PROJECT SUMMARY Advances in macrophage biology have revealed that metabolic pathways play key roles to control their activation phenotype and effector function during host defense and tissue homeostasis. In vitro studies led to fundamental insights into immunometabolism, however our understanding of the functional relevance of metabolic changes in macrophages within native interstitial tissues remains limited. Here, we propose that fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD is a powerful imaging-based tool to probe the temporal and spatial changes in intracellular metabolism in situ in a live organism. Imaging-based approaches allow for maintaining highly plastic macrophages in their native microenvironment and examine their intracellular metabolism in physiological and clinically relevant contexts. Zebrafish is an established in vivo model of immunity and inflammation, with high similarity to the human immune system and genome. Due to the optical clarity at larval stage it readily combines with most imaging modalities. Clinical treatment and management of cutaneous wounds caused by thermal injury is difficult, and patients are at high risk to encounter further complications due to common nosocomial pathogens, such as Pseudomonas aeruginosa. Here, we will employ FLIM to study the metabolic regulation of activation and function of macrophages responding to Pseudomonas aeruginosa-infected burn wounds, using larval zebrafish as our in vivo system. In Aim I, we will develop tools to assess the spatial and temporal changes in macrophage metabolism in situ in a live organism. In Aim II, we will investigate the mechanisms Pseudomonas aeruginosa employs, such as recently identified microbial oxylipins, to modulate macrophage inflammation during host defense at damaged tissues, and impact overall wound healing. In vitro studies in immunometabolism support the prospects of modulating metabolic pathways for therapeutic benefits. However, the therapeutic potential remains unclear without understanding how metabolism regulates immune cell function in vivo. This study will demonstrate that imaging the endogenous fluorescence of metabolic coenzymes is a valuable non-invasive and label-free approach to fill these gaps in our understanding and better inform the development of new therapies.