Focused Ion Beam Milling for Cryo-electron Tomography

描述(申请人提供)：冷冻电子断层扫描(CET)是纳米尺度研究天然(冷冻-水合)细胞整体结构的唯一方法。这是唯一能够识别和确定大分子在其细胞环境中的结构和取向的方法。CET的标本最初是通过玻璃体冷冻准备的；它们保持充分的水合，不使用化学固定剂或着色剂。然而，由于只有厚度小于几百纳米的样品才能在电子显微镜下有效成像，CET的使用在很大程度上局限于分离的细胞器或小细胞，如细菌。真核细胞只有在特殊情况下才能成像，而且只在它们的外围。为了将CET扩展到广泛的细胞和组织，我们第一次使用冷冻-超微切割法(机械切片)来制备足够薄的组织切片，以满足CET的需要。然而，我们发现冰冻材料的切片是一项困难且重复性差的技术，充满了不可避免的人工制品。因此，我们引入了一种全新的方法：低温聚焦离子束(Cryo-FIB)球磨用于CET的玻璃化冷冻样品。自那以后，其他实验室已经证实了我们开创性的概念验证实验。我们在这项建议中的目标是将冷冻-FIB铣削发展成一种实用、可靠的生物CET工具，并使研究人员能够通过相关的光学和电子显微镜捕捉样本中感兴趣的区域。 公共卫生相关性：我们建议提供一种方法，能够通过冷冻电子断层扫描(CET)来确定任何细胞在其自然的、冷冻的状态下的三维结构。与CET的传统应用不同，CET仅限于非常小的细胞、细胞的薄边或细菌和分离的细胞成分，新方法将使单层细胞培养、细胞悬液和组织中的细胞得到研究。这将对生物医学研究产生广泛而重大的影响。

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. PUBLIC HEALTH RELEVANCE: We propose to provide a method that enables determination of the 3-D structure of any cell in its native, frozen, state by means of cryo-electron tomography (CET). Unlike traditional applications of CET, which are limited to very small cells, just the thin edges of cells, or bacteria and isolated cellular components, the new method will enable monolayer cell cultures, cell suspensions, and cells in tissue to be studied. This will have a broad and major impact on biomedical research.