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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他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。 相关光电子显微镜、活体成像和功能分析新探针和新方法的开发与应用 在该子项目中，我们的努力旨在（1）扩展和推广四半胱氨酸/双砷标记技术，该技术已被证明是相关LM和EM的强大且通用的方法，以及（2）开发其他互补且独特的标记方法。我们正在探索增加四半胱氨酸/双砷标记的灵敏度的方法，同时最大限度地减少其对宿主蛋白正常行为的影响。我们将双砷标记的应用扩展到化学固定的组织，并提出利用这些方法来探索病毒转导动物和转基因模型中复杂组织的中尺度结构和功能。与Roger Y. Tsien，我们将开发一种新的基因编码的小型单线态氧发生器（miniSOG），专门用于EM标记的光氧化。 miniSOG标签可以单独使用或与其他分子标签组合使用，并显示其对相关LM/EM研究的适用性。此外，我们继续开发我们成功的分子标记蛋白质的存储库和数据库，作为“细胞生物学工具包”，以促进使用这些探针的新项目，并将其可用性和实用性扩展到更大的科学界。该核心项目的最终目标是扩展我们的工具集，通过进一步开发和改进工具和方法（如四半胱氨酸系统），以最小限度地干扰多尺度相关LM和EM的天然标本超微结构来标记细胞和组织中的一种或多种蛋白质。

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.