Simultaneous NAD(P)H and FAD Fluorescence Lifetime Microscopy reveals long UVA–induced metabolic stress in reconstructed human skin

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess overtime the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of the two endogenous biomarkers nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)⁺/NAD(P)H and FAD/FADH2 redox ratios We measure NAD(P)H and FAD fluorescence lifetime and fraction of bound coenzymes both in keratinocytes in the epidermis basal layer and in fibroblasts in the dermis superficial layer. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to OXPHOS or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamics and sensitivities to skin cell types and UVA1 dose. While FAD biomarker is UVA1 dose-dependent in keratinocytes, NAD(P)H biomarker shows earlier time points modulation in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter MCE prevents UVA1 metabolic stress response from occurring.