Multiphoton Microscopy Targeting Tissue Development. Towards the real-time imaging of epidermal differentiation

Abstract

The development of novel organ-on-a-chip models calls for methodologies, which will allow the study of tissue development and pharmacological outcome in real-time. Multiphoton microscopy (MPM) has been proposed as a suitable technique for noninvasive imaging of live specimens, utilizing intrinsic tissue fluorescence. In combination with fluorescence lifetime imaging (FLIM), the fluorescence intensity at each pixel can be complemented with the fluorescence lifetime of the fluorophore, possibly enabling fluorophore differentiation. In this work, the potential of MPM for longitudinal imaging of tissue development was explored in the context of epidermal differentiation. In Paper I, it was shown that MPM can be used for the recognition of epidermal morphology in reconstructed human epidermis (RHE), utilizing the intensity of intrinsic tissue fluorescence based on two-photon excitation. MPM-FLIM has been previously used for the recognition of cellular metabolism based on the fluorescence of metabolic co-enzymes, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). However, other cellular components, such as keratin, might exhibit intrinsic fluorescence, leading to spectral crosstalk. Therefore, the cellular system required a detailed characterization considering the intrinsic fluorophore composition and the contribution of NADH, FAD, and keratin to the recorded lifetime, as demonstrated in Paper II. After the characterization, a metabolic imaging utilizing MPM-FLIM was undertaken on a simpler 2D differentiation model, combined with the biochemical validation of the studied system with respect to the differentiation, presented in Paper III. While the increase in the differentiation markers and shift from glycolysis to oxidative phosphorylation were confirmed in the model, MPM-FLIM data were rendered inconclusive. In the final part of the thesis, MPM-FLIM was combined with an on-stage incubation system for the studies of tissue development in real-time. Taken together, this work presents MPM as a tool for real-time imaging of epidermal differentiation. While the MPM-FLIM studies have provided additional spectroscopic information, the presented work suggests that the MPM-FLIM data interpretation with respect to the metabolism may be more challenging than anticipated, and that biochemical validation of the FLIM findings is of utmost importance.