Cryo-FIB processing of vitreous biological specimen for electron tomography

用于电子断层扫描的玻璃体生物标本的冷冻 FIB 处理

• 批准号: 7939814
• 负责人: Peijun Zhang
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-26 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939814
• 关键词: 3-Dimensional; Antiviral Response; Architecture; Area; Biological; Cells; Complex; Confocal Microscopy; Cryoelectron Microscopy; Development; Discipline; Electrons; Fluorescent Probes; Freezing; Frozen Sections; HIV; Image; Ions; Life; Life Cycle Stages; Light; Mammalian Cell; Mechanics; Methodology; Methods; Microscope; Microtomy; Monitor; Morphologic artifacts; Organelles; Pathogenesis; Pharmaceutical Preparations; Process; Research Personnel; Resolution; Roentgen Rays; Sampling; Specimen; Staging; Structure; System; Techniques; Technology; Temperature; Therapeutic; Thick; Time; Tissues; Tomogram; Translational Research; Ultramicrotomy; Virus; cancer cell; cellular imaging; computerized; cryogenics; data acquisition; design; electron tomography; instrument; light microscopy; particle; pressure; protein complex; public health relevance; structural biology; success

DESCRIPTION (provided by applicant): The emerging discipline of cryo-electron tomography provides unique opportunities to determine 3-dimensional cellular architectures in their native conditions at resolutions one to two orders of magnitude higher than what is currently achieved using light microscopy. It bridges the critical gap between high resolution structure determination of protein complexes by NMR or X-ray crystallographic techniques and single-particle living cell imaging by light microscopy using fluorescent probes. Advances made in recent years to automate data acquisition using modern computerized microscopes have enabled this technology to image complex assemblies within the native cells and to determine the 3D architecture of cells in their native states. Despite the acknowledged potential of this methodology, one major limitation, specimen thickness, has hindered its broader application in cellular and structural biology. Until now, most of the cryo-electron tomographic studies have been confined to bacterial cells and viruses or thin areas at the leading edge of cells, where useful information can be recovered from cellular tomograms. Extension of this technology to large cells, mammalian cells in particular (>1 um), and even high-pressure frozen tissues requires thinning of the cryo-specimen to less than a half micron in thickness. We will explore using a focused ion beam (FIB) to thin the frozen-hydrated specimen to a degree suitable for cryo-electron tomography. This method could eliminate common difficulties and artifacts associated with cryo-ultramicrotomy and significantly advance cryo-electron microscopy (cryoEM), particularly cryo-electron tomography, in studying the 3D architectures of cellular assemblies, organelles, cells and tissues in their native state. To this end, we propose the following specific aims: Aim 1: Develop a cryo-stage for thinning frozen-hydrated specimens within the FIB instrument; Aim 2: Characterize the cryo-FIB milling process with plunge-frozen bacterial cells and large mammalian cells; Aim 3: Analyze the 3D architectures of cryo-FIB thinned bacterial and mammalian cells using cryo-electron tomography. The development and implementation of this methodology to thin biological specimens preserved in a frozen-hydrated native state will overcome the major limitation on using such specimens in cryoEM and permit use of cryo-electron tomography as a standard technology for high-resolution 3D imaging of native cells and tissues. The proposed methodology could then be applied to a wide range of biomedical and translational research initiatives, such as spatial localization of tagged therapeutic drugs within the cancer cells in its native state and analysis of structural and morphological changes of cancer cells upon various drug treatments. Public Health Relevance Statement: The development and implementation of this methodology will overcome the major limitation in imaging of biological specimens preserved in a frozen-hydrated native state using cryo-electron microscopy and permit use of cryo-electron tomography as a standard technology for 3D imaging of native cells and tissues at resolution 10 to 100 times higher than that obtained with confocal microscopy. The proposed methodology could then be applied to a wide range of biomedical and translational research initiatives, such as spatial localization of tagged therapeutic drugs within the cancer cells in their native state and analysis of structural changes of cancer cells upon various drug treatments; study the intricate interplays between HIV and its host cellular components that are essential for HIV pathogenesis and providing structural information on how virus utilizes the host machinery both to promote its replication and, at the same time, to subvert and evade the antiviral responses of the cell.

描述（由申请人提供）：低温电子断层扫描的新兴学科提供了独特的机会，以确定三维细胞结构在其天然条件下的分辨率比目前使用光学显微镜实现的高一到两个数量级。它弥合了通过NMR或X射线晶体学技术对蛋白质复合物进行高分辨率结构测定与通过使用荧光探针的光学显微镜进行单粒子活细胞成像之间的关键差距。近年来，使用现代计算机显微镜自动化数据采集方面取得的进展使该技术能够对天然细胞内的复杂组装体进行成像，并确定细胞在天然状态下的3D结构。尽管公认的潜力，这种方法，一个主要的限制，标本厚度，阻碍了其更广泛的应用在细胞和结构生物学。到目前为止，大多数冷冻电子断层成像研究都局限于细菌细胞和病毒或细胞前缘的薄区域，在这些区域可以从细胞断层图像中恢复有用的信息。将该技术扩展到大细胞，特别是哺乳动物细胞（>1 μ m），甚至高压冷冻组织，需要将冷冻标本薄化至厚度小于半微米。我们将探讨使用聚焦离子束（FIB）薄冷冻水合标本到一定程度适合冷冻电子断层扫描。这种方法可以消除与冷冻超微切片术相关的常见困难和伪影，并显着推进冷冻电子显微镜（cryoEM），特别是冷冻电子断层扫描，在研究细胞组装体，细胞器，细胞和组织在其天然状态下的3D架构。为此，我们提出了以下具体目标：目标1：开发一种用于在FIB仪器内稀释冷冻水合标本的冷冻台;目标2：用急冻细菌细胞和大型哺乳动物细胞表征冷冻FIB研磨过程;目标3：使用冷冻电子断层扫描分析冷冻FIB稀释的细菌和哺乳动物细胞的3D结构。这种方法的开发和实施，以薄的生物标本保存在冷冻水合的天然状态将克服使用这样的标本在cryoEM的主要限制，并允许使用cryo-electron断层扫描作为一个标准技术的高分辨率的3D成像的天然细胞和组织。然后，所提出的方法可以应用于广泛的生物医学和转化研究计划，如标记的治疗药物在其天然状态下的癌细胞内的空间定位，以及分析各种药物治疗后癌细胞的结构和形态变化。 公共卫生相关性声明：这种方法的开发和实施将克服保存在冷冻水合天然状态下使用冷冻电子显微镜的生物标本成像的主要限制，并允许使用冷冻电子断层扫描作为天然细胞和组织的3D成像的标准技术，其分辨率比共聚焦显微镜高10至100倍。然后，所提出的方法可以应用于广泛的生物医学和转化研究计划，例如标记的治疗药物在其天然状态下的癌细胞内的空间定位和分析各种药物治疗后癌细胞的结构变化;研究艾滋病毒及其宿主细胞成分之间错综复杂的相互作用，这些成分对艾滋病毒的发病机制至关重要，并提供有关病毒如何利用宿主机制来促进其复制，同时破坏和逃避细胞的抗病毒反应。