Focused Ion Beam Milling for Cryo-electron Tomography

描述（由申请人提供）：冷冻电子断层扫描（CET）是唯一可用于天然（冷冻-水合）状态下细胞整体结构纳米级研究的方法。它是唯一能够识别和确定大分子在其细胞环境中的结构和方向的方法。CET的标本最初是通过玻璃体冷冻制备的;它们保持完全水合，不使用化学固定剂或染色剂。然而，由于只有薄于几百纳米的样品才能在电子显微镜中有效成像，因此CET的使用主要限于分离的细胞器或小细胞，如细菌。真核细胞仅在特殊情况下成像，并且仅在其外围成像。为了将CET扩展到广泛的细胞和组织，我们首先使用冷冻超微切片术（机械切片）制备足够薄的组织切片用于CET。然而，我们发现冷冻材料的切片是一种困难且重现性差的技术，充满了不可避免的伪影。因此，我们引入了一种全新的方法：低温聚焦离子束（cryo-FIB）铣削玻璃冷冻标本CET。此后，其他实验室也证实了我们突破性的概念验证实验。我们在这个建议中的目标是开发冷冻FIB铣削成一个实用的，可靠的工具，生物CET，并使研究人员能够捕获相关的光学和电子显微镜的标本中的感兴趣的区域。

DESCRIPTION (provided by applicant): Cryo-electron tomography (CET) is the only method available for nanoscale study of the overall architecture fo cells in their native (frozen-hydrated) state. It is the only method capable of identifying and determining the structure and orientation of macromolecules in their cellular context. Specimens for CET are initially prepared by vitreous freezing; they remain fully hydrated, and no chemical fixatives or stains are used. However, because only specimens thinner than a few hundred nanometers can be effectively imaged in the electron microscope, use of CET has largely been limited to isolated cell organelles or small cells such as bacteria. Eukaryotic cells have been imaged only in special cases, and only at their peripheries. In order to extend CET to a broad range of cells and tissue, we were the first to use cryo-ultramicrotomy (mechanical sectioning) to prepare tissue sections sufficiently thin for CET. However, we have found sectioning of frozen material to be a difficult and poorly reproducible technique, fraught with unavoidable artifacts. Therefore, we have introduced an entirely new approach: cryo-focused-ion-beam (cryo-FIB) milling of vitreously frozen specimens for CET. Other laboratories have since confirmed our ground-breaking proof-of-concept experiments. Our goal in this proposal is to develop cryo-FIB milling into a practical, reliable tool for biological CET, and to enable the researcher to capture regions of interest in the specimen by correlative light and electron microscopy.