Multimodal nonlinear optical microscopic imaging provides new insights into acetowhitening mechanisms in live mammalian cells without labeling.

We developed a multimodal nonlinear optical microscopy imaging (e.g., third-harmonic generation (THG) and two-photon excited fluorescence (TPEF)) platform based on a femtosecond laser pumped photonic crystal fiber to investigate the acetowhitening phenomenon induced by acetic acid in live mammalian cells without labeling. After treated by acetic acid with concentrations of higher than 0.2%, THG images show that light scattering is remarkably increased inside the nucleus and cytoplasm in cells. Co-localized TPEF and THG imaging on tryptophan and NADH in cells indicates that the change of scattering property is largely originating from the morphological change of metabolic proteins induced by acetic acids. Further TPEF imaging on NADH and FAD in cells confirms that this change is irreversible when acetic acid concentration is higher than 1.2%. These subcellular-level THG/TPEF imaging results reveal that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.