The significance of the impact of cryo-electron microscopy on the ability to image new biological structures was recognized with the 2017 Nobel Prize in Chemistry for ‘developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution’.1 Cryo-EM is a significant development in biological imaging and relies on flash-freezing a sample for imaging, rather than requiring high-quality crystalline samples that can be challenging to grow.
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Cryo-EM is a widely adopted technique by the scientific community. It is predicted that by 2024 the number of structures identified with EM methods will exceed crystallography.2
The widespread uptake of cryo-EM has also pushed method development of the technique including the combination of cryo-EM with cryo-variations of super-resolution (SR) microscopy methods.3
The combination of cryo-SR and cryo-EM methods has led to a significant enhancement in the quality and wealth of information that can be obtained on cellular structures.3 While it can be challenging to find suitable flash-freezing approaches that do not degrade the cell quality and still produce samples suitable for imaging, recent work in correlative cryo-SR and cryo-EM has offered significant new insights into the global cellular ultrastructure.
Cells are highly complex biological materials; a single cell can contain thousands of proteins. Much of the structure in a cell is on the nanometer length scale and so can be challenging to image with traditional optical microscopy approaches. This is because the diffraction limit of visible light is on the order of several hundred nanometers and so different experimental approaches, such as super-resolution methods, need to be implemented to get a spatial resolution that is better than this.
One of the key ideas in biology is that there is a close relationship between the structure and function of biological species. One of the clearest examples of this is with enzymes. Enzymes generally catalyze only a very specific subset of chemical reactions and their specificity often comes from the shape of their active site, that only allows for interactions with the desired class of chemical species.
Understanding and measuring biological structures is crucial in developing our understanding of how certain diseases affect the human body and designing pharmaceutical treatments that can interact with the correct receptors to have the desired effect. For cells, a potential treatment needs to be delivered to the correct region of the cell and this may require passing through cell membranes.
Microscopic methods have been one of the most widely adopted traditional tools in advancing our understanding of cell structure. Some microscopy methods are capable of performing live-cell imaging whereas others require immobilization of the cells before imaging.
Phase-contrast microscopy and differential interference-contrast microscopy are popular choices for live-cell imaging.4 The advantage of live-cell imaging for biological studies is it makes it possible to visualize cellular dynamics in real-time and is somewhat less artifact-prone than fixed-cell studies.
Biochemistry also has an important role to play in understanding cell composition and to some extent structure. DNA, RNA, and protein structural studies using techniques such as fluorescence for sequencing or even crystallography for proteins can help understand the composition of the potential structure of cellular components.
As well as making it possible to record structural information on biological species that could not be crystallized, one significant advantage of cryo-EM and SR-EM for cell structure studies is the spatial resolution.
Higher spatial resolution makes it possible to identify higher levels of detail on cell structures, for example, not just locating the endoplasmic reticulum but also the presence of intranuclear vesicles that are thought to play an important role in the transportation of material across the cell nucleus.3
Although cooling cells to cryogenic temperatures does immobilize them, it is still possible to recover dynamical information in such studies.5 As proteins in cells can be measured in situ with cryo-EM, if the protein folding processes can be identified, this is an excellent way to understand the mechanisms of some diseases that interfere with the protein folding process.
There are still developments and scope for improvement in cryo-EM methodologies such as finding ways to reduce the sample damage from the electron beam, or in cryo-SR methods to find brighter fluorophores that allow for higher contrast imaging.
Both methods have already had a significant impact on understanding the shapes and structures of cells, in particular some of the small connective regions between cells that cannot be visualized with poorer resolution techniques.
References and Further Reading
- Shen, P. S. (2018). The 2017 Nobel Prize in Chemistry: cryo-EM comes of age. Analytical and Bioanalytical Chemistry, 410(8), 2053–2057. https://doi.org/10.1007/s00216-018-0899-8
- Callaway, E. (2020). The protein-imaging technique taking over structural biology. Nature, 578, 201. https://media.nature.com/original/magazine-assets/d41586-020-00341-9/d41586-020-00341-9.pdf
- Hoffman, D. P., Shtengel, G., Xu, C. S., Campbell, K. R., Freeman, M., Wang, L., Milkie, D. E., Pasolli, H. A., Iyer, N., Bogovic, J. A., Stabley, D. R., Shirinifard, A., Pang, S., Peale, D., Schaefer, K., Pomp, W., Chang, C. L., Lippincott-Schwartz, J., Kirchhausen, T., … Hess, H. F. (2020). Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells. Science, 367(6475). https://doi.org/10.1126/science.aaz5357
- Cooper G. M. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Tools of Cell Biology. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9941/
- Murata, K., & Wolf, M. (2018). Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Biochimica et Biophysica Acta - General Subjects, 1862(2), 324–334. https://doi.org/10.1016/j.bbagen.2017.07.020