The National Center for In-situ Tomographic Ultramicroscopy (NCITU)

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Summary We propose to establish the National Center for In-situ Tomographic Ultramicroscopy (NCITU) to be based at the Simons Electron Microscopy Center (SEMC), which is a part of the New York Structural Biology Center (NYSBC), based in New York, NY. NCITU will be dedicated to developing and providing access to both standard and advanced methods for in-situ cryoET specimen preparation, and to disseminating this expertise through a cryoET cross-training program designed to serve a wide variety of skill levels and career goals. The primary goal of NCITU will be to provide user access and service towards preparing specimen for direct visualization of macromolecular associations and organization, particularly in native cells. A highly qualified technical team will provide direct hands-on service, guidance, and assistance to ensure that samples and grids are prepared with the highest quality and in the shortest time possible. An already well established cross-training program will be expanded and extended to provide training in every aspect of the cryoET specimen preparation pipeline. The operations at NCITU will build on already established practices at SEMC/NYSBC and benefit from the overall vibrant environment as well as the expertise of the highly trained staff already in place. NCITU will provide a full range of services for in-situ sample preparation for tomography including: culturing biological samples, plunge freezing and high pressure freezing, cryo-light microscopy (cryoLM), cryo- ultramicrotomy, focused ion beam milling (FIB-milling) including lift-out, and initial cryoTEM screening prior to submission of specimens to the network Hub for high-resolution cryoET data collection. We have already established workflows for many of these activities and have active research projects focused on expanding capabilities for automating and streamlining the entire workflow. We will continue to stay at the forefront of new developments in this area and we will incorporate new methods, technologies and workflows into the standard NCITU operating procedures as soon as these are stable and robust. While the goals of NCITU are not innately innovative, it is our intention to set the standard as one of the first world class, nationally accessible cryoET sample preparation facilities, to provide access and training that is freely and fairly available to all researchers in the US, and to continually strive to improve methods and enable science of the highest quality and value.