权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Confocal Microscope for Mutant-GFP-Based FRET

用于基于突变 GFP 的 FRET 的共焦显微镜

基本信息

批准号：
6440962
负责人：
DAVID T YUE
金额：
$ 21.35万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-01 至 2004-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6440962
关键词：
biomedical equipment purchase confocal scanning microscopy electrophysiology

项目摘要

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.

本申请要求蔡司LSM 510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两个关键的优势，在所有的核心设施共聚焦显微镜在约翰霍普金斯大学。首先，显微镜将拥有必要的灵活性，在选择性的激发和发射波长，以允许实施一种新的3（3）-FRET算法，快速和非破坏性指定的FRET效率（E）之间的供体和受体荧光团。（方便地用GFP颜色突变体如CFP和YFP完成），E的测定可以指定10-100埃之间的荧光团间距离，从而提供活细胞中蛋白质-蛋白质相互作用的有力和定量测量。 3（3）-FRET算法克服了这种方法的主要障碍，尽管通常观察到标记蛋白质表达水平的变化，但仍允许测定E。因此，拟议的共聚焦仪器将提供蛋白质-蛋白质相互作用的空间分辨地图。第二，没有核心设施共聚焦显微镜目前容纳同时成像和膜片钳记录;电生理仪器的附件将被证明是破坏性的传统用户，这样的设备将很容易被损坏的高容量的非电生理参与者。然而，许多科学问题关键在于同时图像采集和膜电压的测量/控制。通过从重视这种双重能力的用户群中汲取经验，所提出的具有专用膜片钳的共聚焦仪器将解决这一空白。 Zeiss LSM 510平台可实现3（3）-FRET和电生理学的最佳实施。该显微镜将锚由3个主要和3个次要用户，所有的研究将从根本上先进的平台的独特功能的部门设施的支持。