Project Summary We request funds to support the upgrade of our existing Nikon A1R confocal microscope, which is now almost 10 years old and lacks many of the newer features that have been developed since its purchase in 2012. The upgraded instrument will be housed within the Program in Membrane Biology (PMB) Microscopy Core at the Massachusetts General Hospital (MGH) in the multidisciplinary Simches Research Center building. The mandate of this MGH-approved Core facility is to provide access to a full range of microscopy techniques to 2 NIH funded P30 Centers and individual research groups within the MGH community and beyond. The Core has considerable expertise in static and live cell imaging techniques used by our extensive user-base over the years. Our imaging capabilites were enhanced 4 years ago by the acquisition of a Zeiss LSM800 Airyscan confocal platform to function alongside the older Nikon A1R. Together these instruments have provided sufficient high level imaging bandwidth to serve our users. The Nikon A1R is extensively used for longer term, live cell imaging and has an inverted microscope and an environmental chamber to faciliate this need. The LSM800, associated with an upright microscope and no environmental chamber, provides consistent and timely access to a high resolution confocal that is mostly used to perform static imaging of fixed specimens. With the increase in demand for imaging of living cells expressing various types of fluorescent molecules and tagged proteins, and the increasing need to image these cells over time using 3D reconstructions, the purpose of this application is to provide our users with access to modern confocal microscopy options that are better adapted for specialized imaging purposes than the current older A1R system. In particular, the new high speed HD resonant scanning mode with associated AI image processing capabilities provides a remarkable increase in image size and resolution when compafed to the old resonant scanning system. Coupled with replacement of the old PMT detectors with new GaAsp detectors and the incorporation of stable solid state lasers, these upgrades will allow specimens to be exposed to much lower laser intensities, reducing photobleaching and photodamage, while providing a significant increase in resolution compared to the old system. In addition, images and multicolor 3D stacks can be collected much more rapidly in resonant scanning mode, while not sacrificing quality, meaning that experiments can be conducted quickly and efficiently, saving time and money for our users. The PMB Core has supported hundreds of peer-reviewed publications and dozens of grant applications over the years. The Core has an ongoing quest to constantly improve and upgrade Core facilities in support of its user base. This has been achieved using funds from NIH SIG applications as well as considerable institutional support from MGH leadership. Providing our Core users with continued access to up-to-date c...