Multiphoton imaging coupled with steady-state and time-resolved fluorescence spectroscopy reveals protein-bound FAD in femtosecond-laser-injured regenerating muscle.

Regeneration of skeletal muscle involves tightly coupled structural and redox processes that remain difficult to characterize in vivo or in situ with molecular specificity. Here, we introduce a label-free spectroscopic imaging framework that integrates multiphoton imaging-including second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF)-with steady-state and time-resolved fluorescence spectroscopy to visualize sarcomere remodeling and redox dynamics in living zebrafish muscle. SHG imaging revealed a transient disruption and gradual restoration of sarcomeric order, indicating an asynchronous, sequential reassembly of myofibrillar structures following localized femtosecond laser ablation. In parallel, TPEF imaging detected a pronounced and spatially confined increase in intrinsic emission at the injury site. Single-photon fluorescence spectroscopy and lifetime analysis identified this emission as protein-bound, oxidized flavin adenine dinucleotide (FAD), matching the photophysical signature of mitochondrial flavoproteins and revealing localized redox activation during the early phase of repair. Complementary reactive‑oxygen-species (ROS) imaging confirmed localized oxidative stress associated with elevated electron-transport activity, linking mitochondrial metabolism to structural recovery. Together, these multimodal observations establish a quantitative, label-free framework for correlating mitochondrial FAD-mediated redox activity with sarcomeric remodeling, establishing a versatile optical platform for investigating flavin photophysics, oxidative metabolism, and regenerative physiology in living muscle tissues.