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Accurate imaging of ischemic penumbra is crucial for improving the management of acute stroke patients. T2* magnetic resonance imaging (MRI) combined with a T2*oxygen challenge (T2*OC) is being developed to detect penumbra based on changes in blood deoxyhemoglobin. Using 100% O2, T2*OC-defined penumbra exhibits ongoing glucose metabolism and tissue recovery on reperfusion. However, potential limitations in translating this technique include a sinus artefact in human scans with delivery of 100% OC and relatively small signal changes. Here we investigate whether an oxygen-carrying perfluorocarbon (PFC) emulsion can enhance the sensitivity of the technique, enabling penumbra detection with lower levels of inspired oxygen. Stroke was induced in male Sprague-Dawley rats (n=17) with ischemic injury and perfusion deficit determined by diffusion and perfusion MRI, respectively. T2* signal change was measured in regions of interest (ROIs) located within ischemic core, T2*OC-defined penumbra and equivalent contralateral areas during 40% O2+/-prior PFC injection. Region of interest analyses between groups showed that PFC significantly enhanced the T2* response to 40% O2 in T2*-defined penumbra (mean increase of 10.6+/-2.3% compared to 5.6+/-1.5% with 40% O2, P<0.001). This enhancement was specific to the penumbra ROI. Perfluorocarbon emulsions therefore enhances the translational potential of the T2*OC technique for identifying penumbra in acute stroke patients.