A group of six major NIH-funded users requests funds for a spinning-microlens array laser-scanning confocal microscope. The Perkin Elmer Ultraview system requested includes a broad selection of excitation laser lines, including a special-purpose 442-nm HeCad laser for specific excitation of CFP fusion proteins and CFP/YFP ratio imaging, which is not available on an other system at UCHC. In addition, the spinning microlens array allows high-frame-rate acquisition using the full resolution of a high quantum-yield cooled CCD camera. This excitation/collection strategy has been shown to result in reduced photobleaching and photodamage in imaged samples when compared to point-scanned galvanometer confocal systems, probably because of sub-saturating instantaneous excitation and the higher signal-to-noise of the CCD detector. An additional computer-controlled dye-tunable photobleaching laser system is included for high-time/space-resolution studies of fluorescence recovery after photobleaching (FRAP), as well as dequenching measurements of fluorescence resonance energy transfer (FRET). The instrument will be housed in the Center for Biomedical Imaging Technology (CBIT) at the UConn Health Center, which offers use of high-level fluorescence microscopy instrumentation and image analysis resources to the research community both at UCHC and outside institutions, through a well-established user facility. The six major users have well-developed projects that each require some aspects that are unique the to requested system and unavailable elsewhere at UCHC. William Mohler will record high-speed 4D data and perform FRAP analysis to understand the mechanism of cell fusion in C. elegans. Vladimir Rodionov will use fluorescent speckle imaging study the cytoskeletal basis for self-centering of the centrosome. Elizabeth Eipper will study the dynamics of peptide vesicle secretion via high-speed confocal imaging and FRAP. Dianqing Wu will study the interactions of Wnt-signalling molecules in vivo via FRET. Steven Pfeiffer will study the dynamic transport of myelin proteins and their localization to lipid rafts via FRET. Bruce Mayer will visualize actin cytoskeletal changes induced by aggregation of signaling oroteins, as well as dvnamic colocalization and orotein interaction bv FRET in vivo.
|Ditlev, Jonathon A; Michalski, Paul J; Huber, Greg et al. (2012) Stoichiometry of Nck-dependent actin polymerization in living cells. J Cell Biol 197:643-58|
|Sobota, Jacqueline A; Mohler, William A; Cowan, Ann E et al. (2010) Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation. BMC Neurosci 11:32|