Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM

Macrophages (M{Phi}s) are important immune effector cells that promote (M1 M{Phi}s) or inhibit (M2 M{Phi}s) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, is not fully understood because of the lack of non-invasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free non-invasive method, can visualize M{Phi}s in human dermis in vivo. We demonstrate in vitro that human dermal M{Phi}s exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized M{Phi}s, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 M{Phi}s with a sensitivity of 0.88{+/-}0.04 and 0.82{+/-}0.03 and a specificity of 0.89{+/-}0.03 and 0.90{+/-}0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of M{Phi}s in health and disease.