Cryogenic electron microscopy

Cryogenic electron microscopy (cryo-EM) is a form of Transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. Cryo-EM is primarily used to observe fine cellular structures, viruses, and large biomolecules that are difficult to study at room temperature. The technique has revolutionized structural biology by allowing scientists to visualize biological complexes in their native state without the need for dyes or fixatives that can alter their properties.

Overview

Cryo-EM involves rapidly cooling a sample to cryogenic temperatures, typically using liquid ethane, to vitrify the water within the sample. This rapid cooling prevents the formation of ice crystals, which can damage the structure of the sample. The vitrified sample is then transferred to a TEM for imaging. Cryo-EM encompasses several techniques, including single-particle analysis (SPA), cryo-electron tomography (cryo-ET), and microcrystal electron diffraction (microED).

Single-Particle Analysis

Single-particle analysis (SPA) is a cryo-EM technique used to determine the high-resolution structures of proteins and protein complexes. In SPA, images of individual particles are aligned and averaged to increase the signal-to-noise ratio, allowing for the reconstruction of a three-dimensional model of the molecule.

Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) is a technique that collects a series of two-dimensional images at different angles relative to the electron beam. These images are then combined to produce a three-dimensional reconstruction of the sample, providing insight into the organization of complex cellular structures in a near-native state.

Microcrystal Electron Diffraction

Microcrystal electron diffraction (microED) is a method that uses cryo-EM to collect diffraction patterns from microcrystals. This technique is particularly useful for determining the structures of small organic molecules and proteins that are difficult to crystallize for X-ray diffraction.

Applications

Cryo-EM has a wide range of applications in structural biology, including the study of viruses, the mapping of protein-protein interactions, and the understanding of the mechanisms of large molecular machines. Its ability to visualize proteins in their native state has been instrumental in drug discovery and the development of new therapeutics.

Advantages and Limitations

The main advantage of cryo-EM is its ability to study complex biological structures in their native state without the need for staining or crystallization. However, the technique requires expensive equipment and significant expertise to perform. Additionally, while cryo-EM can achieve high resolution, it is not always suitable for studying very small molecules.

See Also

• Transmission electron microscopy
• Structural biology
• Protein structure
• Vitrification