Focused Ion Beam Milling for Cryo-electron Tomography

用于冷冻电子断层扫描的聚焦离子束铣削

• 批准号: 8712509
• 负责人: MICHAEL MARKO
• 金额: $ 50.95万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8712509
• 关键词: 3-Dimensional; Address; Architecture; Area; Bacteria; Biocompatible Materials; Biological; Biomedical Research; Cell Culture Techniques; Cell Cycle Stage; Cells; Chemicals; Cultured Cells; Custom; Development; Devices; Educational workshop; Electron Microscope; Electron Microscopy; Engineering; Ensure; Environment; Eukaryotic Cell; Fixatives; Freezing; Frozen Sections; Geometry; Goals; Image; Ions; Knowledge; Label; Laboratories; Life; Mechanics; Methods; Microtome - medical device; Microtomy; Microtubules; Morphologic artifacts; Organelles; Process; Prokaryotic Cells; Protocols documentation; Publications; Reporting; Research Personnel; Resolution; Scanning; Secure; Side; Site; Specimen; Staging; Staining method; Stains; Structure; Surface; Suspension substance; Suspensions; Synaptic Vesicles; Techniques; Temperature; Thick; Tissues; Tomogram; Training; Translating; Ultramicrotomy; cell type; cellular imaging; design and construction; electron tomography; improved; innovation; instrument; interest; irradiation; light microscopy; macromolecule; monolayer; nanometer; nanoscale; novel strategies; pressure; reconstruction; research study; tool; transmission process

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.

描述（由申请人提供）：冷冻电子断层扫描（CET）是唯一可用于对其天然（冷冻水合）状态的整体结构FO细胞进行纳米级研究的方法。它是唯一能够识别和确定大分子在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污渍。但是，由于在电子显微镜中只能有效成像比几百个纳米的标本薄，因此CET的使用很大程度上仅限于分离的细胞细胞器或小细胞（例如细菌）。真核细胞仅在特殊情况下才成像，仅在其外围。为了将CET扩展到广泛的细胞和组织，我们是第一个使用冷冻 - 粉状切开术（机械切片）来准备足够薄的组织切片的人。但是，我们发现冷冻材料的切片是一种困难且可再现的技术，充满了不可避免的伪影。因此，我们引入了一种全新的方法：针对CET的玻璃体冷冻样品的冷冻焦点束（冷冻纤维）铣削。此后，其他实验室已经确认了我们开创性的概念验证实验。我们在该提案中的目标是将冷冻铣削开发成生物CET的实用，可靠的工具，并使研究人员能够通过相关光和电子显微镜捕获样品中感兴趣的区域。