In vivo staining-free visualization of macrophages in human skin using two-photon tomography with fluorescence lifetime imaging
Macrophages (MФs) are immune cells that promote (M1 MФs) or inhibit (M2 MФs) inflammation and are involved in numerous physiological and pathogenic immune responses. In skin, MФs are mainly located in the dermis near blood vessels. Here, we show for the first time the possibility of non-invasive staining-free visualization of M1 and M2 MФs in human skin in vivo using two-photon-excited fluorescence lifetime imaging (TPE-FLIM). We demonstrate in vitro that human dermal ΜΦs exhibit specific TPE-FLIM properties (fluorescence lifetime τ1=225±84 ps and τ2=1,289±278 ps for M1 and τ1=807±250 ps and τ2=2,352±229 ps for M2 MФs) that distinguish them from the extracellular matrix and other dermal cells. Thus, based on determined TPE-FLIM parameters, we visualized ΜΦs, their phenotypes and phagocytosis in skin of healthy individuals in vivo. The developed machine learning algorithm identified M1 and M2 MФs with high sensitivity and specificity. The TPE-FLIM may become an important tool for non-invasive in vivo diagnostics and therapy control in dermatology that target ΜΦs and can advance the understanding of their role in health and disease.
Maxim E. Darvin