We propose to purchase a Leica TCS SP2 AOBS laser scanning confocal microscope equipped with special 405 nm diode and 594 nm orange HeNe lasers to be housed in our existing light microscopy Imaging Facility. This facility was used by 77 different researchers in calendar year 2002, including 56 using our fiveyear- old Leica SP1 UV confocal. The growth and increasing sophistication of our users have revealed two significant shortcomings of the existing microscopes. First is the inability to perform key experiments, including 1) fluorescence recovery after photobleaching (FRAP), 2) photo-activation of fluorophores in defined regions of interest, and 3) time-resolved imaging of more than one component at rates faster than one two-component image every 1-2 seconds. Second is a debilitating lack of sensitivity from our existing SP1 confocal for even moderately bright samples. This leads many experiments better suited for a confocal to be done on our deconvolution microscope, despite the optical flaws, simply to get adequate sensitivity. A straightforward way of addressing all of these concerns is the replacement of our existing confocal with one of contemporary design. Of contemporary confocals, the Leica TCS SP2 AOBS and Zeiss LSM 510 META are best suited to meet the needs of our most sophisticated users while still allowing a multi-user facility. We have selected the Leica TCS SP2 AOBS because 1) the Leica can meet our experimental needs and has superior sensitivity as evidenced during demos by Leica and Zeiss; 2) the superior Leica """"""""filter-free"""""""" optical design; 3) the continuity of user interface for our large user group, making extensive retraining unnecessary; 4) continuity of our strong relationship with our Leica field service engineer, and 5) the trade in value of our existing Leica SP1. Major NIH-funded users will be Jodi M. Nunnari (""""""""Regulation of Mitochondrial Fission""""""""), Jonathon M. Scholey (""""""""Roles of Microtubule-Based Motility in Mitosis and Intracellular Transport""""""""), and Carol A. Erickson (""""""""Direct Observation of Neural Crest Cell Emigration in a Living Embryo""""""""). These users will account for approximately 50% of the instrument time, with the balance being offered to our large imaging community. This use will continue to be under the direction of one PhD and one MS scientist and administered under the current policies of an existing departmental Imaging Committee. A letter showing strong institutional support for maintenance and continued operation of the confocal is included.
