COBRE: UVM MED: CORE B: IMAGING & PHYSIOLOGY CORE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Cobre成像和生理学核心为固定和活细胞免疫荧光准备提供了广泛的成像技术。我们拥有Noran/Prairie Technologies LSCM(激光扫描共聚焦显微镜)、DeltaVision RT修复显微镜系统、Zeiss/BioRad专用多光子LSCM、尼康TIRF(全内反射荧光)系统、Lamda DG-4比率成像系统、Andor Ixon/奥林巴斯宽场荧光显微镜和尼康SMZ-1500荧光立体显微镜。膜片钳电子器件可用于大多数显微镜系统，并且每个系统都有一个集成的灌流设置。 Noran能够高速采集图像，主要用于对整个组织和分离的细胞进行钙成像。DeltaVision系统有一个温度和二氧化碳可控的孵化器，允许对培养细胞进行延时实验，还擅长对固定样本进行免疫荧光和共定位实验。BioRad Radiance 2100专用多光子具有相干变色龙飞秒脉冲红外激光器和固定式立式奥林巴斯显微镜。脉冲红外激发，加上非扫描探测器，使其有可能在500微米深的地方进行数小时的成像。它目前被用于脑切片上的钙成像和去势实验，以及对发育中的鸡胚胎脊髓中的神经元进行逆行标记。该比率成像系统用于Fura-2负载细胞或转染FRET为基础的钙指示剂Cameleon的细胞的成像。TIRF系统用于囊泡运输研究以及细胞黏附和迁移实验。目前，它正被建立在膜片钳啮齿动物细胞的细胞膜附近进行钙成像。Andor Ixon系统是一台配备奥林巴斯显微镜的高速EM电荷耦合器件，旨在对组织制剂中的快速钙事件进行成像。 对于数据去卷积和分析，我们有两个专用的工作站，运行SoftWoRx(应用精度)来进行去卷积，Metamorph(通用成像)，Volocity(Perkin Elmer)和ImageJ(NIH)来进行一般分析和渲染。EM CCD工作站包括Andor IQ-CORE分析软件。设备调度在线，用户工时记录在核心数据库中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The COBRE Imaging and Physiology Core provides a wide range of imaging techniques for fixed and live-cell immunofluorescence preparations. We maintain a Noran/Prairie Technologies LSCM (laser scanning confocal microscope), a DeltaVision RT restoration microscopy system, a Zeiss/BioRad dedicated multiphoton LSCM, a Nikon TIRF (total-internal reflection fluorescence) system, a Lamda DG-4 ratiometric imaging system, an Andor iXon/Olympus widefield fluorescence microscope, and a Nikon SMZ-1500 fluorescence stereoscope. Patch-clamp electronics are available for use on most microscope systems, and each system has an integrated perfusion setup. The Noran is capable of high-speed image acquisition and is used primarily for calcium imaging on whole tissues and isolated cells. The DeltaVision system has a temperature and CO2-controlled incubator allowing for time-lapse experiments on cultured cells, and also excels at immunofluorescence and colocalization experiments on fixed samples. The BioRad Radiance 2100 Dedicated Multiphoton features a Coherent Chameleon femtosecond pulsed IR laser and a fixed-stage upright Olympus microscope. The pulsed infrared excitation, coupled with non-descanned detectors, make it possible to image at depths of up to 500 microns for several hours. It is currently being used for calcium-imaging and uncaging experiments in brain-slices as well as retrograde labeling of neurons in the spinal cord of developing chick embryos. The ratiometric imaging system is used for imaging of fura-2 loaded cells or cells transfected with the FRET-based calcium indicator Cameleon. The TIRF system is used in vesicular trafficking studies as well as cell adhesion and migration experiments. Currently, it is being set up for calcium imaging near the cell membrane on patch-clamped rodent cells. The Andor iXon system is a high-speed EM CCD coupled with an Olympus microscope, designed to image fast calcium events in tissue preparations. For data deconvolution and analysis, we have two dedicated workstations running softWoRx (Applied Precision) for deconvolution, MetaMorph (Universal Imaging), Volocity (Perkin Elmer) and ImageJ (NIH) for general analysis and rendering. The EM CCD workstation includes Andor iQ-CORE analysis software. Equipment scheduling is online, and user hours are recorded in the core database.