Imaging of intracellular calcium signaling is a key approach used by each Investigator on this PPG. This PPG employs.extensive imaging of a variety of cell types (smooth muscle, endothelium, astrocytes), at the level of isolated single cells (endothelial and smooth muscle cells) and multicellular preparations (isolated arteries, brain slices), each of them exhibiting calcium signals with different spatio-temporal dynamics (Ca^* pulsars, Ca^"""""""""""""""" sparks, Ca^"""""""""""""""" waves, global Ca^* changes). In addition, some studies require Ca^^ imaging combined with simultaneous electrophysiological and arterial diameter measurements. To address the complexity of imaging different calcium signals/tissues we use a variety of synthetic (fura-2, fluo-4) and genetically encoded (GCaMP2, GCaMP2-mCherry) calcium indicators. To adequately image such a diverse assortment of tissues requires the use of specialized imaging instruments with different features. The Imaging Core provides access for each investigator to expensive (over $1,000,000), state-of-the-art equipment that will address the needs of each proposal. By consolidating the imaging equipment into a single Core we will reduce expenses for collecting imaging data. The Core equipment consists of four wide-field epi-fluorescent systems, three laser scanning confocal microscopes and one multiphoton imaging system. Each setup is housed in a separate dark room in the connected Given and HSRF buildings of the University of Vermont. The specific objectives of the Imaging Core are to maintain the instruments in optimal condition, coordinate scheduling to maximize the use of each instrument, train users unfamiliar with software or instrumentation, provide expertise to investigators in experimental design, processing, analysis and interpretation of the experimental data, and create and maintain custom software for data analysis.
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