Collagen-Coating Modulates Femtosecond Laser-Induced Autofluorescence and Morphological Changes in Human Fibroblasts

This follow-up in vitro study aimed to determine how collagen-coated substrates modulate the response of human fibroblasts to femtosecond laser irradiation-using our previously published uncoated-glass data as control-with particular focus on cell viability, morphology, and autofluorescence of metabolic cofactors. Human fibroblasts cultured on collagen-coated glass plates were exposed to an 800 nm, 90 fs laser (320 mW average power, 0.07 cm² spot) for 5, 20, or 100 s, delivering radiant exposures of 22.6, 90.6, and 452.9 J/cm² (photon densities 6.4 × 10¹⁸, 2.6 × 10¹⁹, and 1.3 × 10²⁰ photons/cm²), respectively. Cell viability, morphology, and autofluorescence were assessed by laser-scanning microscopy at 0, 1, 25, and 45 h post-irradiation. Compared to uncoated glass, collagen-coated substrates showed markedly accelerated and more severe damage, particularly after 100 s exposure, including extensive cellular swelling, cytoplasmic granularity, and pyknotic nuclei. Autofluorescence intensity increased dramatically on collagen-coated surfaces, with spectral signatures consistent with elevated contributions from endogenous flavins, lipopigments, and porphyrins. These findings demonstrate that the presence of a collagen extracellular matrix substantially enhances fibroblast susceptibility and metabolic stress responses to femtosecond laser irradiation, highlighting a critical role of the substrate in ultrafast laser-cell interactions relevant to laser-based therapeutics, tissue remodeling, and wound healing.