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.