The long-term objectives of our 12 federal grants (R01-type) are to address important and unique problems in neuroscience and cardiac physiology, including a better understanding how living cells in the nervous system and heart respond to drugs or other changes in the environment. The current application is in response to a significant need of our 8 Major Users to obtain confocal images and electrophysiological recordings, often simultaneously, from live neural, sensory and cardiac cells or tissue slices. Specific needs include the following major functional capabilities: Confocal imaging of Ca2+ transients and fluorescent markers in live tissue slices;ratiometric Fura-2 imaging of Ca2+ transients;high temporal and spatial resolution of Ca2+ imaging during concomitant patch clamp recordings of ion channel activity;and UV Flash photolysis of caged compounds. All of the Major Users have extensive experience with live cell calcium imaging, electrophysiology, and/or confocal microscopy. Collectively, we have published over a hundred papers using one or more of these techniques, including 15 papers that simultaneously used functional confocal imaging and electrophysiological recording in live cells. An inherent problem of laser confocal microscopes is the very intense excitation light, leading to substantial dye decomposition (photobleaching) during live-cell imaging. The proposed Olympus BX- DSU Live-Cell Disk Scanning Imaging System overcomes this problem with optimization of signal detection. It uses spinning disk confocal technology with a new generation, super-cooled EM-CCD camera to generate images with high temporal and spatial resolution. The broad spectrum light source produces substantially less photobleaching than do lasers. Furthermore, this system adds a UV flash illuminator for photolysis of photoactivatable (caged) compounds, complete integration of patch-clamp instrumentation for concomitant electrophysiological recordings, and an upright stand preferred for tissue slice work. The result is a very cost-effective and flexible solution that responds to all described needs of the Major Users. No other instrument at or near the University of Kentucky can provide this constellation of features. The University will provide appropriate space, salary support for experienced technical staff, and maintenance for the system. The system will be set up within a large well-established Imaging Facility at the University of Kentucky by experts in scanning disk technology and electrophysiology. This system will allow the Major Users to continue to achieve new discoveries towards the better understanding and treatment of chronic pain, obesity, auditory disorders, olfactory disorders, pulmonary disease, consequences of brain injury, and cardiovascular disease.
