Mast cells (MCs) and macrophages (ΜΦs) are important multifunctional immune cells found in all tissues of the body. In the skin, resting and activated MC populations and M1- and M2-polarized ΜΦs are located in the dermis. Histomorphometric analysis of skin biopsies is used to determine the quantity of these cells and their activation and polarisation state. Non-invasive in vivo visualization of MCs and ΜΦs in the skin is currently not possible.
We show for the first time that two-photon excited fluorescence lifetime imaging (TPE-FLIM) can be used for label-free non-invasive visualization of resting and activated MCs and M1- and M2-polarized ΜΦs in human skin in vivo with high sensitivity and specificity. First, we recorded TPE-FLIM parameters (lifetime and intensity) of human MCs and ΜΦs in vitro and demonstrate that they have specific TPE-FLIM parameters that are distinct from major components of the extracellular matrix and other dermal cells. Second, we confirmed the visualization of MCs and ΜΦs in the skin biopsies ex vivo based on their known TPE-FLIM parameters and subsequent cell-specific immune staining. Finally, we found cells with previously determined TPE-FLIM parameters for resting and activated MC populations  and M1- and M2-polarized ΜΦs  in the human skin in vivo.
The developed non-invasive method can advance the understanding of the role of MCs and MФs in health and disease diagnostics and therapy monitoring in dermatology and immunology.
 Kröger, Scheffel, Nikolaev, Shirshin, Siebenhaar, Schleusener, Lademann, Maurer, Darvin. In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging. Sci Rep. 2020;10:14930.
 Kröger, Scheffel, Shirshin, Schleusener, Meinke, Lademann, Maurer, Darvin. Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM. bioRxiv. 2021;2021.11.29.470361.