CORE--EM TOMOGRAPHY

核心--EM断层扫描

• 批准号: 6589300
• 负责人: John RICHARD MCINTOSH
• 金额: $ 49.81万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6589300
• 关键词: analytical method; biomedical automation; biomedical facility; cells; cytoskeleton; electron microscopy; fluorescence resonance energy transfer; fluorescent dye /probe; freeze etching; immunoelectron microscopy; macromolecule; membrane structure; method development; peptide chemical synthesis; protein isoforms; structural biology; tissue /cell preparation; tomography

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.

现在，电子显微镜（EM）断层扫描是一种以大约5nm分辨率研究细胞结构的验证方法。 我们寻求在高尔基体配合物和细胞骨架的成分上进行工作，并实现冷冻水分的大分子的高分辨率EM。我们的核心研究将追求三个技术目标。我们将自动层析成像自动化，使生成大量细胞结构的3 d重建变得方便。这一进步将为Boulder和其他地方的结构生物学家的科学家提供硬件和计算工具，这些工具将允许3D重建广泛的细胞结构，例如细胞膜系统和细胞骨架的一部分。我们还将努力通过冻结固定性和保存完好的材料中生物大分子的标记来改善固定方法。现有的方法（例如免疫系统）具有重要的局限性，尽管我们将使用这些方法进行某些工作，但我们提出了四种新型方法来标记可以提高细胞中大分子局部性的分辨率，易度和敏感性。我们还将努力使断层图的建模更加客观，更有效，因此我们可以以更高的速度提取可靠的信息，以量化更高的速度对可靠信息进行定量检查，从而产生数据以更高的速度对可靠信息进行定量检查，从而产生数据以在蜂窝精细结构中对可变性进行定量检查。这些进步中的每一个都将有助于结构细胞生物学在回答重要的科学问题方面提升到新的可靠性和有效性。