CORE--EM TOMOGRAPHY

电子显微镜(EM)断层扫描现在是一种被证实的研究细胞结构的方法，分辨率约为5 nm。我们寻求开展关于高尔基复合体和细胞骨架组件的工作，并使冷冻水合大分子的高分辨率EM成为可能。我们的核心研究将追求三个技术目标。我们将使断层扫描自动化，从而方便地生成大量细胞结构的3-D重建。这一进展将为博尔德的科学家和其他地方的结构生物学家提供硬件和计算工具，使其能够对广泛的细胞结构进行3D重建，如细胞膜系统和部分细胞骨架。我们还将努力改进冷冻替代固定的方法和保存良好的材料中生物大分子的标记方法。现有的方法，如免疫EM，有重要的局限性，虽然我们将使用这些方法进行一些工作，但我们提出了四种新的标记方法，可能会提高细胞中大分子定位的分辨率、易用性和敏感性。我们还将努力使断层图像的建模更加客观和高效，以便我们能够更快地提取可靠信息，以更快的速度产生用于可靠信息的定量检查的数据，以更快的速度产生用于可靠信息的定量检查的数据，从而为细胞精细结构的可变性的定量检查产生数据。这些进展中的每一项都将帮助结构细胞生物学在回答重要科学问题方面达到一个新的可靠性和有效性水平。

Electron microscope (EM) tomography is now a proven method for the study of cellular architecture at approximately 5nm resolution. We seek to carry out work on the Golgi complex and components of the cytoskeleton and to enable high resolution EM of frozen-hydrated macromolecules. Our CORE research will pursue three technological goals. We will automate tomography, making it convenient to generate 3- D reconstructions of large volumes of cellular structure. This advance will provide both scientists at Boulder and structural biologist elsewhere with the hardware and computational tools that will allow 3-D reconstructions of extensive cellular structures, like cytomembrane systems and parts of the cytoskeleton. We will also work to improve methods for fixation by freeze-substitution and for the labeling of biological macromolecules in well preserved material. Existing methods, such as immunoEM, have important limitations, and while we will use these approaches for some work, we propose four novel approaches to labeling that may improve the resolution, ease, and sensitivity of macromolecular localizations in cells. We will also work to make the modeling of tomograms more objective and efficient, so we can extract reliable information with greater speed, producing data for a quantitative examination of reliable information with greater speed, producing data for a quantitative examination of reliable information with greater speed, producing data for a quantitative examination of variability in cellular fine structure. Each of these advances will help structural cell biology move to a new level of reliability and effectiveness in answering important scientific questions.