Nonlinear nano-optics of a single cell

在这个研究项目中，我们开发了测量光和组织细胞的生物分子之间的非线性相互作用的技术。近红外超短脉冲激光照射生物样品时，产生二次谐波(SHG)和相干反斯托克斯拉曼散射(CARS)等非线性光学散射。散射光的波长和强度提供了蛋白质分子种类和取向的信息。我们研制了倍频显微镜和CARS显微镜，对活细胞进行了观察，利用倍频显微镜对大鼠心肌细胞进行了观察，得到了细胞的倍频图像。检测到肌球蛋白细丝特异的SHG。心肌细胞的倍频图像显示肌球蛋白细丝在细胞内的分布。倍频成像不需要细胞的前处理，如染料标记。用倍频显微镜观察HeLa细胞。只有在有丝分裂过程中，微管蛋白才会产生强烈的二次谐波光。我们认为这是因为在有丝分裂过程中产生的致密微管蛋白具有很强的各向异性，从而提供了有效的SHG。我们开发了时间门控成像系统。由于倍频光与激发光具有几乎相同的脉宽，同步检测倍频光提高了倍频图像的S/信噪比。通过调节两束入射激光的频差对脂分子的振动进行调节，得到了细胞内脂分子的分布。利用倍频显微镜和CARS显微镜对心肌细胞进行了观察，得到了肌球蛋白和脂肪分子的分布，表明非线性光学效应可以用于生物分子的直接检测和生物样品的显微观察，而不需要常规的染料标记技术。

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.