Bioscience Method 2024, Vol.15, No.2, 50-57 http://bioscipublisher.com/index.php/bm 51 1 Fundamentals of Cryo-Electron Microscopy 1.1 Principle and workflow of cryo-electron microscopy Cryo-electron microscopy, also known as cryo-transmission electron microscopy, is a high-resolution imaging technique used for structural analysis of biological macromolecules under near-physiological conditions. Its principles and workflow are closely linked, ensuring high-quality and accurate imaging of biological samples (Angel and Marta, 2019). The core of cryo-electron microscopy lies in its "freezing" step. In a low-temperature environment (typically around -180 °C), biological samples are rapidly frozen to stabilize their bioactivity and structure. This freezing process minimizes radiation damage and ice crystal formation in the sample, thereby preserving its original state. The frozen samples are then placed under an electron microscope. Instead of visible light used in conventional optical microscopes, an electron microscope uses a high-energy electron beam to observe the sample. As the electron beam passes through the sample, it interacts with atoms within, causing scattering and absorption effects. These effects are captured by an electron detector and converted into visible images. Clare et al. (2017) noted that during the image formation process, the electron microscope adjusts parameters such as the electron beam focus, exposure time, and detector sensitivity to capture the fine structure of the sample. Further processing and analysis of the electron images provide three-dimensional information about the structure of biological macromolecules. The workflow of cryo-electron microscopy requires highly precise instruments and strict operating procedures, as well as accurate sample preparation and in-depth data analysis. The advantage of this technique is its ability to perform high-resolution imaging of biological macromolecules under conditions close to physiological, thereby revealing details of the interactions between drugs and biological macromolecules, and providing important insights into the mechanisms of drug action. 1.2 Application of cryo-electron microscopy in the structural analysis of biological macromolecules Cryo-electron microscopy plays a crucial role in the structural analysis of biological macromolecules. Because it can image biological samples at low temperatures close to physiological conditions, it has become a powerful tool for studying the structures of biological macromolecules, especially those samples that are difficult to crystallize. In the structural analysis of biological macromolecules, cryo-electron microscopy provides high-resolution three-dimensional images, allowing for the direct observation of the fine structures and dynamic changes of biological macromolecules (Figure 1). This includes not only the structures of individual biological macromolecules such as proteins and nucleic acids but also their interactions and the formation of complexes. Figure 1 CryoEM technique for imaging (Adopted from Sara et al., 2021) Image caption: a: lower distribution of biomolecules; b: higher magnification reveals their relationship with the surface of nanoparticles (Adopted from Sara et al., 2021)
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