The objective of this application is to establish a novel state-of-the-art facility termed the Cell Dynamics Microscope for the observation and manipulation of molecules within living cells using fluorescence microscopy. The envisioned workstation is centered on a Zeiss Axiovert 200M inverted microscope with two major accessories. The first is a Perkin Elmer Ultraview Live Cell Imager for imaging fluorescence in living cells with minimal photobleaching and phototoxicity. The second accessory is a Digital Diaphragm laser illumination system from Photonic Instruments. This new instrument allows intense laser illumination of any subregion, set of subregions or pattern within the field of view. Setting of subregions or patterns is controlled by drawing or importing patterns on a computer monitor image of the target. This ability permits unprecedented control of illumination for photochemical techniques including fluorescence recovery after photobleaching (FRAP), Fluorescence loss in photobleaching (FLIP), fluorescence resonance energy transfer (FRET), photoactivation of caged compounds, direct photoablation, dyesensitized photoablation (sometimes called chromophore-assisted laser inactivation or CALl), and ion imaging. The members of the major user group will apply these techniques in several areas: Microtubule and Kinetochore Dynamics in Mitosis (Gorbsky), Protein Translocation in Photoreceptor Cells (McGinnis), Microtubules in Insulin-mediated Glut4 Translocation (Olson), NF-KB Dynamics in Bladder Epithelium (Saban), and Polycystin-2 in Primary Cilia and Mitotic Spindles (Tsiokas). These projects will be supplemented by nine projects from a group of additional users. The analyses performed with this new instrument will complement the molecular studies ongoing in each laboratory. The Cell Dynamics Microscope will become a pivotal resource for the community of researchers seeking elegant and userfriendly methods to observe and manipulate target molecules within living cells. ? ?
