This application requests a BioRad two-photon microscope optimized for quantitative imaging with living cells and tissues. The BioRad two-photon laser scanner will be mounted on an Olympus fixed stage upright microscope with water immersion objectives for working either with or without coverslips in aqueous environments, and two-photon excitation will be via a Coherent mode-locked Ti-Sa laser. The facility will serve investigators from multiple departments throughout the Johns Hopkins University Medical Institutions, who need the instrument to perform live cell and live tissue fluorescence microscopy that is beyond the capabilities of confocal microscopy. Our 4 major users require two-photon microscopy to (a) provide high resolution imaging of cellular and subcellular events within tissue at focal depths greater than 50um from the surface (at these depths, confocal microscopy can neither provide high spatial resolution nor avoid photodamage), (2) permit localized deposition of energy for uncaging or photobleaching experiments so that events can then be tracked by conventional confocal microscopy on the same microscope, and (3) perform spectral analysis of fluorophores reporting from subcellular structures. Three major users have experience with two- photon microscopy and have documented that two-photon microscopes based on the BioRad scanner provide a significant advance beyond confocal microscopy in addressing their experimental needs. A fourth major user has no direct experience with the two-photon microscope, but the benefit of two-photon microscopy to his project is documented by two-photon work in other laboratories with the same preparation and fluorescent indicators. Two-photon microscopy needs of our users can not be met by equipment at our institution, because there is no two-photon microscope at the entire Johns Hopkins University, and confocal microscopy is inadequate to address the questions of the users. The two-photon microscope would be installed in an existing centralized core facility. The application is supported by a broad base of interest, need, and financial support at the University, and will serve a large NIH-funded scientific community.
