Project Summary/Abstract (Center Overview) From its inception as an NIDDK-funded P50 Center in 2002, the mission of the Indiana O’Brien Center for Advanced Renal Microscopy has been to provide renal researchers with effective access to advanced forms of microscopy. Over the past 20 years of continuous funding, the IU O’Brien Center has pioneered the development and dissemination of novel methods of multiphoton intravital microscopy, quantitative tissue cytometry and digital image analysis that have been applied in collaborative studies of renal physiology and pathophysiology with investigators from around the world. Studies conducted with the P30 and P50 IU O’Brien Center have generated data used in over 190 publications and numerous grant applications that have helped launch the careers of a new generation of investigators. We look forward to continuing our work within the consortium framework of the new U54 mechanism. The goals and projects proposed for the Center are organized into three general aims – (1) Provide quantitative intravital microcopy of mice and rats as a service to renal researchers. Provide hands-on training to encourage dissemination of intravital microscopy as an effective research tool. (2) Provide quantitative, large-scale microscopy of the kidney as a service to renal researchers. Three-dimensional confocal tissue cytometry, CODEX multiplexed cytometry and Spatial Transcriptomics will be offered individually or in combination, supporting uniquely comprehensive molecular characterizations. (3) Develop and implement methods of tissue clearing, light-sheet microscopy and digital image analysis to expand the scale of quantitative microscopy of renal tissues, based upon a unique multi- scale/multi-resolution strategy and state-of-the-art methods of digital image analysis. The Indiana O’Brien Center will include an Administrative Core, and two Biomedical Resource cores - an Intravital Microscopy Core, and a Molecular Imaging Core, both hosted by the Indiana Center for Biological Microscopy. Insofar as these two cores effectively extend services currently offered by the Indiana O’Brien Center, they will benefit from the existing infrastructure, expertise and experience developed over the past twenty years by the Center. The Center will also support a Resource Development Core, in collaboration with investigators at the University of Washington, University of Nebraska and Purdue University that will develop technologies that will start to be implemented at the beginning of the second year of funding.