A MULTI-SCALE APPROACH TO CELL STRUCTURE & FUNCTION

这是一份更新P41研究资源拨款的提案，该拨款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研究。我们在技术研究（R&D）方面的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，旨在解决细胞器和大分子组件的结构和功能。我们的设备在某种意义上是独一无二的，我们正在研究最先进的冷冻技术之间的接口，这些技术应用于冷冻水合标本，揭示出最佳的3-D分子分辨率，以及通常通过冷冻替代和细胞和组织的塑料包埋提供的大规模3-D成像。通过这种方式，我们提供了细胞结构与大分子细节和原子尺度解释之间的直接联系。冷冻替代和塑料包埋是大型复杂系统和整个细胞的高通量三维成像的优秀方法，分辨率约为。4 - 5纳米。这足以明确地检测细胞结构，如微管、肌动蛋白丝、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物。对冷冻水合标本的层析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，这需要专注于规模小得多的业务。在这一更新中，我们提出了一系列新的方法，通过采用新的高电子密度标记技术，专门为玻璃化的大分子和细胞样品设计，以及将荧光显微镜与三维电子显微镜相结合的相关方法，来提高我们对冷冻水合标本的解释。我们在我们的软件包中提出了新的计算程序，例如从层析成像中提取的三维数据的体积平均，以及在显微镜数据采集过程中以及在对准和图像校正过程中提高层析三维重建分辨率的各种过程。

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.