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Nonlinear nano-optics of a single cell

单细胞的非线性纳米光学

基本信息

批准号：
14205013
负责人：
FUJITA Katsumasa
金额：
$ 26.54万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

在这个研究项目中，我们开发了测量光与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，会产生二次谐波和相干反斯托克斯拉曼散射等非线性光学散射。散射光的波长和强度带来了分子种类和蛋白质取向的信息。我们研制了二次谐波显微镜和汽车显微镜，对活细胞进行了观察，并利用二次谐波显微镜对大鼠心肌细胞进行了观察，获得了心肌细胞的二次谐波图像。检测到肌球蛋白丝特异性SHG。心肌细胞的SHG图像显示肌球蛋白丝在细胞内的分布。SHG成像不需要细胞的预处理，例如染料标记。用二次谐波显微镜观察HeLa细胞。只有在有丝分裂期间，微管蛋白才能产生强的二次谐波光。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，从而产生了有效的二次谐波。由于二次谐波的脉冲宽度与激发光的脉冲宽度几乎相同，因此同步检测二次谐波可以提高二次谐波图像的信噪比。将两束激光的频率差调节到脂质分子的振动，得到了脂质分子在细胞中的分布。我们用SHG和汽车显微镜观察了心肌细胞，得到了肌球蛋白和脂质分子的分布，这些结果表明，非线性光学效应可以应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。