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<0.001), while persistent reduced complexity of mitochondrial network in high-density cultures were observed. Compartment-specific metabolic analysis demonstrated differential responses in cytosolic versus mitochondrial regions, resolving apparent contradictions reported in the literature regarding whole-cell measurements and subcellular energetics. A correlation analysis performed between the metabolic indexes revealed reorganization of cellular metabolism following PBM, with the high-confluency condition showing more consistent strengthening of the relationships between redox parameters. In conclusion, this research establishes a novel model of studying PBMthat integrates immediate photochemical effects with subsequent adaptive responses, while accounting for confluency effects. The strong confluency dependence of both the PBM effects on the metabolic indexes and the mitochondria response patterns highlights the critical importance of the cellular environment on the PBM outcome. These findings provide a better understanding of the PBMmechanisms from a novel angel, while provide a foundation for optimizing PBM.