Funds are requested to purchase a Zeiss LSM MP multiphoton microscope with a Coherent Chameleon Ultra II Ti:Sapphire pulsed laser. This instrument will replace our current BioRad Radiance Multiphoton Microscope, which no longer meets the needs of our users. In addition, Carl Zeiss is phasing out their legacy BioRad systems and no longer stocks replacement hardware or provides software upgrades for the Radiance 2300 system. The dedicated multiphoton microscope will be housed in the Imaging/Physiology Core Facility supported by the NCRR Center of Biomedical Research Excellence (COBRE) in Neuroscience grant (5P20 RR16435, 07/01/06 - 06/30/11). The competitive renewal of the COBRE grant is expected to start 7/1/01/2011 and provide 5 additional years of Core support. The Neuroscience COBRE Imaging/Physiology Core is administered by a full-time, highly trained microscopist. Acquisition of the Zeiss LSM 7 MP dedicated multiphoton microscope, a modern, modular, and actively supported system, would greatly enhance the capabilities of the Core and would meet the emerging needs of the present Core investigators and provide the opportunity for additional funded investigators to significantly expand the scope of their research programs. The new Zeiss multiphoton microscope, model LSM 7MP, offers impressively rapid acquisition speed, about 4 times higher than the speed of our BioRad Radiance 2100 MPD two-photon microscope. The high acquisition speed combined with the superior sensitivity and lower noise levels makes LSM 7 MP perfect for studying the activity of living neurons and other cell types in the brain. In recent years, multiphoton photolysis of caged compounds (uncaging) is increasingly used as a tool to quickly deliver calcium and other substances intracellularly or in the extracellular space with great precision in time, location and volume. Furthermore, multiphoton uncaging allows deeper penetration into live tissues, with smaller volumes compared to single photon photolysis. Our highly innovative research projects that investigate the neuro-vascular coupling in the brain and cerebral arteriole smooth muscle and endothelium function absolutely require the use of photolysis of caged calcium, IP3, glutamate and ATP. The Zeiss LSM 7 MP is capable of two photon photolysis localized within multiple arbitrarily defined regions of interest without slowing down the acquisition speed. This will allow us to simultaneously uncage and image living cells or tissue with a speed that is adequate for monitoring and recording the changes in cellular activity that occur as a result of uncaging. Moreover, this will enable us to simultaneously or consecutively uncage compounds in more than one cell or location thus providing us with the unique opportunity to study the interactions and communications between different cells or structures in the brain.
This instrument would support research designed to understand the development and normal functioning of the brain and autonomic organs and their alteration in response to disease. Imaging of cellular responses within brain slices and organs will provide key insight into the regulation of neuronal, vascular, and connective tissue function and their interdependence. This insight will inform development of therapeutic approaches for diseases that strike these tissues.
