Probes:Quantitative Optical and Electron Microscopy(RMI)

探针：定量光学和电子显微镜（RMI）

• 批准号: 6831134
• 负责人: David W Piston
• 金额: $ 68.49万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6831134
• 关键词: angiotensin receptor; benzodiazepine receptor; bioimaging /biomedical imaging; cell membrane; electron microscopy; fluorescent dye /probe; infrared radiation; nanotechnology; optics; rare earth element; technology /technique development; visible light

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.

描述（由申请人提供）：该提案旨在并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，以实现活体组织和整个动物内单分子的成像。 我们将根据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）三个领域的既定专业知识，开发可见光和红外光谱区域的新型荧光探针。 这些方法中的每一种都将在“同类”比较中进行测试，以对质膜表面靶标（使用血管紧张素受体作为我们的模型系统）和细胞内靶标（使用线粒体外周型苯二氮卓受体（PBR）进行成像。 ）作为模型系统）。 我们提出的大部分工作将集中在通过宽场、反卷积、共焦和多光子激发显微镜进行亚细胞分辨率荧光成像，为此我们已经准备好了所有必需的仪器。 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。 为了达到极高分辨率，我们还将确定使用这些探针通过电子显微镜直接检测进行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，这些方法将通过我们的新探针成为可能。 这项研究将为从特定分子动力学和相互作用方面描述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。