Multiphoton FLIM imaging of NADH and FAD to analyze cellular metabolic activity of reconstructed human skin in response to UVA light

Abstract

Human skin is constantly exposed to environmental stresses such as UV light and pollution. These agents cause oxidative stress associated with reactive oxygen species (ROS) generation, that will interfere with the normal cellular redox equilibrium. As ROS are mainly produced within mitochondria, the cellular metabolic activity could be impacted by UV light. We dynamically assessed UVA light (representing the majority of solar UV rays reaching Earth surface) effects on cellular metabolic activity of reconstructed human skin using multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton FLIM offers non-invasive, label-free quantitative functional information on cellular metabolic activity based on the endogenous two-photon excited fluorescence (2PEF) of NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavine adenine dinucleotide) metabolic coenzymes. The experiments were performed in both stratum granulosum and spinosum layers (T-Skin™ model, Episkin™), before and after (30 min and 2 h) UVA exposure (20 J/cm²; 20 min exposure; 320 – 400 nm). We observed quasi similar effects in both epidermal layers after UVA exposure: • Decrease of RedOx ratio NADH / (NADH + FAD) at 30 min and 2 h; • Increase in the proportion of protein-bound NADH at 2 h, and in the proportion of free FAD as early as 30 min after UVA exposure; This study shows that the effects of UVA light on epidermis, can be non-invasively evidenced and followed overtime using NADH/FAD multiphoton FLIM imaging method. Altogether, these data suggest that epidermal cells respond to UVA light by promoting oxidative phosphorylation, the most efficient metabolic pathway for ATP production.