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，用于研究细胞结构，分辨率约为5nm。 我们寻求开展工作的高尔基复合体和组件的细胞骨架，并使高分辨率的冷冻水合大分子的EM。我们的核心研究将追求三个技术目标。我们将实现断层扫描自动化，从而方便地生成大量细胞结构的3D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，这些工具将允许对广泛的细胞结构进行三维重建，如细胞膜系统和细胞骨架的一部分。我们还将努力改进冷冻置换固定方法和在保存良好的材料中标记生物大分子的方法。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，我们提出了四种新的方法来标记，可以提高分辨率，易用性和灵敏度的大分子定位在细胞中。我们还将努力使断层图像的建模更加客观和有效，因此我们可以以更快的速度提取可靠的信息，以更快的速度产生用于定量检查可靠信息的数据，以更快的速度产生用于定量检查可靠信息的数据，产生用于定量检查细胞精细结构变异性的数据。这些进展将有助于结构细胞生物学在回答重要科学问题时达到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.