Funds are requested for a multiphoton microscope (MPM) to be located in the Imaging Core Laboratory of The J. David Gladstone Institutes (JDGI) at the San Francisco General Hospital (SFGH) campus of the University of California, San Francisco (UCSF). We request a MPM because many NIH-funded investigators at the JDGI and at UCSF need to image deep structures in living tissue and cannot be accommodated on existing MPMs in the area due to logistical or experimental limitations. The projects of the 10 major users and 5 minor users in this application will occupy 80% of the available time on the instrument. The Imaging Core Laboratory has a single-photon confocal microscope (Bio-Rad MRC-600), but it is overused and it fails to produce quality images of deep structures in living tissue owing to light scattering. Based on our own preliminary data and performance comparisons, we propose a Zeiss LSM 510 model with a Meta Detector and a Coherent Chameleon laser for multiphoton illumination. The Zeiss system was easier for us to use than the Leica system and that it performed better than a system by Bio-Rad. The Meta Detector permitted us to simultaneously discriminate multiple variants of green fluorescent protein - common application of our major users. The Chameleon laser was much easier to operate than """"""""2-box models"""""""" models and so it should be better utilized in our multi-user facility. We preferred the longevity of the diode construction of the Coherent laser to the Mai Tai model by Spectraphysics. The PI, who has extensive imaging experience, will oversee the set up and management of the instrument. An advisory council will be formed to set policy, to resolve disputes, and to monitor the effectiveness of our management. The council will include the head of a multiuser imaging facility at UCSF and the head of the multi-user flow cytometry facility at the JDGI. Day-to-day management, operation, and training will be carried out in an established, well-staffed imaging core facility. The Core uses a recharge business model and has been providing histology and microscopy services for 15 years. In addition, we will use a three-tier MPM training program implemented at a UCSF multi-user core imaging facility. The JDGI has strongly supported the effort to obtain this microscope by providing substantial additional funds needed for its initial purchase and by providing the infrastructure and ongoing support needed for its continued operation. The NIH will benefit because the microscope will significantly expand the abilities of the major users, enabling them to achieve funded aims and to perform previously unfeasible experiments.
