Kaplan Mohammed, Nicolas William J, Zhao Wei, Carter Stephen D, Metskas Lauren Ann, Chreifi Georges, Ghosal Debnath, Jensen Grant J
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
Methods Mol Biol. 2021;2215:83-111. doi: 10.1007/978-1-0716-0966-8_4.
Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).
电子冷冻断层扫描（cryo-ET）是一种能够在完整的大分子复合物处于细胞环境时对其进行研究的技术。多年来，cryo-ET对我们理解大型生物分子复合物的外观、组装、拆卸、功能及进化产生了巨大影响。近期的硬件和软件开发以及将cryo-ET与其他技术（如聚焦离子束铣削（FIB铣削）和冷冻光显微镜）相结合，已将cryo-ET的领域扩展到包括嵌入厚样品（如真核细胞）深处的瞬态分子复合物，并提高了通过cryo-ET获得的结构分辨率。在本章中，我们将概述如何对包括原核和真核细胞以及生物植物组织在内的各种生物样品进行cryo-ET研究。该概述将包括样品制备、数据采集和数据处理，以及诸如FIB铣削、冷冻切片和冷冻相关光电子显微镜（cryo-CLEM）等混合方法。
