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

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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 facility is designed primarily for live-cell imaging and electrophysiology. It contains a refurbished Noran/ Prairie Technologies laser scanning confocal microscope, a single-level Nikon TIRF (total-internal reflection fluorescence) system, a ratiometric imaging system, a DeltaVision restoration microscopy system and a BioRad dedicated multiphoton laser scanning microscope. We also have patch-clamp electronics available for use on most microscope systems. The Noran is used primarily for calcium imaging on whole tissues and isolated cells. The DeltaVision system has a temperature and humidity-controlled incubator allowing for time-lapse experiments on cultured cells (such as migration assays), as well as immunofluorescence studies on fixed samples. The BioRad Radiance 2100 Dedicated Multiphoton includes a Coherent Chameleon pulsed femtosecond IR laser and a fixed-stage upright Olympus microscope. The 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 in brain-slices and 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 and also cell adhesion and migration experiments. In the future, it may also be used for calcium imaging near the cell membrane. For data deconvolution and analysis, we have two dedicated workstations running softWoRx (Applied Precision) for deconvolution, MetaMorph (Universal Imaging), Volocity (Improvision) and ImageJ (NIH) for general analysis and rendering.