CORE--EM TOMOGRAPHY

电子显微镜（EM）断层扫描现在是一种经过验证的方法，用于研究大约5nm分辨率的细胞结构。我们寻求开展高尔基复合体和细胞骨架成分的工作，并使冷冻水合大分子的高分辨率EM成为可能。我们的CORE研究将追求三个技术目标。我们将实现断层扫描的自动化，从而方便地生成大量细胞结构的三维重建。这一进展将为博尔德大学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，使广泛的细胞结构（如细胞膜系统和细胞骨架的一部分）的三维重建成为可能。我们还将致力于改进冷冻取代固定方法和保存良好的生物大分子的标记方法。现有的方法，如免疫电镜，具有重要的局限性，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，这些方法可以提高细胞中大分子定位的分辨率，易用性和灵敏度。我们还将努力使层析图的建模更加客观和高效，以便我们能够以更快的速度提取可靠信息，以更快的速度为可靠信息的定量检查生成数据，以更快的速度为可靠信息的定量检查生成数据，以更快的速度为细胞精细结构的可变性的定量检查生成数据。每一项进步都将帮助结构细胞生物学在回答重要科学问题时达到可靠性和有效性的新水平。

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.