Editorial Feature

Applications of Cryo-Electron Microscopy

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A higher resolution of any image is achieved by the wavelength of the radiation used for imaging. A typical electron microscope uses a beam of electrons in the wavelength of ~ 0.02Å for operation at 300kV. This has achieved resolutions in the range of 2–5Å. Practically one may magnify, to atomic resolution now, and image anything, unlike in light microscopy.

Drawbacks of Typical Electron Microscopy

However, routine performance of a normal electron microscope was unsuccessful due to the damage done to the samples by the high energy electrons. The vacuum needed for the beam of electrons also damages the samples, especially biological or organic ones. To overcome these challenges, Richard Henderson coated a layer of glucose solution onto his sample and for the first time, the structure of bacteriorhodopsin, a protein, was done at a resolution of ~ 7Å, in 1975. Persistent research to obtain better images from electron microscopy led to the arrival of cryoelectron microscopy, where imaging is done by cooling samples with liquid nitrogen.

Normal cooling also caused the formation of ice crystals in the sample that hindered true imaging. This was corrected by vitrification of the water layer on the sample. Frozen samples kept at liquid helium or liquid nitrogen temperatures are used in cryoelectron microscopy.

Cryoelectron Microscopy

Development in the microscopy has led to atomic resolution imaging (even single atom visualization has been possible!); including 3D structures using electron tomography. The images obtained are computationally combined to have ‘tomograms’ (similar to the computerized axial tomography) – which results in 3D images of the sample/specimen. Using high-end software tools, users built high-quality reconstructions, 3D-models, atomic density maps, in a fully automated manner.

In fact, many cryoelectron microscopy images provide structures at atomic detailing, which was previously obtained only from X-ray crystallography. Jacques Dubochet, Joachim Frank and Richard Henderson shared the 2017 chemistry Nobel Prize for cryoelectron microscopy to image molecules.

The phenomenal success and use of this technique are that “Cryoelectron microscopy is a tool at its sharpest, providing accurate results.” This microscopy has exploded the imaging world with innumerable fine quality images that equal none.

Advantages of Cryoelectron Microscopy

Sample preparation in cryoelectron microscopy is simple, without the need for any fixing, dying and other pre-preps that is generally required in other microscopic techniques. This allows to view and study fine structures in small organisms such as viruses and bacteria or other cellular parts and protein complexes that require molecular resolution. The extent of detailing in the imaging and the resulted tomograms is unbelievably sharp; for example, internal bacterial structures, twisted nucleoids, localization of ribosomes at a specific location within the cell, mammalian sub-cellular structures, and single particle imaging and characterization.

Cryoelectron microscopy is often used by complimenting with techniques such as X-ray crystallography and NMR spectroscopy. Analysis of small, dynamic proteins not feasible using NMR spectroscopy and X-ray crystallography may be uniquely suited for study in cryoelectron microscopy. Similar to the Protein Data Bank (PDB) created using the NMR spectroscopy and X-ray crystallography, a worldwide repository for data from cryoelectron microscopy deposited at Electron Microscopy Data Bank (EMDB). EMDB was founded at the European Bioinformatics Institute (EBI) in 2002. It has been managed by the Protein Data Bank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB PDB), and the National Center for Macromolecular Imaging (NCMI).

Among a few challenges are: strikingly, two different users using the same tool do not necessarily get the same result. The current standard metric for determining resolution, Fourier shell correlation, is inconsistently calculated by users and also inadequate for determining map resolvability. Averaging is required from multiple tomograms when with a single tomogram it is not possible to extract the information.

Conclusion

Notwithstanding the hurdles in making use of this microscopy, Cryoelectron microscopy stands apart, revolutionizing molecular biology, materials science, and medicine. The ability to view structures at their native state, focusing on reactive sites on proteins has helped scientists design drugs, such as anti-virals, understand the diseases at the molecular level, bringing into light the processes occurring at the physiological level. Among other sciences, Cryoelectron microscopy brings about a great impact in structural biology and biomedicine therapeutics.

Sources and Further Reading

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Ramya Dwivedi

Written by

Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.

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