AbstractMacrophage infiltration and recruitment in breast tumors has been correlated with poor prognosis in breast cancer patients and has been linked to tumor cell dissemination. Much of our understanding comes from animal models in which macrophages are labeled by expression of an extrinsic fluorophore. However, conventional extrinsic fluorescence labeling approaches are not readily applied to human tissue and clinical use. We report a novel strategy that exploits endogenous fluorescence from the metabolic co-factors NADH and FAD with quantitation from Fluorescence Lifetime Imaging Microscopy (FLIM) as a means to non-invasively identify tumor-associated macrophages in the intact mammary tumor microenvironment. Macrophages were FAD^HI and demonstrated a glycolytic-like NADH-FLIM signature that was readily separated from the intrinsic fluorescence signature of tumor cells. This non-invasive quantitative technique provides a unique ability to discern specific cell types based upon their metabolic signatures without the use of exogenous fluorescent labels. Not only does this provide high resolution temporal and spatial views of macrophages in live animal breast cancer models, this approach can be extended to other animal disease models where macrophages are implicated and has potential for clinical applications.