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）：冷冻电子断层扫描的新兴学科为在本机条件下在本地条件下以比使用光显微镜当前实现的分辨率高一到两个数量级来确定其本地条件下的三维蜂窝结构提供了独特的机会。它弥合了使用NMR或X射线晶体学技术对蛋白质复合物测定的高分辨率结构之间的临界差距，并使用荧光探针通过光学显微镜进行单粒子活细胞成像。近年来，使用现代计算机显微镜自动化数据采集的进步使这项技术能够对天然细胞内的复杂组件进行图像复杂的组件，并确定其本地状态中细胞的3D体系结构。尽管这种方法具有公认的潜力，但标本厚度的一个主要局限性阻碍了其在细胞和结构生物学中的广泛应用。到目前为止，大多数冷冻电子层析成像研究都局限于细菌细胞和病毒或细胞前缘的薄区域，在这些细胞的前缘，可以从细胞断层图中回收有用的信息。将该技术扩展到大细胞，尤其是哺乳动物细胞（> 1 um），甚至高压冷冻的组织都需要将冷冻特异性变薄至厚度小于一半微米。我们将使用聚焦离子束（FIB）探索将冷冻水合的样品稀释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 - 脱毛术相关的常见困难和伪影，并显着提高冷冻电子显微镜（Cryoem），尤其是冷冻电子断层扫描，在研究其天然状态的细胞组件，细胞器，细胞和组织的3D体系结构时。为此，我们提出了以下特定目的：目标1：开发用于稀疏的冷冻水合试样中的冷冻阶段； AIM 2：用凹凸不平的细菌细胞和大型哺乳动物细胞来表征冷冻纤维铣削过程； AIM 3：使用冷冻电子层析成像分析冷冻纤维稀释细菌和哺乳动物细胞的3D结构。这种方法的开发和实施是在冷冻水合的天然状态中保存的薄生物标本，将克服在冷冻中使用此类标本的主要局限性，并允许使用冷冻电子层析成像作为对天然细胞和组织的高分辨率3D成像的标准技术。然后，提出的方法可以应用于广泛的生物医学和转化研究计划，例如在其天然状态下在癌细胞中标记的治疗药物的空间定位，并分析各种药物治疗对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使用冷冻水合的天然状态的生物标本成像的主要局限性，并允许将低温性层析成像作为低温层造影作为标准技术，用于在10至100倍的10到100倍以比copocal microscopy的分辨率10到100倍的天然细胞和组织。然后，提出的方法可以应用于广泛的生物医学和转化研究计划，例如在癌细胞中在其本地状态下对标记的治疗药物进行空间定位，并分析各种药物治疗时癌细胞的结构变化；研究HIV及其宿主细胞成分之间的复杂相互作用，这些相互作用对于HIV发病机理至关重要，并提供有关病毒如何利用宿主机制来促进其复制的结构信息，并同时颠覆和逃避细胞的抗病毒药反应。