Dual Confocal Microscopic Scanner

描述（由申请人提供）：共聚焦系统包括一个显微镜，一组激光器和一个扫描仪，它引导激发光进入显微镜，并将发射光进入一个或多个光敏传感器。在我们寻找的仪器中，蔡司LSM 5-Meta/LIVE组合（或DUO），两个扫描仪，每个都有一组激光器，汇聚成一个显微镜。第二个扫描仪（操作员）的主要目的是为样品提供额外的照射，同时通过第一个扫描仪独立成像荧光。操作者允许对样品进行一些操作，所有这些操作都与光转换有关。没有仪器与所要求的仪器竞争，这是唯一可用的双扫描仪。主要用户是来自拉什大学三个部门和分部的经验丰富的成像仪以及洛约拉大学成像中心的负责人，他们将应用该仪器的显著特点来实现以下目的：(i)监测细胞质、线粒体和骨骼肌、心肌和脑血管内皮的储存细胞器内的钙，（ii）吞噬体和亚膜结构域的质子浓度和进化。（iii）细胞膜曲率和活细胞和细胞组分上的脂筏通过筏优选或筏排除分子之间的FRET， （iv）监测通过双分子层中单个通道的单向通量和(v)监测内皮细胞中的cAMP。这种监测将与操作者诱导的Ca2+、质子或IP3的光释放同时进行。在各种情况下，监测将使用新的SEER模式（由PI开发），以最大限度地提高探头灵敏度。虽然其中一些操作可以用传统的共聚焦显微镜完成，但大多数操作都需要能够同时独立地定义操作光以及激发和发射的成像参数。项目还将受益于独特的激发和发射波长的灵活性，特别是对已经实施的最先进的生物传感器的价值，以及极端的成像速度，对于跟踪快速发展的微域以及移动细胞器和筏非常有用。这些项目与若干疾病实体直接或间接相关，包括渐冻症（一）、艾滋病毒传染性（四）、心力衰竭和心律改变（一、二）、炎症和免疫反应（三、五）。这项提案得到了机构的大力支持，证明了它对拉什大学基础研究和转化研究的重要性。

DESCRIPTION (provided by applicant): Confocal systems include a microscope, a group of lasers, and a scanner, which guides the excitation light into the microscope and the emitted light into one or more photosensors. In the instrument that we seek, Zeiss LSM 5-Meta/LIVE combination (or DUO), two scanners, each with a set of lasers, converge into one microscope. The main purpose of the 2nd scanner (operator) is to provide additional irradiation of the sample, while independently imaging fluorescence via the first scanner. The operator permits a number of operations on the sample, all related to photoconversion. There are no instruments competing with the one requested, which is the only dual scanner available. Major users are experienced imagers from three departments and divisions of Rush University plus the head of Loyola University's Imaging Core, who will apply the salient features of the instrument for the purposes of (i) calcium monitoring inside cytosol, mitochondria and storage organelles of skeletal and cardiac muscle and brain vascular endothelia, (ii) proton concentration and evolution in phagosomes and submembrane domains, (iii) membrane curvature and lipid rafts on living cells and cell fractions by FRET between raft-preferred or raft-excluded molecules, (iv) monitoring of unidirectional flux through individual channels in bilayers and (v) monitoring of cAMP in endothelial cells. This monitoring will be simultaneous with operator-induced photorelease of Ca2+, protons, or IP3. In various cases monitoring will use the new modality of SEER (developed by the PI) to maximize probe sensitivity. While some of these operations can be done with conventional confocal microscopes, most require the ability to simultaneously and independently define the operator light and the imaging parameters of excitation and emission. Projects will also benefit from the unique flexibility of excitation and emission wavelengths, especially valuable for state of the art biosensors already implemented, and the extreme imaging speed, useful for following rapidly evolving microdomains as well as mobile organelles and rafts. The projects are directly or indirectly relevant to a number of disease entities, including ALS (i), HIV infectivity (iv), heart failure and alterations of rhythm (i, ii), inflammatory and immune response (iii, v). The extraordinary level of institutional support bestowed on this proposal attests to its perceived importance for basic and translational research at Rush University.