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

基本信息

批准号：
8080025
负责人：
MICHAEL MARKO
金额：
$ 67.46万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
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/8080025
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 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

项目摘要

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.

描述（由申请人提供）：冷冻电子断层扫描（CET）是可用于纳米级研究细胞天然（冷冻水合）状态的整体结构的唯一方法。它是唯一能够识别和确定细胞环境中大分子的结构和方向的方法。 CET 标本最初是通过玻璃体冷冻制备的；它们保持完全水合，并且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本才能在电子显微镜中有效成像，因此 CET 的使用在很大程度上仅限于分离的细胞器或细菌等小细胞。仅在特殊情况下对真核细胞进行成像，并且仅在其外围进行成像。为了将 CET 扩展到更广泛的细胞和组织，我们率先使用冷冻超薄切片术（机械切片）来制备足够薄的用于 CET 的组织切片。然而，我们发现冷冻材料的切片是一项困难且重复性差的技术，充满了不可避免的伪影。因此，我们引入了一种全新的方法：冷冻聚焦离子束 (cryo-FIB) 铣削玻璃体冷冻标本以进行 CET。此后其他实验室也证实了我们突破性的概念验证实验。我们在此提案中的目标是将冷冻 FIB 铣削开发成一种实用、可靠的生物 CET 工具，并使研究人员能够通过相关光学和电子显微镜捕获样本中的感兴趣区域。公共健康相关性：我们建议提供一种方法，能够通过冷冻电子断层扫描 (CET) 确定任何细胞在其天然、冷冻状态下的 3-D 结构。与 CET 的传统应用不同，CET 的应用仅限于非常小的细胞、细胞的薄边缘或细菌和分离的细胞成分，而新方法将能够研究单层细胞培养物、细胞悬浮液和组织中的细胞。这将对生物医学研究产生广泛而重大的影响。