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

Project Summary/Abstract The National Center for In-situ Tomographic Ultramicroscopy (NCITU) seeks supplemental funding to acquire dedicated access to cryo-fluorescence microscopy capabilities. Currently, NCITU shares access to a Zeiss Airyscan cryo-light microscope within the Simons Electron Microscopy Center (SEMC), but with the recent expansion of focused ion beam (FIB) microscopes at the center, dedicated instrumentation has become essential to meet growing user demand. The requested supplemental funding will support dedicated access to a Leica Stellaris 8 cryo-fluorescence confocal microscope, which offers critical advanced capabilities beyond what is available through our current shared instrumentation. The Leica Stellaris 8 system provides superior flexibility in fluorophore selection and allows optimized detection thanks to the ability to measure the full spectra. This capability becomes crucial when imaging weak markers in vitrified samples, and it allows complete freedom in selecting markers appropriate for their specific biological questions. Notably, the Stellaris 8 can measure fluorescence lifetime, enabling sophisticated multicolor imaging, which reaches up to 11 simultaneous colors on this platform at room temperature. This expanded multicolor capability will open the possibility of investigating complex processes by simultaneously tracking multiple subcellular markers with precise spatial and temporal correlation. This enhanced cryo-fluorescence microscopy capability will directly support NCITU's mission of providing cutting-edge in-situ structural biology services and training to researchers nationwide. With established infrastructure, experienced staff, and a growing user base across 16 states, NCITU is ideally positioned to maximize the impact of this investment. The primary objectives of this supplement are to ensure NIH-funded researchers have access to the most advanced correlative microscopy techniques and to eliminate scheduling bottlenecks that currently limit throughput. This expansion will accelerate biomedical discoveries across multiple fields by enabling researchers to precisely correlate multiple cellular components in space and time before visualization by cryo-electron tomography.