LSM 5 Live Microscope for Cell Imaging Shared Resource

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DESCRIPTION (provided by applicant): We propose to acquire a newly developed high-speed laser scanning_slit-confocal microscope (LSM 5 Live, Zeiss), to support research of five NIH-funded investigators at Vanderbilt University that require imaging of rapid dynamic events in live samples. No other microscope on campus has equivalent capabilities, therefore, this system offers significant potential for a large number of investigators. The new instrument will be incorporated into the Cell Imaging Shared Resource (CISR). The CISR provides expert optical microscopy services for about 250 VUMC laboratories, most are NIH-funded, including seven research centers; trained users can access the facility 24 hours/day, 7 days a week. The CISR has a 12-year history of outstanding service, reliability and self-sufficiency; microscope usage is currently about 9000 hours/year. During a recent evaluation we confirmed that the LSM 5 Live can acquire viable confocal images up to 120 (512x512) frames/sec. We imaged flowing blood cells in microvasculature of mouse muscle at rates of 100- 250 frames/sec. We also imaged motions of all individual cardiac cells throughout the beating heart of an embryonic zebrafish by combining piezo-objective focusing with high frame rates to obtain 3-D images (20slices/40microns) at a rate of three z-series/sec. The signal collection efficiency of the instrument was excellent and photobleaching was minimal under the conditions required for our feasibility experiments. Importantly, we visualized numerous biological processes without obvious, significant photo-damage, e.g., calcium oscillations in pancreatic islets were persistent following >12,000 frames of data. The signal-to- bleaching ratio appeared more favorable compared to a point-confocal laser scanning system. Five Major Users with immediate, essential and appropriate need for the requested instrument will utilize 70% of total instrument activity. They will use it to study: 1) zebrafish models of cellular and molecular mechanisms of cardiovascular development and congenital heart disease; 2) C. elegans models of meiosis and molecular processes of reproduction; 3) stimulus-secretion coupling in intact functioning pancreatic islets; 4) mouse models of fuel metabolism and it's relationship to diabetes and heart disease; and 5) mouse models of neo-vascularization and functionality of transplanted islets. Placing an LSM 5 Live in the established and widely used CISR will not only advance the research of the Major Users in this proposal, but also provide significant opportunity to advance the NIH-sponsored research of many more investigators studying features of living models of biology and disease processes previously inaccessible to scientific observation.