Olympus FV1000 Livecell Confocal System

描述（由申请人提供）：本提案的长期目标是提供最先进的共聚焦成像能力，以推进威斯康星大学麦迪逊分校心脏再生研究计划和细胞与分子心律失常研究计划中的NIH资助项目。这些小组的研究人员正在进行的研究项目目前广泛使用共聚焦显微镜，但由于缺乏服务支持和额外维修的高成本，现有的专用仪器（Bio-Rad Micro radiance）最近已经退役（10年）。此外，当代共焦显微镜的技术进步和新功能提供了显着的进步，成像能力。心脏再生医学研究的一个基本需求是能够跟踪移植的细胞并确定它们在心肌中的命运。FV 1000提供的五通道检测将通过允许同时检测多个不同的细胞系特异性蛋白质来帮助心脏再生研究项目，以更清楚地划分修复和再生心肌中的不同细胞类型。此外，奥林巴斯FV 1000提供的光谱检测系统将允许更准确地检测特定的荧光团，最大限度地减少串扰。基础心律失常研究中的关键问题越来越多地集中在确定离子通道蛋白和相关蛋白到表面膜的运输以及确定这些分子在心肌细胞中的精确亚区室化。申报的Olympus FV 1000将通过使用系统的SIM（同步激光刺激和成像）功能实现光漂白（FRAP）实验后的荧光恢复，实现离子通道和相关蛋白质的膜运输的动态跟踪。此外，进行组合的细胞电生理学和共聚焦成像实验的能力大大增强了功能信息，这些信息可以通过这些实验使用技术，如动态荧光共振能量转移（FRET）在电压钳实验和钙成像与同时电压钳研究。与市场上其他仪器的比较表明，奥林巴斯FV 1000具有经过验证的功能，具有竞争力的价格为指定的需求与优质的服务。必要的空间、行政支持、监督和技术专长已经到位。一个顾问委员会将提供监督，该委员会不仅由用户组成，还包括共聚焦成像和心脏生物学方面的专家。来自医学和公共卫生学院，医学系和心血管医学系的机构支持将提供显微镜管理员和维护和维修所需的资金。总体而言，所要求的奥林巴斯FV 1000显微镜将大大推进多个研究项目，改善当前的成像数据采集，以及提供新的功能，这将使强大的新应用程序不可能在现有的设备。 公共卫生：心脏病仍然是美国最常见的死亡原因。所要求的共聚焦显微镜将为旨在更好地了解心律失常的细胞和分子机制的研究项目提供强大的工具，心律失常是疾病和猝死的主要原因。此外，这种显微镜将使开创性的研究集中在确定新的策略，以修复和再生心肌损伤后，如心肌梗死。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to provide state-of-the-art confocal imaging capabilities to advance NIH-funded projects in the Cardio regenerative Research Program and Cellular and Molecular Arrhythmia Research Program at the University of Wisconsin-Madison. Ongoing research projects by investigators in these groups currently utilize confocal microscopy extensively, but the available dedicated instrument (Bio-Rad Micro radiance) has recently been retired (10 years old) because of lack of service support and high costs associated with additional repair. Furthermore, technological advances and new features in contemporary confocal microscopes provide a significant advance in imaging capabilities. A fundamental need for research in cardio regenerative medicine is to be able to track transplanted cells and determine their fate in cardiac muscle. The five channel detection provided by the FV1000 will assist cardio regenerative research projects by allowing the simultaneous detection of multiple distinct cell lineage-specific proteins to more clearly demarcate distinct cell types in repaired and regenerated myocardium. Furthermore, the spectral detection system provided by the Olympus FV1000 will allow a more accurate detection of specific fluorophores, minimizing cross-talk. Critical questions in basic arrhythmia research are increasingly focused on determining the trafficking of ion channel proteins and associated proteins to the surface membrane and determining the precise sub compartmentalization of these molecules in cardiomyocytes. The proposed Olympus FV1000 will enable dynamic tracking of the membrane trafficking of ion channels and associated proteins by enabling fluorescence recovery after photo bleaching (FRAP) experiments using the SIM (simultaneous laser light stimulation and imaging) functionality of the system. Additionally, the ability to perform combined cellular electrophysiology and confocal imaging experiments greatly enhances the functional information that can be obtained by these experiments using techniques such as dynamic fluorescence resonance energy transfer (FRET) during voltage clamp experiments and calcium imaging with simultaneous voltage clamp studies. Comparisons with other instruments on the market suggested that the Olympus FV1000 has the proven functionality at a competitive price for the specified needs with excellent service available. The necessary space, administrative support, supervision, and technical expertise are already in place. An advisory board consisting not only of users but also of experts in confocal imaging and cardiac biology will provide oversight. Institutional support from the School of Medicine and Public Health, Department of Medicine, and Division of Cardiovascular Medicine will provide a microscope manager and funds needed for maintenance and repair. Overall, the requested Olympus FV1000 microscope will significantly advance multiple research projects improving current imaging data acquisition as well as providing new functionality that will enable powerful new applications not possible on available equipment. PUBLIC HEALTH REVELANCE: Heart disease remains the most common cause of death in the United States. The requested confocal microscope will provide a powerful tool for research projects aimed at better understanding the cellular and molecular mechanisms of arrhythmias, a major cause of illness and sudden death. In addition, this microscope will enable pioneering research focused on identifying new strategies to repair and regenerate cardiac muscle following injury such as myocardial infarction.