Zeiss LSM780 Confocal Microscope for a Core Facility

描述(由申请人提供)：作为已建立的核心设施的一部分，我们要求在倒置显微镜支架上安装蔡司LSM780激光扫描共聚焦显微镜和荧光相关光谱(FCS)。针对多个活细胞/组织研究，我们的需求来自三个来源：现有激光扫描共聚焦仪器的超额认购，需要减少对荧光标记的活细胞的光损伤，以及需要解决分子水平的事件，特别是在活细胞中。虽然我们有三台蔡司激光扫描共焦，但它们的使用量很大，一台仪器每天最高使用20小时。光损伤和光漂白与仪器的信噪比性能有关，LSM780在我们的测试中表现突出，允许使用其他共焦无法实现的技术。为了了解生物途径，活细胞技术变得越来越重要。FCS不仅提供了关于细胞内空间组织调控的关键分子测量，而且还区分了我们系统中信号机制的假说。此外，当地努力构建新的FRET生物传感器，并将不同的生物传感器组合到相同的细胞中，这需要更高的仪器性能。自动光谱分解极大地促进了这些FRET研究。正在进行的活细胞研究使用了所有这些技术，以及FRAP、光激活和基本的时间推移成像。拟议的工具将提供数量级的优越性能，将使NIH资助的关键项目成为可能。

DESCRIPTION (provided by applicant): As part of an established core facility, we request a Zeiss LSM780 laser-scanning confocal microscope with fluorescence correlation spectroscopy (FCS) on an inverted microscope stand. Addressing multiple live-cell/tissue studies, our needs come from three sources: oversubscription on existing laser-scanning confocal instruments, need to reduce photodamage to fluorescently labeled living cells, and need to resolve molecular-level events, particularly in living cells. Although we have three Zeiss laser- scanning confocals, they are heavily used, with peak usage 20h/day on one instrument. Photodamage and photobleaching are related to the instrument's signal-to-noise performance, and the LSM780 is outstanding in our tests, allowing techniques not possible with other confocals. To understand biological pathways, live-cell techniques become ever more important. Not only does FCS provide key molecular measurements about regulation of spatial organization within cells, it also discriminates hypotheses in signaling mechanisms in our systems. In addition, local efforts to construct new FRET biosensors and combine different biosensors into the same cells demand greater performance of instrumentation. These FRET studies are greatly facilitated by automated spectral unmixing. Ongoing live-cell studies use all these techniques, as well as FRAP, photoactivation, and basic time-lapse imaging. The proposed instrument will provide an order-of-magnitude superior performance that will enable key NIH- funded projects.