This application requests funds to purchase a high speed imaging system for confocal fluorescence imaging, subcellular photolysis and patch clamp control of single cells. The confocal imaging system will be used in a new shared instrument facility at the University of Maryland Biotechnology Institute (UMBI). Extensive testing of each element of the system by the users has been carried out and planned work using this novel state-of-the-art imaging system is outlined in the proposed projects. The imaging system involves three components. The confocal instrument is the Zeiss LiveS instrument that is capable of acquiring XY images at 1 kHz at 50 lines by 512 pixels or full frame (512 X 512) at a frame rate of 120 Hz (8.3 ms per image). Linescan images (1 X 512) can be obtained at 62.5 kHz (16 microseconds). The photolysis component of the system is centered on a Spectra Physics HIPPO-355 OEM laser running at 100 kHz with an average power of 5 W. The scan head can be placed on an inverted microscope for optimal single cell patch clamp experiments or on an upright microscope of brain slice or special culturing conditions. The triggering and laser launch optics are customized for use with both the Zeiss Live5 instrument and with the patch clamp electronics. It can be synchronized to microsecond precision, which is adequate for all planned work. The patch clamp component of the system is centered on a HEKA EPC10 patch clamp unit and LIH-1600 computer interface running Patchmaster acquisition software. Researchers at the University System of Maryland in Baltimore (UMBI and UMB) have achieved a three-decade reputation for the study of calcium signaling at high temporal and spatial resolution in living cells. They have discovered Ca2+ sparks in cardiac, skeletal and smooth muscle, have investigated function in these tissues in normal and pathological conditions and have been world- wide leaders in this area. They have a similar reputation in neuronal and brain slice imaging. This proposed new instrumentation is not available anywhere in the Baltimore-Washington area and will allow these scientists to carry out funded research and to make significant advances. ? ? ?
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