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Intravital microscopy has proven to be a powerful tool for studying microvascular physiology. In this study, we propose a gas exchange system compatible with intravital microscopy that can be used to impose gas perturbations to small localized regions in skeletal muscles or other tissues that can be imaged using conventional inverted microscopes. We demonstrated the effectiveness of this system by locally manipulating oxygen concentrations in rat extensor digitorum longus muscle and measuring the resulting vascular responses. A computational model of oxygen transport was used to partially validate the localization of oxygen changes in the tissue, and oxygen saturation of red blood cells flowing through capillaries were measured as a surrogate for local tissue oxygenation. Overall, we have demonstrated that this approach can be used to study dynamic and spatial responses to local oxygen challenges to the microenvironment of skeletal muscle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sové, Milkovich, Nikolov, Holdsworth, Ellis and Fraser.)