Summary The primary goal of the National Center for In-situ Tomographic Ultramicroscopy (NCITU) is to provide user access and service towards preparing specimens for direct visualization of macromolecular associations and organization, particularly in native cells. NCITU is 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. A highly qualified technical team provides 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. As NCITU develops, it 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. This is a request for supplemental funding to purchase instrumentation that will expand and enhance the capabilities of the center. We are requesting: (i) a correlative fluorescence light microscope integrated into the FIB/SEM; (ii) a modern high pressure freezer; and (iii) a dedicated cryo fluorescence confocal light microscope.