A MULTI-SCALE APPROACH TO CELL STRUCTURE & FUNCTION

细胞结构的多尺度方法

基本信息

批准号：
8262156
负责人：
ANDREAS HOENGER
金额：
$ 107.18万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-12-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8262156
关键词：
3-Dimensional Algorithms Area Biological Biological Preservation Biological Process Biomedical Research Cardiac Myocytes Cell physiology Cells Cellular Structures Collaborations Communities Complex Computer software Computers Cryoelectron Microscopy Data Data Set Development Disease Documentation Educational workshop Electron Beam Electron Microscopy Electrons Embryonic Development Environment Fluorescence Microscopy Freeze Substitution Freezing Frozen Sections Generations Goals Grant Human Image Imaging Device Infection Institution Knowledge Label Laboratories Lead Light Link Metals Methods Microfilaments Microscope Microtubules Mitochondria Molecular Molecular Analysis Molecular Structure Multienzyme Complexes NMR Spectroscopy Organelles Organism Parasites Pathologic Pathologic Processes Peptides Plastic Embedding Preparation Procedures Process Property Publications Research Resolution Resources Ribosomes Running Sampling Science Series Services Specimen Speed Structure Structure-Activity Relationship System Techniques Technology Three-Dimensional Imaging Time Tissues Tomogram Training Virus Work X-Ray Crystallography base chromosome movement computerized data processing data acquisition design electron density image processing improved instrumentation interest light microscopy macromolecular assembly meetings method development novel pre-clinical reconstruction research and development sample fixation skills software development tissue/cell preparation tomography trend web site

项目摘要

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.

这是续签P41研究资源赠款的一项建议，该资料支持细胞和大分子结构的3-D电子显微镜的巨石实验室。我们的技术研究目标（R＆D）包括开发可靠标本准备，图像获取和图像处理的方法。此外，我们还向科学界提供了软件和技术的服务，培训和传播。我们的方法包括3-D电子显微镜，旨在解决细胞细胞器和大分子组件的结构和功能。我们的设施是独一无二的，从某种意义上说，我们正在使用应用于冷冻保湿标本的最先进的冷冻技术之间的界面，揭示了3-D中最佳的分子分辨率，以及通常由冻结物体和细胞和组织的塑料嵌入提供的大规模3-D成像。这样，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。冻结物质和塑料嵌入是对大型复合系统和整个细胞进行高通量3-D成像的极好方法，可分辨率约为。 4-5 nm。这足以明确检测细胞结构，例如微管，肌动蛋白丝，线粒体，ER系统，甚至大分子结构，例如核糖体或其他大型酶络合物。冷冻水分标本上的层析成像3-D成像的目的是最精确的分子细节，低至2NM分辨率甚至更高。但是，这需要专注于较小的卷。在此续约中，我们提出了一系列新方法，这些方法应该通过采用新型的高电子密集标记技术来改善对冷冻水合的样品的解释，这些标记技术专为玻璃化的大分子和细胞样品而设计，并将其相关的方法与3-D电子显微镜结合了荧光光学的相关方法。我们在软件包中提出了新的计算程序，例如从横线图中挑选的3-D数据的体积平均数量以及各种过程，这些过程将改善在显微镜上的数据采集期间，以及对齐和图像校正程序的分辨率3-D重建。