Cellular transformations in near-infrared light-induced apoptosis in cancer cells revealed by label-free CARS imaging.

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti-Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro-apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm2 (~58%) and 3 J/cm2 (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis-associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm2 . Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm2 ) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non-irradiated and irradiated with 10 J/cm2 . Furthermore, irradiation of the cells with the 0.3 J/cm2 dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.