Confocal Microscope for Mutant-GFP-Based FRET

本申请要求蔡司LSM510激光扫描共聚焦显微镜，配备专用设备，允许膜片钳电生理测量与共聚焦图像采集同时进行。这种特殊的仪器将具有约翰霍普金斯大学所有核心设备共聚焦显微镜所没有的两个关键优势。首先，显微镜将具有必要的灵活性，在选择性激发和发射波长，以允许实现一种新的3(3)-FRET算法，快速和无损地指定供体和受体荧光团之间的FRET效率(E)。（用gfp颜色突变体如CFP和YFP方便地完成），测定E可以指定10-100埃之间的荧光团间距，从而提供了活细胞中蛋白质-蛋白质相互作用的强大定量测量。3(3)-FRET算法克服了该方法的主要障碍，尽管通常观察到标记蛋白的表达水平存在差异，但仍可以确定E。因此，提出的共聚焦仪器将提供蛋白质-蛋白质相互作用的空间分辨图。其次，目前没有核心设施共聚焦显微镜能够同时成像和膜片钳记录；电生理设备的连接将证明对传统用户具有破坏性，并且这种设备很容易被大量非电生理参与者损坏。然而，许多科学问题关键取决于同时图像采集和测量/控制膜电压。通过吸引重视这种双重能力的用户群，建议使用专用膜片钳的共聚焦仪器将解决这一空白。蔡司LSM510平台适应3(3)-FRET和电生理的最佳实现。该显微镜将锚定一个由3个主要用户和3个次要用户支持的部门设施，所有这些用户的研究都将从根本上通过该平台的独特功能得到推进。

This application requests a Zeiss LSM510 laser scanning confocal microscope, outfitted with dedicated equipment permitting patch-clamp electrophysiological measurements to be made concurrently with confocal image acquisition. This particular instrument will feature two crucial advantages absent in all core-facility confocal microscopes at Johns Hopkins University. First, the microscope will possess the requisite flexibility in selective excitation and emission wavelengths to permit implementation of a novel 3(3)-FRET algorithm that rapidly and nondestructively specifies the FRET efficiency (E) between donor and acceptor fluorophores. (Conveniently done with GFP-color mutants like CFP and YFP), determination of E can specify interfluorophore distances between 10-100 Angstroms, thereby providing a powerful and quantitative measure of protein-protein interaction in living cells. The 3(3)-FRET algorithm overcomes a major obstacle to this approach, allowing E to be determined despite commonly observed variability in the expression levels of labeled proteins. The proposed confocal instrument will thus furnish spatially resolved maps of protein-protein interaction. Second, no core-facility confocal microscope currently accommodates simultaneous imaging and patch-clamp recording; the attachment of electrophysiological apparatus would prove disruptive to conventional users, and such apparatus would be easily damaged by a high-volume of non-electrophysiological participants. Yet, numerous scientific questions hinge critically upon simultaneous image acquisition and measurement/control of membrane voltage. By drawing from a user base that values this dual capability, the proposed confocal instrument with dedicated patch clamp would address this void. The Zeiss LSM510 platform accommodates optimal implementation of 3(3)-FRET and electrophysiology. The microscope will anchor a departmental facility supported by 3 major and 3 minor users, all of whose research would be fundamentally advanced by the platform's unique capabilities.