Probes for Quantitative Optical and Electron Microscopy

描述（由申请人提供）：本提案旨在平行开发新的荧光探针和成像方法，以允许活细胞系统中的定量单分子灵敏度。 长期目标是提高整体检测灵敏度和标记特异性，以允许活组织和整个动物内的单个分子成像。 基于我们在基因编码蛋白、镧系元素螯合物和纳米晶体（也称为量子点）三个领域的专业知识，将开发可见光和红外光谱区域的新荧光探针。 这些方法中的每一种都将在“苹果对苹果”的比较中进行测试，用于质膜表面靶点（使用血管紧张素受体作为我们的模型系统）和细胞内靶点（使用线粒体外周型苯二氮卓受体（PBR）作为模型系统）的成像。 我们提出的大部分努力将集中在亚细胞分辨率荧光成像的宽场，去卷积，共聚焦，和多光子激发显微镜，我们已经到位的所有所需的仪器。 为了进一步提高检测的灵敏度，我们将实施基于光谱和时间选通分辨率的新检测策略。 为了达到极高的分辨率，我们还将确定使用这些探针中的每一个通过电子显微镜直接检测相关成像的实用性和局限性。 最后，展望未来的体内成像应用，我们将开发新的红色和近红外（NIR）成像方法，这将使我们的新探针成为可能。 这项研究将为描述特定分子动力学和相互作用方面的细胞生理学提供工具，这将引导我们进入结构细胞生物学的下一个前沿。

DESCRIPTION (provided by applicant): This proposal is directed towards the parallel development of new fluorescent probes and imaging methodologies to permit quantitative single molecule sensitivity in living cell systems. The long-term goal is to improve the overall detection sensitivity, and labeling specificity to allow imaging of single molecules within living tissues and whole animals. New fluorescent probes in the visible and infrared spectral regions will be developed based on our established expertise in three areas, genetically-encoded proteins, lanthanide chelates, and nanocrystals (also called quantum dots). Each of these approaches will be tested in an "apples-to-apples" comparison for imaging of a plasma membrane surface target (using the angiotensin receptor as our model system) and an intracellular target (using the mitochondrial peripheral-type benzodiazepine receptor (PBR) as the model system). The majority of our proposed effort will focus on sub-cellular resolution fluorescence imaging by widefield, deconvolution, confocal, and multi-photon excitation microscopy, for which we have all the required instrumentation already in place. To further enhance the sensitivity of detection, we will implement new detection strategies based on spectral and time-gated resolution. To reach extremely high-resolution, we will also determine the utility and limitations of using each of these probes for direct detection by electron microscopy for correlative imaging. Finally, looking towards future in vivo imaging applications, we will develop new red and near infra-red (NIR) imaging approaches that will be made possible with our new probes. This research will provide tools for describing cell physiology in terms of specific molecular dynamics and interactions, which will lead us into the next frontier of structural cell biology.