TRD 3: DEVELOP TECHNIQUES FOR IMAGING BIOLOGICAL SPECIMENS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该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。 使用三维冷冻X射线断层摄影术来检测细胞中的特定分子。一旦我们实现了这些目标，我们将 专注于开发同时识别两种或更多种不同分子的方法， 分子共定位和原位相互作用。这些能力将通过相关光的发展而得到增强 和X射线显微镜技术，其中用绿色荧光蛋白（GFP）标记的蛋白质构建体在 然后用X射线断层摄影术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 用于高通量、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，酵母酿酒酵母，与x射线断层扫描; 2）发展的能力， 使用X射线断层摄影术研究复杂真核细胞的细胞结构;以及3）开发用于标记 用X射线断层摄影术来观察细胞。

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.