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(申请人提供)：冷冻电子断层摄影术的新兴学科提供了独特的机会来确定自然条件下的三维细胞结构，分辨率比目前使用光学显微镜获得的分辨率高一到两个数量级。它在通过核磁共振或X射线结晶学技术确定蛋白质复合体的高分辨率结构和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架起了一座重要的桥梁。近年来在使用现代计算机化显微镜自动获取数据方面取得的进步使这项技术能够成像天然细胞内的复杂组件，并确定处于天然状态的细胞的3D结构。尽管这种方法有公认的潜力，但样品厚度的一个主要限制阻碍了它在细胞和结构生物学中的更广泛应用。到目前为止，冷冻电子断层扫描的研究大多局限于细菌细胞和病毒或细胞前沿的薄层区域，在这些区域可以从细胞断层图像中恢复有用的信息。将这项技术扩展到大型细胞，特别是哺乳动物细胞(&gt；1um)，甚至高压冷冻组织，都需要将冷冻标本的厚度减薄到半微米以下。我们将探索使用聚焦离子束(FIB)将冷冻水合样品变薄到适合冷冻电子断层扫描的程度。这种方法可以消除冷冻超微切割术中常见的困难和伪影，并极大地促进了冷冻电子显微镜，特别是冷冻电子断层成像在研究细胞组件、细胞器、细胞和组织的自然状态下的三维结构方面的进展。为此，我们提出了以下具体目标：目标1：开发用于在FIB仪器中稀释冷冻水化样品的冷冻工作台；目标2：利用深度冷冻的细菌细胞和大型哺乳动物细胞来表征冷冻-FIB粉碎过程；目标3：使用冷冻电子断层扫描技术分析冷冻-FIB稀释的细菌和哺乳动物细胞的三维结构。这一方法的开发和实施将克服在冷冻-水合天然状态下保存的薄生物标本在低温电子显微镜中使用的主要限制，并允许使用冷冻电子断层成像作为对天然细胞和组织进行高分辨率3D成像的标准技术。然后，建议的方法可以应用于广泛的生物医学和翻译研究倡议，例如标记的治疗药物在其自然状态的癌细胞内的空间定位，以及分析癌细胞在不同药物治疗时的结构和形态变化。 公共卫生相关声明：这一方法的开发和实施将克服使用冷冻电子显微镜对保存在冷冻水合自然状态下的生物标本进行成像的主要限制，并允许使用冷冻电子断层成像作为标准技术，以比共聚焦显微镜获得的分辨率高10至100倍的分辨率对自然细胞和组织进行3D成像。然后，建议的方法可以应用于广泛的生物医学和翻译研究倡议，如标记的治疗药物在其自然状态的癌细胞内的空间定位和分析癌细胞在各种药物治疗时的结构变化；研究艾滋病毒及其宿主细胞成分之间复杂的相互作用，这些成分对艾滋病毒的发病机制至关重要，并提供关于病毒如何利用宿主机制促进其复制同时颠覆和逃避细胞的抗病毒反应的结构信息。