CORE 3: DEVELOP TECHNIQUES FOR IMAGING BIOLOGICAL SPECIMENS USING CRYO X-RAY TO

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这个核心项目的目标是开发使用三维低温X射线断层扫描检查整个细胞、识别细胞结构和定位细胞中特定分子的方法。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，以便在原位检查分子的共局域化和相互作用。这些能力将通过相关光和X射线显微技术的发展而得到增强，在活细胞中跟踪标记有绿色荧光蛋白(GFP)的蛋白质结构，然后用X射线断层扫描以更高的分辨率定位。实现这些目标将为高通量、高分辨率(优于50纳米)的分析提供强大的新工具，这将补充现有的蛋白质组学工具。这一具体目标分为三个不同的任务：1)发展利用x射线断层摄影术识别简单的真核细胞酿酒酵母细胞结构的能力；2)发展利用x射线断层摄影术鉴定复杂真核细胞的细胞结构的能力；以及3)发展利用x射线断层摄影术标记细胞中分子的方法。

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 capabilities 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