Dual Confocal Microscopic Scanner

描述（由申请人提供）：共聚焦系统包括显微镜、一组激光器和扫描仪，扫描仪将激发光引导到显微镜中，并将发射光引导到一个或多个光电传感器中。在我们寻求的仪器中，蔡司LSM 5-Meta/LIVE组合（或DUO），两个扫描仪，每个扫描仪都有一组激光器，汇聚成一个显微镜。第二扫描仪（操作员）的主要目的是提供样品的额外照射，同时通过第一扫描仪独立成像荧光。操作员允许对样品进行许多操作，所有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系和部门的经验丰富的成像人员加上洛约拉大学成像核心的负责人，他们将应用仪器的显着特征用于（i）细胞溶质，线粒体和骨骼肌和心肌的储存细胞器以及脑血管内皮细胞内的钙监测，（ii）吞噬体和亚膜结构域中的质子浓度和演变，（iii）膜曲率和脂筏对活细胞和细胞组分之间的FRET筏首选或筏排除分子，（iv）监测单向流量通过个别渠道在双层和（v）监测cAMP在内皮细胞。该监测将与操作员诱导的Ca 2+、质子或IP 3的光释放同时进行。在各种情况下，监测将使用SEER（由PI开发）的新模式，以最大限度地提高探头灵敏度。虽然这些操作中的一些可以用传统的共焦显微镜完成，但大多数需要能够同时独立地定义操作者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，对于已经实现的最先进的生物传感器特别有价值，以及极高的成像速度，对于快速发展的微域以及移动的细胞器和筏非常有用。这些项目直接或间接与一些疾病实体有关，包括ALS（i），HIV传染性（iv），心力衰竭和节律改变（i，ii），炎症和免疫反应（iii，v）。该提案获得的机构支持证明了其对拉什大学基础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.