This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The COBRE Imaging and Physiology Core facility is designed primarily for live-cell imaging and electrophysiology. It contains a DeltaVision RT restoration microscopy system, a BioRad/Zeiss Radiance 2100 multiphoton laser scanning confocal microscope, a Noran/Prairie Technologies AOD (acousto-optic deflector) laser scanning confocal microscope, a Nikon TIRFM (total-internal reflection fluorescence microscopy) system, and a Lambda DG-4 ratiometric imaging system. Patch-clamp electronics are available for combined electrophysiology and imaging studies. Each imaging system is equipped with perfusion apparatus to sustain live preparations. The DeltaVision RT Restoration system has a temperature and humidity-controlled incubator allowing for time-lapse experiments on cultured cells. Images from the DeltaVision system are deconvolved on a separate workstation using iterative algorithms. The BioRad Radiance 2100 Dedicated Multiphoton is coupled to a Coherent Chameleon tunable femtosecond-pulsed IR laser and a fixed-stage upright Olympus microscope. The infrared, multiphoton excitation, which allows the use of non-descanned detectors, makes it possible to image at depths of up to 500 microns for several hours with reduced bulk photobleaching. The Coherent's pulsed laser wavelength tunability permits the use of a wide variety of fluorescent probes as well. The Noran LSCM uses fast AOD laser scanning for up to 50 image/second acquisition rates, facilitating fast cellular imaging. The ratiometric imaging system uses a Xenon illuminator and fast filter-changer to collect the differential ion-dependent emissions of selected probes. The TIRF system has a Nikon 1.45 NA lens which allows through-the-lens TIRF using a Melles-Griot Kr/Ar multiline laser. 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.
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