Photobiomodulation Effects on Cellular Metabolism: An In Vitro Study Using NAD(P)H and FAD Fluorescence Lifetime Imaging

Photobiomodulation (PBM) therapy, a minimally-invasive treatment based on the application of red and/or near-infrared light at sub-thermal irradiances, presents a promising potential for modulating cellular metabolismin various clinical applications. Unfortunately, its precise mechanisms remain incompletely characterized and understood. This thesis presents the first application of dual-cofactor Nicotinamide Adenine Dinucleotide-Phosphate (NAD(P)H) and Flavin Adenine Dinucleotide (FAD) Fluorescence Lifetime ImagingMicroscopy (FLIM) to investigate PBMeffects, providing novel insights into cellular metabolic responses to PBM. Following specific modifications of a commercially-available FLIM setup and on the basis of historical works performed by our group, Human Umbilical Vein Endothelial Cells (HUVECs) were exposed to 808 nmlaser light (15 mW/cm², 180 seconds, 2.7 J/cm²) at different confluency levels (50 % and 90 %), and analyzed at immediate (0 h) or delayed (18 h) timepoints. Through an integrated analytical approach developed in the context of this work, combining wide-field measurements with high-resolution single-cell imaging, this study revealed consistent shifts toward a more oxidized cellular redox state of the metabolic cofactors NAD(P)H and FAD following PBM treatments, with significantly stronger effects in the high-confluency condition (effect sizes (d) of 0.84 and 0.63 for the optical redox ratio (ORR)). In addition, "high-resolution" subcellular analysis uncovered a biphasic, confluency-dependent mitochondrial network response. Interestingly, initial mitochondria fragmentations were observed across all conditions, followed by robust mitochondrial biogenesis in sub-confluent cultures (117.8 % increase in network length, p