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）：新兴的冷冻电子断层扫描学科提供了独特的机会来确定自然条件下的 3 维细胞结构，其分辨率比目前使用光学显微镜所达到的分辨率高一到两个数量级。它弥补了通过 NMR 或 X 射线晶体学技术确定蛋白质复合物的高分辨率结构与通过使用荧光探针的光学显微镜进行单颗粒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实现数据采集自动化方面取得的进展，使该技术能够对天然细胞内的复杂组件进行成像，并确定细胞在天然状态下的 3D 结构。尽管这种方法的潜力得到公认，但样本厚度这一主要限制阻碍了其在细胞和结构生物学中的更广泛应用。到目前为止，大多数冷冻电子断层扫描研究仅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照片中恢复有用的信息。将该技术扩展到大细胞，特别是哺乳动物细胞（>1微米），甚至高压冷冻组织，需要将冷冻标本的厚度减薄至半微米以下。我们将探索使用聚焦离子束 (FIB) 将冷冻水合样本减薄至适合冷冻电子断层扫描的程度。这种方法可以消除与冷冻超薄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、细胞和组织处于自然状态的 3D 结构方面。为此，我们提出以下具体目标： 目标 1：开发一个低温阶段，用于在 FIB 仪器内稀释冷冻水合标本；目标 2：用深度冷冻的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。开发和实施这种方法来薄化保存在冷冻水合天然状态下的生物样本将克服在冷冻电镜中使用此类样本的主要限制，并允许使用冷冻电子断层扫描作为天然生物高分辨率 3D 成像的标准技术。细胞和组织。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，例如标记治疗药物在天然状态下的癌细胞内的空间定位，以及分析癌细胞在各种药物治疗后的结构和形态变化。 公共卫生相关性声明：该方法的开发和实施将克服使用冷冻电子显微镜对冷冻水合自然状态下保存的生物样本进行成像的主要限制，并允许使用冷冻电子断层扫描作为 3D 成像的标准技术天然细胞和组织的分辨率比共聚焦显微镜获得的分辨率高 10 至 100 倍。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，例如标记治疗药物在天然状态下的癌细胞内的空间定位，以及分析癌细胞在各种药物治疗后的结构变化；研究 HIV 与其宿主细胞成分之间复杂的相互作用，这对于 HIV 发病机制至关重要，并提供有关病毒如何利用宿主机制促进其复制并同时破坏和逃避细胞抗病毒反应的结构信息。