NEW PROBES & METHODS FOR CORRELATED LIGHT & EM, LIVE IMAGING, & FUNCT ANALYSIS

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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. DEVELOPMENT AND APPLICATIONS OF NEW PROBES AND METHODS FOR CORRELATED LIGHT AND ELECTRON MICROSCOPY, LIVE IMAGING, AND FUNCTIONAL ANALYSIS In this subproject, our efforts are directed at (1) extending and generalizing the tetracysteine/biarsenical labeling technology already shown to be a powerful and versatile method for correlated LM and EM, and (2) developing other complementary and unique labeling methods. We are exploring approaches to increase the sensitivity of the tetracysteine/biarsenical tag while minimizing its impact on the host protein normal behavior. We extend the application of biarsenical labeling to chemically fixed tissues and propose the utilization of these methods to explore mesoscale structure and function in complex tissues in virally transduced animals and in transgenic models. In collaboration with the laboratory of Roger Y. Tsien, we will develop a new genetically encoded small Singlet Oxygen Generator (miniSOG) specifically for improved photooxidation for EM labeling. The miniSOG tag can be used alone or in combination with other molecular tags, and shows its applicability to correlated LM/EM studies. In addition, we continue to develop a repository and database of our successful molecular-tagged proteins as a "cell biology toolkit" to facilitate new projects using these probes and expand their availability and usefulness to a larger segment of the scientific community. The ultimate objective of this core project is to expand our toolset to label one or more proteins in cells and tissues with minimal perturbation of the native specimen ultrastructure for multi-scale-correlated LM and EM through further development and refinement of tools and methods like the tetracysteine system.