Nonlinear nano-optics of a single cell

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In this research project, we developed techniques to measure nonlinear interaction between light and biomolecules that organizes cells. When near-infrared ultrashort pulsed laser are irradiated on biological specimen, nonlinear optical scatterings such as second-harmonic generation(SHG) and coherent anti-Stokes Raman scattering(CARS) are generated. The wavelength and intensity of the scattered light bring information of molecular species and orientation of proteins. We developed an SHG microscope and a CARS microscope and observed living cells.Using SHG microscope, we observed rat cardiomyocytes and obtained SHG image of the cells. SHG specific to myosin filament was detected. SHG images of the cardiomyocytes show distribution of myosin filament in the cell. No pre-treatment of the cell such as dye labeling is needed for SHG imaging. we also observed HeLa cells using SHG microscope. Strong second-harmonic light is generated from tubulin only during mitosis. We think this is because dense tubulin that is generated during mitosis has strong anisotropy and gives efficient SHG.We developed time-gated imaging system. Since SHG have nearly identical pulse width as excitation laser light, synchronous detection of SHG light improves the S/N ratio of SHG images.We observed HeLa cells with a CARS microscope. Tuning the frequency difference of two incident lasers to vibration of lipid molecules, we obtained distribution of the lipid molecules in the cell. We observed cardiomyocytes with SHG and CARS microscope and obtained distribution of myosin and lipid molecules.From these results, it appears that nonlinear optical effect can be applied to direct detection of biomolecules and microscopic observation of biological samples without conventional dye labeling technique.