Insights into metabolic changes during epidermal differentiation as revealed by multiphoton microscopy with fluorescence lifetime imaging

<p>Rapid developments in the field of organotypic cultures has generated a growing need for effective quality control measures during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied as a non-invasive approach to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. &nbsp;Keratinocytes grown at 1.5 mM Ca2+ exhibited increased expression of differentiation markers KRT1 and KRT10 compared to 60 &mu;M Ca2+, and a metabolic shift from glycolysis to mitochondrial respiration. Fitting the fluorescence decay with a biexponential model revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Using these two parameters, the epidermal differentiation process could be monitored over a 96 h period. Implementing discriminating analysis based on k-means clustering generated clusters that correlated well with culturing time, suggesting that this methodology can be employed as part of an automated pipeline for monitoring keratinocyte differentiation.</p>