A MULTI-SCALE APPROACH TO CELL STRUCTURE & FUNCTION

描述（由申请人提供）：这是一项更新P41研究资源补助金的提案，该补助金支持博尔德实验室进行细胞和大分子结构的三维电子显微镜检查。我们的技术研究（R&D）目标包括开发可靠的样品制备、图像采集和图像处理方法。此外，我们还为科学界提供软件和技术的服务、培训和传播。我们的方法包括三维电子显微镜，旨在解析细胞器和大分子组装体的结构和功能。我们的设施是独一无二的，在某种意义上说，我们正在研究最先进的冷冻技术之间的接口应用于冷冻水合标本揭示最好的分子分辨率在3-D，和大规模的3-D成像通常由冷冻替代和塑料包埋的细胞和组织。通过这种方式，我们提供了细胞结构和大分子细节与原子尺度解释之间的直接联系。冷冻置换和塑料包埋是大的复杂系统和整个细胞的高通量3-D成像的优秀方法，分辨率约为1000。4-5 nm.这足以明确地检测细胞结构，如微管，肌动蛋白丝，线粒体，ER系统，甚至大分子结构，如核糖体或其他大的酶复合物。冷冻水化标本的断层扫描三维成像旨在获得最精确的分子细节，分辨率可达2nm甚至更高。然而，这需要专注于更小的数量。在这次更新中，我们提出了一系列新的方法，应提高我们的解释冷冻水化标本采用新的高电子密度标记技术，专门设计用于玻璃化的大分子和细胞样品，和相关的方法相结合的荧光显微镜与3-D电子显微镜。我们提出了新的计算程序，在我们的软件包，如体积平均的3-D数据从断层图像和各种过程，这将提高分辨率的断层3-D重建，无论是在数据采集的显微镜，并为对齐和图像校正程序。公共卫生相关性：治愈一种病理状态需要对健康状态有详细的分子知识，这是临床前生物医学研究的主要目标，就像我们实验室的工作一样。然而，我们的一些合作与人类病理过程的理解直接相关，例如我们对心肌细胞和病毒进入和复制的大规模结构分析。我们将获得的数据将构成对细胞过程的基本理解，可能导致许多疾病的新疗法和治疗方法。

DESCRIPTION (provided by applicant): This is a proposal to renew a P41 Research Resource grant that supports the Boulder Laboratory for 3-D Electron Microscopy of Cells and Macromolecular Structures. Our goals for research in technology (R&D) include the development of methods for reliable specimen preparation, image acquisition, and image processing. In addition, we provide service, training and dissemination of software and technology to the scientific community. Our methods comprise 3-D electron microscopy aimed to resolve the structure and function of cellular organelles and macromolecular assemblies. Our facility is unique in a sense that we are working on the interface between most advanced cryo-technologies applied to frozen-hydrated specimens revealing best possible molecular resolution in 3-D, and large-scale 3-D imaging typically provided by freeze substitution and plastic embedding of cells and tissue. This way we provide a direct link between cellular structures and macromolecular detail and atomic-scale interpretation. Freeze-substitution and plastic embedding are excellent method for high-throughput 3-D imaging of large complex systems and entire cells to a resolution of approx. 4-5 nm. This is sufficient to unambiguously detect cellular structures such as microtubules, actin filaments, mitochondria, ER systems, and even macromolecular structures such as ribosomes or other large enzyme complexes. Tomographic 3-D imaging on frozen-hydrated specimens aims for most accurate molecular detail down to 2nm resolution or even beyond. That, however, requires focusing on much smaller volumes. In this renewal we propose a series of new methods that should improve our interpretations of frozen-hydrated specimens by employing novel high-electron dense labeling techniques, specifically designed for vitrified macromolecular and cellular samples, and correlative approaches combining fluorescence light microscopy with 3-D electron microscopy. We propose new computational procedures in our software packages such as volume-averaging of 3-D data picked from tomograms and a variety of processes that will improve the resolution of tomographic 3-D reconstructions, both, during data acquisition on the microscope, and for the alignment and image correction procedures. Public Health Relevance: Curing a pathologic condition requires detailed molecular knowledge of the healthy state, which is a major goal of pre-clinical biomedical research like the work of our lab. Nevertheless, several of our collaborations are directly relevant to an understanding of pathologic processes in humans, such as our large-scale structural analyses of cardiomyocytes and of virus entry and replication. The data we will obtain will constitute an essential understanding of cellular processes that may lead to new cures and treatments of many diseases.