TRD 3: DEVELOP TECHNIQUES FOR IMAGING BIOLOGICAL SPECIMENS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个核心项目的目标是开发检查整个细胞、识别细胞结构和定位的方法 细胞中特定分子的三维低温x射线层析成像。一旦我们实现了这些目标，我们将 专注于开发同时识别两个或更多不同分子的方法，以便检查 原位分子共局域化和相互作用。这些能力将随着相关光的发展而增强 以及x射线显微镜技术，其中标记有绿色荧光蛋白(GFP)的蛋白质结构被跟踪到 然后用X射线断层扫描以更好的分辨率定位活细胞。实现这些目标将提供强大的新的 高通量、高分辨率(优于50 nm)分析的工具，将补充现有的蛋白质组学 工具。这一具体目标分为三个独立的任务：1)发展识别细胞结构的能力 简单的真核细胞，酿酒酵母，用X射线断层扫描；2)培养鉴定能力 利用x射线断层摄影术研究复杂真核细胞的细胞结构；以及3)发展标记分子的方法。 使用X射线断层扫描的细胞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this core project is to develop methods for examining whole cells, identifying cellular structures, and localizing specific molecules in cells using three dimensional cryo x-ray tomography. Once we have accomplished these goals, we will focus on developing the methodology to simultaneously identify two or more different molecules in order to examine molecular co-localizations and interactions in situ. These apabilities will be enhanced by the development of correlated light and x-ray microscopy techniques, in which protein constructs tagged with green fluorescent protein (GFP) are tracked in live cells then localized at better resolution with x-ray tomography. Accomplishing these goals will provide powerful new tools for high throughput, high resolution (better than 50 nm) analyses that will complement the existing proteomics tools. This specific aim is divided into three separate tasks: 1) to develop the ability to identify cellular structures in a simple eukaryotic cell, the yeast Saccharomyces cerevisiae, with xray tomography; 2) to develop the ability to identify cellular structures of complex eukaryotic cells using x-ray tomography; and 3) to develop methods for labeling molecules in cells using x-ray tomography.