NON LINEAR OPTICAL METHODS IN NEAR FIELD SCANNING

在这项研究计划中，双光子激发(TPE)荧光和二次谐波(SHG)的非线性光学方法将与近场扫描光学显微镜(NSOM)相结合来研究膜生理方面的问题。由于现有的膜染色染料的非线性增强和远场背景贡献的降低，这些方法将在保持亚光学分辨率的同时，提供比现有的线性激发方法更强的对比度。化学成分和物理性质对细胞功能很重要，但光学显微镜不能提供必要的分辨率来揭示这些细节。在局部分子水平上，膜电位、弹性或亲水性等性质会发生变化，这些变化可能对细胞功能有重要影响。NSOM仪器将被用来将这些特性与活细胞中的局部化学成分(例如胆固醇与脂肪的比率)相关联。荧光寿命测量和含时荧光各向异性测量将被用来探测荧光团周围的局部环境。二次谐波的产生来自激发极化而不是吸收，潜在的敏感染料将被用来产生良好的对比度，而不会出现明显的光漂白。倍频结合偏振分析将用于研究膜中荧光团的取向。倍频还将作为测量膜电位的一种替代方法。最初的实验将在模型细胞系上进行，例如3T3成纤维细胞和N1E-115未分化和分化的神经母细胞瘤细胞，但一旦投入使用，该设备将成为用于活细胞成像的通用高分辨率工具。该方案的一个关键方面将是构建腔倾倒的钛蓝宝石振荡器，以提供更高的脉冲能量，以便为倍频实验获得高信号电平。

In this research program, the non-linear optical methods of two-photon excitation (TPE) fluorescence and second harmonic generation (SHG) will be combined with near-field scanning optical microscopy (NSOM) to study aspects of membrane physiology. Due to the enhanced non-linear properties of available membrane staining dyes and decreased far-field background contributions, these approaches will provide increased contrast over existing linear excitation methods while maintaining sub- optical resolution. Chemical composition and physical properties are important in cell function but optical microscopy cannot provide the necessary resolution to uncover these details. At he local molecular level, properties such as membrane potential, elasticity, or hydrophilicity, will vary and these variations may be important in cell function. The NSOM apparatus will be used to correlate such properties to local chemical composition, e.g. cholesterol to lipid ratio, in living cells. Fluorescence lifetime measurements and time-dependent fluorescence anisotropy measurements will be used to probe the local environment around fluorophores. Second harmonic generation arises from an induced polarization rather than absorption and potential sensitive dyes will be used to generation good contrast without appreciable photobleaching. SHG combined with polarization analysis will be used study the orientation of fluorophores in membranes. SHG will also be used as an alternative method to measure membrane potential. The initial experiments will be on model cell line, e.g. 3T3 fibroblast and N1E-115 undifferentiated and differentiated neuroblastoma cells but once operational, the apparatus will be general purpose high resolution tool for live cell imaging. A key aspect of this proposal will be constructing cavity dumped titanium sapphire oscillator to provide higher pulse energy so that high signal levels can be obtained for the SHG experiments.