Our understanding of fundamental cell biological processes is both driven by and limited by our ability to visualize the organization of structures and molecules within cells and tissues. The development of """"""""super-resolution"""""""" fluorescence imaging methods that circumvent the resolution limits of conventional light microscopy, achieving lateral resolution of d100 nm, are transforming cell biological research. Access to super-resolution fluorescence imaging technology is now essential for scientists wishing to push the frontiers of cell biological research. This proposal requests funds to purchase the OMX BLAZE 2D, 3D-SIM fast super-resolution imaging system (Applied Precision, Inc.). This super-resolution system can achieve both lateral and axial resolution at twice the diffraction limit of conventional light microscopy and is capable of multi- channel super-resolution far beyond the cover glass. The fast imaging capability of OMX BLAZE is also designed to overcome speed limitations for 3D live-cell imaging. This advanced imaging system will be a shared resource, located in a well-established, multi-user microscopy facility at Stanford: the Cell Sciences Imaging Facility. The requested OMX BLAZE 3D-SIM imaging system will support NIH funded projects from 14 researchers. These projects investigate a wide range of topics, including: meiotic chromosome segregation (Villeneuve);genetic recombination (Villeneuve);centrosome structure, function and duplication (Stearns);centromere and kinetochore assembly (Straight);biogenesis and function of the primary cilium (Nachury, Rohatgi, Stearns);architecture of neural circuits (Smith);molecular mechanisms underlying epithelial cell rearrangements during gastrulation (Nelson);actin filament assembly and dynamics (Nelson);molecular mechanisms of secretory vesicle docking and fusion (Pfeffer);development of neural synapses (Shen);mechano-electrical transduction in touch receptor neurons (Goodman);recombination and genetic engineering (Porteus);Hedgehog pathway signaling in development and cancer (Chen, Scott, Beachy, Rohatgi). These studies investigate critical functional and structural questions regarding fundamental cell biological processes and cover NIH research areas with implications for diverse aspects of human health and disease, including cancer, birth defects, obesity, kidney cysts (in the inherited Bardet-Biedl Syndrome) and neurodegenerative disease. All these projects require multi-channel super-resolution imaging and simultaneous multi-channel fast imaging;this combination of capabilities is most effectively provided by the requested OMX BLAZE 3D SIM system.
