The development of Total Internal Reflection Fluorescence Microscopy, or TIRFM has been a tremendous advance in the study of dynamics events near the cell membrane. The main advantage of TIRFM is the unrivaled axial localization (compared to other types of fluorescence microscopes), whereby only fluorophores within a few hundreds of nanometers of the coverslip-water interface are excited and visualized. We propose to design, build, test and characterize a novel fluorescence microscope that will provide increased spatial-temporal information about the molecular function and regulation of phosphoinositide- anchored protein complexes. This novel microscope system will combine Total Internal Reflection Fluorescence (TIRF) microscopy with Epi-fluorescence and Structured Illumination Microscopy, thus T-E-S- M. TIRF will be used to acquire highly localized (depth <500 nm) information about membrane-associated (plasma membrane, pits, endosomes, vesicles) fluorescent molecules. TIRF provides diffraction-limited lateral resolution, with depth (axial) information encoded in the fluorescence intensities. Structured-light epi- illumination will be used to acquire and compute optical-sections with axial resolution (-500-700 nm) covering the same spatial domain of the TIRF. The computed optical section images will be provide depth- independent intensity information on these molecules without contamination from out-of-focus fluorescence. The relative axial position of these membrane-associated molecules is computed from the exponential relationship of the two sources of intensity information. Computer simulations of TIRF imaging and wide-field, epi-fluorescence imaging of GFP-fusion molecules (such as clathrin, EEA1, GLUT4, Rabs) and tagged ligands (transferrin, EGF), demonstrate that the axial localizations of 10 nm are achievable with known signal levels and system characteristics. The TESM will be able to follow molecular dynamics at speeds of as fast as 25 Hz. The 3-D localization information that will be available from the TESM will provide superior data for image analysis of vesicle mobility, fusion, fission, of the co-localization of vesicles, receptors and other molecules, and vesicles with the different components of the cytoskeleton, and where in the TIRF domain these interaction occur.
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