Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermisin vivousing two-photon excited FLIM

Abstract

Macrophages (MΦs) are important immune effector cells that promote (M1 MΦs) or inhibit (M2 MΦs) 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 MΦs in human dermis in vivo. We demonstrate in vitro that human dermal MΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized MΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MΦs 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 MΦs in health and disease.

Tattoo Pigments Are Localized Intracellularly in the Epidermis and Dermis of Fresh and Old Tattoos: In vivo Study Using Two-Photon Excited Fluorescence Lifetime Imaging

“…5e–h), characterized by the short fluorescence lifetime and in the TPE-AF images by their intense TPE-AF intensity, as presented in Figure 5 as bright fluorescent green areas inside the fibroblasts. Identification of dermal cells by their morphology, location, and the specific TPE-FLIM parameters was described elsewhere by our group [48, 49].…”

“…In vivo identification and classification of mast cells [49] and macrophages [48] were shown by our workgroup using TPE-FLIM, compared in online supplementary Table S2 [48]. Fibroblasts can be identified by morphologic features, highly dendritic features (online suppl.…”

“…The TPE-FLIM signatures of the ECM and dermal cells [48, 49], shown in online supplementary Table S2, are not superimposed by fluorescence lifetime of carbon black nanoparticles excited at 760 nm. Carbon black particles can be separated from melanin-containing melanosomes by the granular-like inhomogeneous appearance in the FLIM image (Fig.…”

“…Two-photon excited fluorescence lifetime imaging (TPE-FLIM) [4,45] is superior in selectiveness and reproducibility compared to single-photon methods [46,47]. The TPE-FLIM method is capable of noninvasively separating dermal components as recently shown by our group for M1 and M2 macrophages [48], resting and activated mast cells [49], and the main extracellular matrix (ECM) proteins and capillaries of the papillary dermis [49][50][51] in vivo. Being a non-invasive imaging method, TPE-FLIM enables ethically unproblematic investigations of tattoos on different skin sites.…”

Non-Invasive classification of macrophage polarisation by 2P-FLIM and machine learning

“…Most often macrophage behaviour is assessed using end-point assays using cytokine measurements, gene analysis and staining of surface markers but there is a gathering shift towards non-invasive modalities to speed up this process and obtain spatio-temporal analysis. Two recent examples of this include the use of raman microscopy to map the lipidomic spatial signature of polarised macrophages (Feuerer et al 2021), and the use of average fluorescent lifetime parameters to discern phenotype (Kröger et al 2021).…”