissue sections, individual bacteria, proteins and viruses. Cryo-electron tomography, single-particle cryo-electron microscopy and electron crystallography have been used on their own, and in combination. Furthermore, information from cryo-electron microscopy has been used in concert with data from other imaging techniques, such as X-ray crystallographic and NMR.
Recent Developments in Cryo-EM
In recent years, the use of Cryo-EM has expanded considerably. The rising popularity of this technique has been fuelled by streamlining and automation of various steps within the Cryo-EM process.
Process refinements have allowed for the use of Cyro-EM with a broader spectrum of samples. Thanks in part to processing refinements and a broader range of applications, national scale facilities now routinely provide access to Cyro-EM technology, expanding access on an unprecedented scale.
References and Further Reading