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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 COBRE成像和生理学核心为固定和活细胞免疫荧光制剂提供了广泛的成像技术。 我们维护了Noran/Prairie Technologies LSCM（激光扫描共聚焦显微镜）、DeltaVision RT恢复显微镜系统、Zeiss/BioRad专用多光子LSCM、Nikon TIRF（全内反射荧光）系统、Lamda DG-4比率成像系统、Andor iXon/Olympus宽视场荧光显微镜和Nikon SMZ-1500荧光立体镜。 膜片钳电子可用于大多数显微镜系统，每个系统都有一个集成的灌注设置。 Noran能够高速采集图像，主要用于整个组织和分离细胞的钙成像。 DeltaVision系统具有温度和CO2控制的培养箱，允许对培养细胞进行延时实验，并且还擅长对固定样品进行免疫荧光和共定位实验。 BioRad Radiance 2100 Dedicated Multiphoton具有相干变色龙飞秒脉冲IR激光器和固定台直立Olympus显微镜。 脉冲红外激发，加上非去扫描探测器，使其能够在长达500微米的深度成像数小时。 它目前被用于脑切片中的钙成像和解开实验，以及发育中鸡胚脊髓中神经元的逆行标记。 比率成像系统用于对负载fura-2的细胞或用基于FRET的钙指示剂Cameleon转染的细胞进行成像。 TIRF系统用于囊泡运输研究以及细胞粘附和迁移实验。目前，它被设置为在膜片钳啮齿动物细胞上的细胞膜附近进行钙成像。 Andor iXon系统是一种与Olympus显微镜耦合的高速EM CCD，旨在对组织制备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（Applied Precision）进行去卷积，MetaMorph（Universal Imaging），Volocity（Perkin Elmer）和ImageJ（NIH）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，用户小时数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.