Dual Confocal Microscopic Scanner

描述(申请人提供)：共聚焦系统包括显微镜、一组激光器和扫描器，扫描器将激发光引导到显微镜中，并将发射的光引导到一个或多个光传感器中。在我们寻找的仪器中，蔡司LSM 5-Meta/LIVE组合(或Duo)，两台扫描仪，每台都有一套激光，汇聚到一个显微镜中。第二扫描仪(操作员)的主要目的是为样品提供额外的照射，同时通过第一扫描仪独立地成像荧光。操作员允许对样品进行多项操作，所有操作都与光转化有关。没有与所要求的仪器竞争的仪器，这是唯一可用的双扫描仪。主要用户是来自拉什大学三个院系的经验丰富的成像员以及Loyola大学成像核心的负责人，他们将为以下目的应用该仪器的显著功能：(I)监测骨骼肌、心肌和脑血管内皮细胞的胞浆、线粒体和储存细胞器内的钙离子；(Ii)吞噬体和亚膜域中的质子浓度和演化；(Iii)活细胞和细胞组分上的膜曲率和脂筏；(Iii)通过RAFT偏好或RAFT排除的分子之间的FRET来监测活细胞和细胞组分；(Iv)监测双层中单个通道的单向通量；以及(V)监测内皮细胞中cAMP的含量。这种监测将与操作员诱导的钙、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使用传统的共焦显微镜完成，但大多数操作需要同时和独立地定义操作员光以及激发和发射的成像参数。项目还将受益于激发和发射波长的独特灵活性，这对已经实施的最先进的生物传感器特别有价值，以及极高的成像速度，有助于跟踪快速演变的微域以及移动细胞器和木筏。这些项目与许多疾病直接或间接相关，包括肌萎缩侧索硬化症(I)、艾滋病毒传染性(IV)、心力衰竭和节律改变(I、II)、炎症和免疫反应(III、V)。这项提议获得了非同寻常的机构支持，证明了它对拉什大学基础研究和翻译研究的重要性。

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.