Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM

Macrophages (ΜΦs) are important immune effector cells that promote (M1
ΜΦs) or inhibit (M2 ΜΦs) inflammation and are involved in numerous
physiological and pathogenic immune responses. Their precise role and
relevance, however, are not fully understood for 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 ΜΦs in the human dermis in vivo. We demonstrate in vitro that
human dermal ΜΦs exhibit specific TPE-FLIM properties that distinguish
them from the main components of the extracellular matrix and other dermal
cells. We visualized ΜΦs, their phenotypes and phagocytosis in the skin of
healthy individuals in vivo using TPE-FLIM. Additionally, machine learning
identified M1 and M2 MФs 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 MФs in
health and disease.