当前位置: X-MOL 学术Nat. Protoc. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.
Nature Protocols ( IF 14.8 ) Pub Date : 2020-05-13 , DOI: 10.1038/s41596-020-0320-x
Felix R Wagner 1, 2 , Reika Watanabe 1 , Ruud Schampers 3 , Digvijay Singh 1 , Hans Persoon 3 , Miroslava Schaffer 4 , Peter Fruhstorfer 3, 5 , Jürgen Plitzko 4 , Elizabeth Villa 1
Affiliation  

Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.

中文翻译:

使用聚焦离子束铣削制备全细胞样品以进行冷冻电子断层扫描。

最近的进展使低温电子显微镜成为实现生化分离大分子复合物的近原子分辨率结构的关键技术。冷冻电子断层扫描 (cryo-ET) 可以在自然环境中对这些复合物进行前所未有的洞察。然而,冷冻电子断层扫描的应用仅限于比大多数细胞更薄的样品,从而大大降低了其适用性。冷冻聚焦离子束 (cryo-FIB) 铣削已用于在完整冷冻细胞中雕刻出(微加工)100-250 nm 的薄区域(称为薄片)。该过程为细胞打开了一扇窗口,用于在其天然环境中进行高分辨率冷冻电子断层扫描和生物分子的结构测定。与荧光显微镜的进一步结合使用户能够确定感兴趣的细胞或区域,以有针对性地制造片层和冷冻电子断层成像。在这里,我们描述了如何使用配备 FIB 和扫描电子显微镜模式的显微镜制备薄片。此类显微镜(Aquilos Cryo-FIB/Scios/Helios 或 CrossBeam)通常称为双光束显微镜,它们配备了冷冻台,可在低温条件下进行所有操作。所述方法的基本原理也适用于配备低温台的其他类型的双光束显微镜。我们还简要描述了如何整合荧光显微镜数据以进行定向研磨以及片层冷冻 ET 数据采集的关键考虑因素。熟悉冷冻电子显微镜并接受过双束显微镜基本培训的用户可以在 2-3 天内完成该协议,从而在手术过程中允许有几个暂停点。
更新日期:2020-05-13
down
wechat
bug