Spinning Microlens Confocal Microscope

一个由美国国立卫生研究院资助的六个主要用户组成的团体要求为旋转微透镜阵列激光扫描共聚焦显微镜提供资金。要求的Perkin Elmer Ultraview系统包括多种激发激光线路，包括用于特定激发CFP融合蛋白和CFP/YFP比率成像的专用442 nm HeCad激光器，这是UCHC的其他系统所不具备的。此外，旋转微透镜阵列允许使用高量子产率冷却CCD相机的全分辨率进行高帧速率采集。与点扫描检流计共焦系统相比，这种激发/收集策略已被证明减少了成像样品中的光漂白和光损伤，这可能是因为亚饱和瞬时激发和CCD探测器的较高信噪比。一个额外的计算机控制的染料可调谐光漂白激光系统被包括在内，用于高时间/空间分辨率的光漂白后荧光恢复(FRAP)的研究，以及荧光共振能量转移(FRET)的去猝灭测量。该仪器将安置在康涅狄格州大学健康中心的生物医学成像技术中心(CBIT)，该中心通过完善的用户设施，为UCHC和外部机构的研究社区提供高水平荧光显微镜仪器和图像分析资源的使用。六个主要用户都有完善的项目，每个项目都需要某些方面，这些方面对于所要求的系统来说是独一无二的，在UCHC的其他地方是无法获得的。William Mohler将记录高速4D数据并进行FRAP分析，以了解线虫细胞融合的机制。弗拉基米尔·罗迪奥诺夫将使用荧光散斑成像技术研究中心体自我中心的细胞骨架基础。伊丽莎白·埃珀将通过高速共聚焦成像和FRAP研究多肽囊泡分泌的动力学。吴殿青将通过FRET研究Wnt信号分子在体内的相互作用。Steven Pfeiffer将通过FRET研究髓鞘蛋白的动态运输及其在脂筏上的定位。Bruce Mayer将在体内可视化由信号oroteins聚集引起的肌动蛋白细胞骨架的变化，以及动态共定位和orotein相互作用。

A group of six major NIH-funded users requests funds for a spinning-microlens array laser-scanning confocal microscope. The Perkin Elmer Ultraview system requested includes a broad selection of excitation laser lines, including a special-purpose 442-nm HeCad laser for specific excitation of CFP fusion proteins and CFP/YFP ratio imaging, which is not available on an other system at UCHC. In addition, the spinning microlens array allows high-frame-rate acquisition using the full resolution of a high quantum-yield cooled CCD camera. This excitation/collection strategy has been shown to result in reduced photobleaching and photodamage in imaged samples when compared to point-scanned galvanometer confocal systems, probably because of sub-saturating instantaneous excitation and the higher signal-to-noise of the CCD detector. An additional computer-controlled dye-tunable photobleaching laser system is included for high-time/space-resolution studies of fluorescence recovery after photobleaching (FRAP), as well as dequenching measurements of fluorescence resonance energy transfer (FRET). The instrument will be housed in the Center for Biomedical Imaging Technology (CBIT) at the UConn Health Center, which offers use of high-level fluorescence microscopy instrumentation and image analysis resources to the research community both at UCHC and outside institutions, through a well-established user facility. The six major users have well-developed projects that each require some aspects that are unique the to requested system and unavailable elsewhere at UCHC. William Mohler will record high-speed 4D data and perform FRAP analysis to understand the mechanism of cell fusion in C. elegans. Vladimir Rodionov will use fluorescent speckle imaging study the cytoskeletal basis for self-centering of the centrosome. Elizabeth Eipper will study the dynamics of peptide vesicle secretion via high-speed confocal imaging and FRAP. Dianqing Wu will study the interactions of Wnt-signalling molecules in vivo via FRET. Steven Pfeiffer will study the dynamic transport of myelin proteins and their localization to lipid rafts via FRET. Bruce Mayer will visualize actin cytoskeletal changes induced by aggregation of signaling oroteins, as well as dvnamic colocalization and orotein interaction bv FRET in vivo.