Probing the influence of low-level laser therapy on cellular and standard proteins by light emitting diode -induced autofluorescence spectroscopy

Photobiomodulation (PBM) is widely reported to influence cellular function through light-induced biochemical signaling; however, the contribution of direct protein–photon interactions to PBM-associated optical responses remains insufficiently understood. The current study investigated whether PBM-relevant wavelengths can modulate intrinsic protein autofluorescence independent of cellular photoreceptors. Purified human serum albumin (HSA) and fibrinogen were irradiated with 632.8 nm and 830 nm lasers using fluences selected to preserve protein photostability. Autofluorescence spectroscopy was performed under ultraviolet excitation to assess wavelength- and dose-dependent changes in intrinsic fluorophore emission. In parallel, SH-SY5Y cells were irradiated under comparable conditions, followed by protein extraction, SDS-PAGE, and fluorescence fingerprinting. Both purified proteins exhibited significant modulation of autofluorescence intensity without detectable spectral shifts, indicating preservation of the local fluorophore environment and absence of gross conformational denaturation. HSA displayed a dose-dependent biphasic response characterized by fluorescence enhancement at lower fluences and attenuation at higher doses, whereas fibrinogen showed predominantly fluorescence quenching, with a partial biphasic trend observed only under near-infrared irradiation. In contrast, cellular protein extracts demonstrated a largely monotonic decrease in autofluorescence intensity with increasing irradiation dose, despite preserved electrophoretic profiles. These findings demonstrate that PBM wavelengths can directly influence intrinsic protein photophysics in a protein-specific manner, independent of cellular signaling pathways. While such direct light–protein interactions may contribute to baseline optical changes observed during PBM, the distinct responses observed in cellular systems highlight the critical role of cellular organization in shaping functional PBM outcomes. This work provides experimental evidence supporting a protein-level component in PBM-associated optical responses.