双光子激发时间分辨荧光光谱测量技术

Time-resolved two-photon excitation fluorescence spectroscopy

建立了一台基于高重复频率扫描相机的双光子激发时间分辨荧光光谱测量系统,能够同时测量样品的荧光光谱和寿命.该系统的时间分辨率为6·5—200ps,光谱分辨率为1—3nm,能够实现快速数据采集以及可靠和可重复的寿命和光谱测量.利用标准荧光染料(若丹明6G、香豆素314)及其混合溶液对该系统进行了测试,所得到的荧光光谱分布和寿命值与文献报道一致.实验结果表明,该系统能有效区分多组分荧光团.这为鉴别多荧光团或多组分生物组织提供了一种独特的对比方法,可用于多光谱分辨荧光寿命成像和荧光共振能量转移成像等方面.

A novel time-resolved two-photon excitation fluorescence spectroscopic system based on a high repetition rate streak camera and capable of simultaneous measurement of fluorescence spectrum and lifetime of the samples is presented. The system has high temporal （6.5-200 ps） and spectral （1-3 nm） resolutions and permits rapid data acquisition and reliable and reproducible lifetime and spectrum determinations. The system is tested with standard fluorescent dyes （rhodamine 6G, coumarin 314 and their mixtures） and the lifetimes and spectra obtained agree well with those reported in the literatures. Experimental results indicate that our system provides a unique contrast mechanism for discriminating multi-fluorophore or multi-component of biological tissues through spectrally and temporally resolved information and may find extensive applications in muhispectral fluorescence lifetime imaging and fluorescence resonance energy transfer imaging.