Kuba Jakub, Mitchels John, Hovorka MiloŠ, Erdmann Philipp, Berka LukÁŠ, Kirmse Robert, KÖnig Julia, DE Bock Jan, Goetze Bernhard, Rigort Alexander
Thermo Fisher Scientific Brno s.r.o., Brno, Czech Republic.
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
J Microsc. 2021 Feb;281(2):112-124. doi: 10.1111/jmi.12939. Epub 2020 Jul 2.
Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.
冷冻电子断层扫描（cryo-ET）是一项开创性技术，用于在细胞结构背景下对生物分子进行三维可视化和分析。它能够对单个蛋白质进行结构研究，并分析其在细胞内的空间排列。冷冻断层扫描提供了复杂、异质且瞬态的亚细胞环境的快照。由于在非晶冰中能出色地保存结构，因此可以研究蛋白质在天然状态下的相互作用和空间关系，而不受化学固定剂或造影剂的干扰。随着聚焦离子束（FIB）技术的引入，用于电子断层扫描的细胞样品制备变得更加简便快捷。最新一代专门为冷冻应用设计的集成FIB和扫描电子显微镜（SEM）仪器（双束显微镜），在自动化、成像以及使用冷冻剥离技术制备高压冷冻块状样品方面取得了进展。此外，相关冷冻荧光显微镜通过集成的软件和硬件接口提供细胞靶向信息。基于相关冷冻显微镜、冷冻FIB和冷冻ET的结合所取得的快速进展，已经为细胞过程带来了丰富的新见解，并提供了新的细胞三维图像数据。在此，我们介绍了我们在冷冻断层扫描工作流程方面的最新进展，并讨论了未来面临的挑战。分层描述：本文描述了我们在冷冻电子断层扫描（cryo-ET）工作流程方面的最新进展。冷冻ET具有卓越的结构保存能力，并能以分子分辨率提供玻璃化细胞内部的三维快照。在通过冷冻ET对细胞样品进行成像之前，必须使其适用于透射电子显微镜观察。这是通过使用冷冻聚焦离子束（cryo-FIB）显微镜从细胞样品制备出200 - 300纳米厚的冷冻薄片来实现的。在工作流程中使用冷冻相关光电子显微镜（cryo-CLEM）来引导冷冻薄片制备到感兴趣的细胞区域。我们涵盖了冷冻ET工作流程的基本介绍，并展示了冷冻CLEM的新进展，这有助于冷冻光学显微镜和冷冻FIB之间的连接。接下来，我们展示了我们在冷冻FIB软件自动化方面的进展，以简化冷冻薄片制备。在最后一部分，我们展示了冷冻FIB如何用于三维成像，以及如何使用新开发的冷冻剥离技术处理块状冷冻细胞样品（通过高压冷冻获得）。
