A focused group of 4 researchers at the University of Arkansas for Medical Sciences (UAMS), all affiliated with the Department of Physiology and Biophysics, request funds for a Zeiss LSM 510 laser scanning confocal microscope. The Zeiss LSM 510 provides a rapid scanning microscope capable of photoactivation/uncaging, molecular interaction studies using acceptor photobleaching FRET, multiple color live cell tracking experiments, organelle-specific photobleaching (FRAP, fluorescence recovery after photobleaching and FLIP, fluorescence loss in photobleaching) as well as excellent standard confocal microscopy and multicolor co-localization. The requested instrument has four confocal channel configurations for fluorescence and one for transmitted light (DIG), and individual adjustable pinholes for correct 3-dimensional measurements. We believe that the Zeiss LSM 510 with multiple lasers (405 nm to 633 nm) offers the best-integrated system available. The system will be operated as a satellite to the Digital and Confocal Microscope Laboratory (DCML) at UAMS and will be open for general use 1 day a week. ? ? The unique advantages of the confocal microscope are several-fold. First, optical sectioning can be performed much more rapidly with hardware (the confocal pinhole) than with a computer (digital deconvolution). This is most important for colocalization of molecules of interest with other compartment markers. Second, with the confocal microscope, acquisition of images in multiple channels is nearly simultaneous provided that the excitation and emission spectra of the probes have distinct spectra. Thus, we can follow molecules in living cells. Third and perhaps most importantly, as a laser scanning confocal microscope, laser illumination can be accurately controlled permitting localized ? photoactivation/uncaging, irregular object photobleaching/uncaging, rapid line scan, and even irregular region of interest (ROI) studies. For example, by coupling laser scanning with selective beam attenuation, irregular profiles, such as a Golgi apparatus or subregions of oocytes, can be photobleached/uncaged. A laser scanning confocal microscope with acousto-optical filters (AOTF) is more than an imaging tool. It is also an optical bench for control of laser placement. A selection of objectives (10-63x) including a water immersion objective will be fitted to the microscope. The system will be configured with 405 nm laser for planned uncaging/photoactivation studies and for FRET using CFP and YFP. A bottom port is included for future fluorescence correlation spectroscopy (FCS). Open usage of the new LSM 510 will give first preference to investigators needing the advanced capabilities of the instrument. ? ?
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