Integrated perfusion and confocal imaging system

 描述（由申请方提供）：解剖小管的微灌注是一种成熟的技术，可以在不存在混淆反馈变量的情况下提供有关分离肾单位节段内细胞功能的信息。这些信息无法通过其他方法获得，包括活体显微镜。在表达异源构建体以及有效敲低肾脏和膀胱中内源性蛋白的转导方法的最新进展已经扩大了使用活细胞方法（包括FRET）在分离的小管中真实的时间中研究膜运输和信号传导途径的生理调节的机会。该申请是为了获得资金，以购买灌注成像系统，从而能够对微灌注的肾小管和血管以及其他组织和细胞进行高速、高分辨率成像。该系统包括一个单光子尼康A1 R共聚焦显微镜与共振扫描仪，光谱检测器和FRET模块，双显微操作器，和一个环境室。该系统将被安置在P30资助的乔治M。匹兹堡大学医学院的奥布莱恩匹兹堡肾脏研究中心。该中心的任务是促进与肾脏生理学、细胞生物学、药理学和病理生理学相关的多学科研究、培训和信息传递。该中心包括肾脏成像，细胞生理学，单肾单位和代谢组学以及模型生物学的核心，并为匹兹堡大学和其他机构的NIH资助的研究人员提供服务。我们的中心缺乏用于灌注小管实时成像的专用显微镜，拟议系统的可用性将显着提高我们能够为中心用户提供的服务。相关性-拟议的系统将为研究疾病的细胞基础的众多生物医学研究人员的研究提供一个至关重要的工具。特别是，研究孤立的 用该系统引导的小管和血管对于阐明与正常细胞功能和疾病过程相关的信号转导过程是独特的强大的。

 DESCRIPTION (provided by applicant): Microperfusion of dissected tubules is an established technique that can provide information about cellular function within isolated nephron segments in the absence of confounding feedback variables. This information cannot be obtained using other approaches, including intravital microscopy. Recent advances in transduction approaches to express heterologous constructs as well as to efficiently knock down endogenous proteins in kidney and bladder have expanded the opportunity to use live cell approaches, including FRET, to investigate physiologic regulation of membrane trafficking and signaling pathways in real time in isolated tubules. This request is for funds to purchase a perfusion imaging system to enable high speed, high resolution imaging of microperfused kidney tubules and blood vessels as well as other tissues and cells. The system consists of a single photon Nikon A1R confocal microscope with a resonant scanner, spectral detector and FRET module, dual micromanipulators, and an environmental chamber. The system will be housed within the P30-funded George M. O'Brien Pittsburgh Center for Kidney Research at the University of Pittsburgh School of Medicine. The mandate of this center is to facilitate multidisciplinary research, trainin and information transfer related to kidney physiology, cell biology, pharmacology, and pathophysiology. The Center includes cores for Kidney Imaging, Cellular Physiology, Single Nephron and Metabolomics, and Model Organisms, and serves a large group of NIH-funded investigators at the University of Pittsburgh and at other institutions. Our center lacks a dedicated microscope for live imaging of perfused tubules, and the availability of the proposed system will significantly enhance the services we are able to provide to center users. Relevance- The proposed system will provide a crucial tool for the research of numerous biomedical investigators studying the cellular basis of disease. In particular, studies of isolated tubules and vessels conducted with this system are uniquely powerful for elucidating signal transduction processes associated with normal cell function and disease processes.