The current request is for a Nikon AX Confocal microscope, which is needed in order to complete the aims of the parent R35. The goal of the current R35 proposal is to understand the underlying pathogenesis of necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal disease in premature infants, and to develop novel treatments for this devastating disease. The typical patient with NEC is a premature infant who rapidly progresses from mild feeding intolerance to systemic sepsis and then death within 24 hours. NEC disproportionately affects communities of color, and half of all patients will require laparotomy, which reveals patchy intestinal inflammation and necrosis. There is no specific therapy for NEC, increasing the urgency for the development of novel therapies for this devastating disease. In studies in both mice and humans, the PI has discovered that the receptor for gram negative bacterial lipopolysaccharide, namely toll-like receptor 4 (TLR4), on the intestinal epithelium, is required for NEC development. We will now extend these studies by identifying four key unanswered questions in the field: 1. What causes NEC, and why is NEC a patchy disease in the intestine? 2. How can we prevent NEC? 3. Can we predict NEC earlier? 4. What causes the long term complications of NEC on the lung, brain and gut? The requested equipment (Nikon AX Confocal microscope) will replace our existing microscope (Nikon A1 Ti Eclipse) which is over 10 years old, has reached the end of its lifespan, and is beset by frequent breakdowns. The existing confocal is also well beyond its allowable service contract, and given its age, replacement parts are very difficult to purchase. These limitations were not anticipated at the time of the initial proposal, and the Nikon AX was not readily available to demo. We have come to realize that a replacement instrument is critically needed for completion of the parent award. We have had the opportunity to demo the new Nikon AX in the lab, and were impressed by its additional capabilities, which include [1] twice the field of view (FOV), which is especially useful for the capture of live cells and tissue organoids to avoid photobleaching (critical for Aims 1-3), and [2] significantly faster image acquisition time, which is especially useful for brain sections and critical for Aim 4. These enhanced features will allow for cleaner data due to less photobleaching, and improved productivity due to increased capture time (from 6h to 30 min for a whole neonatal mouse brain scan). We will use existing funds to cover ongoing costs associated with maintenance and training, and purchase costs not covered by this administrative award. Our Nikon sales team indicates that it takes 4-6 weeks for delivery and assembly, and that we will have a system installed and fully operational within a week of delivery.