A MULTI-SCALE APPROACH TO CELL STRUCTURE & FUNCTION

描述(由申请人提供)：这是一项更新P41研究资源资助的建议，该资助支持博尔德实验室细胞和大分子结构的三维电子显微镜。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，我们还向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，旨在解析细胞细胞器和大分子组件的结构和功能。我们的设备是独一无二的，因为我们正在研究应用于冷冻水合标本的最先进冷冻技术与大规模3D成像之间的接口，这些冷冻技术可显示最佳的3-D分子分辨率，通常由细胞和组织的冷冻替代和塑料嵌入提供。通过这种方式，我们提供了细胞结构、大分子细节和原子尺度解释之间的直接联系。冷冻置换和塑料包埋是对大型复杂系统和整个细胞进行高通量三维成像的极佳方法，分辨率约为。4-5 nm。这足以明确地检测细胞结构，如微管、肌动蛋白细丝、线粒体、内质网系统，甚至大分子结构，如核糖体或其他大型酶复合体。在冷冻水合标本上进行层析三维成像的目的是获得最精确的分子细节，分辨率可达2纳米，甚至更高。然而，这需要专注于规模小得多的业务。在这次更新中，我们提出了一系列新的方法，通过使用新的高电子密度标记技术，特别是为玻璃化的大分子和细胞样本设计的高电子密度标记技术，以及结合荧光显微镜和3-D电子显微镜的相关方法，来改进我们对冷冻水化样本的解释。我们在我们的软件包中提出了新的计算程序，例如从断层图像中挑选出的3-D数据的体积平均，以及在显微镜上采集数据期间，以及在对准和图像校正过程中，将提高断层三维重建的分辨率的各种过程。公共卫生相关性：治愈一种病理状态需要详细的健康状态的分子知识，这是像我们实验室的工作一样的临床前生物医学研究的主要目标。然而，我们的几项合作与理解人类的病理过程直接相关，例如我们对心肌细胞以及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，这些过程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.