We are requesting funds for the purchase of a Leica SP8 WLL confocal microscope. The instrument will be placed in the University of Washington's Keck Imaging Facility. The Keck Facility is a well- established, very heavily used facility providing confocal microscope instrumentation for 20 years, serving over 50 UW research laboratories in 2012. The facility now has two outdated and well-worn confocal microscopes, one of which is no longer supported by its manufacturer. The requested instrument is required to sustain microscope access for our group of 50 users and to provide expanded technical capabilities that are necessary to a group of 6 major users for advancing research projects ranging from dynamics within single cells to development of entire embryos. Thus, the proposed instrument will be required to analyze a vast diversity of specimens, in both fixed and living conditions. Significantly, the proposed instrument will make unique capabilities and badly needed additional confocal capacity available to the entire Keck user base and larger UW research community. It is to achieve these goals that we are requesting the Leica SP8 WLL confocal microscope with an inverted, DMI 6000 microscope stand. The system is specified with a white light laser (WLL) providing selection of any excitation wavelengths (up to 8 simultaneously) from 470 - 670 nm. Combined with Leica's extremely flexible spectral detection, fluorescent imaging parameters are user-customizable to a degree not currently available at the University of Washington. The system includes two hybrid (HyD) detectors for fluorescence. These new-generation detectors provide significantly increased sensitivity, wide dynamic range and low noise characteristics compared to standard detectors. A resonance scanner provides rapid imaging necessary for capturing dynamic events. A motorized stage and adaptive focus correction module are included for automated sampling and maintaining correct focus during time-lapse studies, respectively. A temperature-controlled stage insert is included for live-cell experiments. The Leica SP8 WLL was evaluated by the Keck Facility staff and major users at a recent demonstration and found to meet and exceed all of our expectations. In summary, the proposed instrument will permit users to optimize confocal imaging parameters, in ways currently impossible, to overcome the specimen's limitations rather than forcing experimental compromises necessary to overcome current instrument limitations.
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