Olympus FV1000 Livecell Confocal System

描述(由申请人提供)：该提案的长期目标是提供最先进的共聚焦成像能力，以推进NIH资助的威斯康星大学麦迪逊分校心脏再生研究计划和细胞和分子心律失常研究计划中的项目。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焦显微镜，但可用的专用仪器(Bio-Rad Micro Radiance)最近已退役(使用10年)，原因是缺乏服务支持和与额外维修相关的高昂成本。此外，当代共焦显微镜的技术进步和新功能提供了成像能力的重大进步。心脏再生医学研究的一个基本需求是能够追踪移植细胞并确定它们在心肌中的命运。FV1000提供的五通道检测将通过允许同时检测多个不同的细胞谱系特定蛋白来更清楚地区分修复和再生心肌中的不同细胞类型，从而帮助心脏再生研究项目。此外，奥林巴斯FV1000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。在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心肌细胞表膜的运输以及确定这些分子在心肌细胞中的精确亚室隔化。拟议的奥林巴斯FV1000将使用该系统的SIM(同时激光刺激和成像)功能，通过实现光漂白(FRAP)实验后的荧光恢复，实现对离子通道和相关蛋白质膜运输的动态跟踪。此外，执行细胞电生理学和共聚焦成像联合实验的能力大大增强了这些实验可以通过使用电压钳实验期间的动态荧光共振能量转移(FRET)和同时进行电压钳研究的钙成像等技术获得的功能信息。与市场上其他仪器的比较表明，奥林巴斯FV1000具有成熟的功能，价格具有竞争力，可满足特定需求，并提供出色的服务。必要的空间、行政支持、监督和技术专长已经到位。一个咨询委员会将提供监督，该委员会不仅由用户组成，还包括共焦成像和心脏生物学方面的专家。来自医学与公共卫生学院、医学系和心血管医学部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FV1000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，并提供新功能，使现有设备无法实现的强大新应用成为可能。 公共卫生评论：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.