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Cryo-electron Microscopy
Cryo-electron microscopy or cryo-EM examines frozen biological samples at cryogenic temperatures below −150 °C.
Unlike conventional EM, cryo-EM does not require fixing and dehydrating a sample which helps to preserve the native state of biological structures.
The high-energy electrons used in EM can damage a sample’s chemical bonds. Freezing the sample helps to prevent this damage, allowing for near atomic-resolution images.
In cryo-EM, small biological samples in solution are vitrified, meaning rapidly frozen, in liquid ethane. Vitrification fixes the sample in its native state in a thin water layer, and the water in the sample forms a glass-like state without the appearance of ice crystals.
Thicker samples, such as cells and tissues, cannot be vitrified in a thin liquid layer. Therefore, these samples can be studied using the cryo-EM of vitreous sections method. In this method, a sample is first vitrified by high-pressure freezing and then cut into ultrathin sections at −140 °C before being observed using cryo-EM.
33.16:
Cryo-electron Microscopy
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for which they won the Nobel Prize in Chemistry in 2017.
X-ray diffraction and nuclear magnetic resonance (NMR) techniques are also used for obtaining high-resolution structures of biomolecules or studying their conformational changes. However, to get the X-ray structure, a molecule needs to be crystallized, which may not be possible every time. Even if a molecule is crystallizable, the crystallization process can alter its biomolecular structure, which is no longer representative of the native structure. Cryo-EM does not require a crystallized sample, and also enables visualization of molecular movements or interactions of biomolecules in their native state. Similarly, NMR technique is only limited to relatively small soluble proteins and is not suited for the non-polar proteins embedded in cell membranes. On the other hand, cryo-EM enables the study of larger proteins, membrane-bound receptors, or biomolecular complexes.
Cryo-EM is extensively being used in biochemistry to study the structure, function, and interaction of biomolecules. The infectious agents, such as viruses, can be identified and studied using cryo-EM. For example, during the Zika virus outbreak in Brazil, cryo-EM was utilized to create a 3D image of the virus structure so that the potential anti-viral drug targets could be identified.