A diverse user-group of 8 investigators, encompassing one research institute and two departments from two Schools at The Johns Hopkins University, request funds to purchase a Zeiss LSM 510 Meta confocal microscope to replace a heavily used, antiquated Leica TCS-NT, which has been the primary workhorse of the integrated Imaging Center (IIC). This state-of-the-art Zeiss confocal system will replace and constitute a significant upgrade from our present Leica system in terms of capabilities and, most importantly, reliability. IIC investigators, funded primarily through the NIH, work on a host of cell biological/bioengineering related questions including, though not limited to, chromatin structure and gene expression; membrane organization and dynamics; microtubule-based motors; membrane trafficking; germ cell development and migration; sensory neuron development; and mechanisms of endocytosis. In the requested configuration, the LSM 510-META will afford our investigators the capability to precisely image multiple fluorophores simultaneously, and the ability to accurately modulate laser power to minimize phototoxicity and photobleaching. Additionally, our investigators variously require the advanced live cell imaging techniques of FLIP, FRET, FRAP, sequential/multi-track scanning, photo-activation (un-caging) of GFP, and 4D imaging. These capabilities required by our investigators are all absent or deficient on our current Leica TCS-NT. Of greater import in this submission has been the consistent unreliability of our current Leica system; and the inability of Leica-Service to maintain our system in competent working condition. Though maintained under service contract since the instrument was acquired in 1998, it has been non-functional 25% of its useful life. Moreover, even when operable, the system performed less than optimally nearly 40% of the time due to ongoing and unresolvable laser/pinhole/ objective problems. Consequently, given the central importance of confocal microscopy to IIC investigators, and escalating concerns pertaining to reliability and performance limitations we proactively undertook the lease/rental of an LSM 510 META in order 1) to ensure reliable access to confocal microscopy for our users thus supporting NIH funded research; and, 2) in anticipation of this grant application, have the instrument readily available for evaluation as to performance and suitability in our research. Importantly, renting the system did not obligate its purchase; but rather allowed for thorough evaluation of the LSM 510-META 'side-by-side' against other competitive instruments available in the Baltimore area, including the Leica TCS-SP2 and the Perkin-EImer Ultraview. In the evaluation of our user-group, the LSM-510 Meta is clearly the best in terms of versatility, capability, and suitability for the proposed research.
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