实用的冷冻断裂-冷冻微割技术方法及其在细胞冷冻电子断层成像技术中的应用。
Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography.
机构信息
University of California, San Diego, La Jolla, CA, USA.
出版信息
Methods Mol Biol. 2021;2215:49-82. doi: 10.1007/978-1-0716-0966-8_3.
Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to ∼400 nm in thickness, narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella) through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a near-native state. This approach has proved to be robust and relatively simple for new users and exhibits minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users with prior cryo-EM experience, with extensive notes on operation and troubleshooting.
冷冻电子断层扫描（cryo-ET）是一种强大的技术，可以在原位检查细胞结构。然而，TEM 直接对 cryo-ET 的成像限制在厚度约为 400nm 的样本内，这限制了其在细胞突起或小细菌和病毒等领域的应用。近年来，冷冻聚焦离子束（cryo-FIB）铣削已成为一种从细胞样本中生成薄样本的方法，以便为 cryo-ET 做准备。在该技术中，使用镓离子束逐渐去除细胞材料，以将薄的、电子透明的部分（薄片）穿过块状材料。该薄片可用于高分辨率 cryo-ET，以在近天然状态下可视化 3D 中的细胞。这种方法已被证明对有 cryo-EM 经验的新用户来说是稳健且相对简单的，并且表现出最小的切片伪影。在本章中，我们为有 cryo-EM 经验的用户描述了一种用于 cryo-FIB 铣削的通用方法，并附有关于操作和故障排除的详细说明。
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