As part of an established core facility, we request a Zeiss LSM780 laser-scanning confocal microscope with fluorescence correlation spectroscopy (FCS) on an inverted microscope stand. Addressing multiple live-cell/tissue studies, our needs come from three sources: oversubscription on existing laser-scanning confocal instruments, need to reduce photodamage to fluorescently labeled living cells, and need to resolve molecular-level events, particularly in living cells. Although we have three Zeiss laser- scanning confocals, they are heavily used, with peak usage 20h/day on one instrument. Photodamage and photobleaching are related to the instrument's signal-to-noise performance, and the LSM780 is outstanding in our tests, allowing techniques not possible with other confocals. To understand biological pathways, live-cell techniques become ever more important. Not only does FCS provide key molecular measurements about regulation of spatial organization within cells, it also discriminates hypotheses in signaling mechanisms in our systems. In addition, local efforts to construct new FRET biosensors and combine different biosensors into the same cells demand greater performance of instrumentation. These FRET studies are greatly facilitated by automated spectral unmixing. Ongoing live-cell studies use all these techniques, as well as FRAP, photoactivation, and basic time-lapse imaging. The proposed instrument will provide an order-of-magnitude superior performance that will enable key NIH- funded projects.
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