Lin Chun-Yu, Chiu Kuo-Chih, Chang Chia-Yuan, Chang Shih-Hui, Guo Tzung-Fang, Chen Shean-Jen
Department of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan.
Opt Express. 2010 Jun 7;18(12):12807-17. doi: 10.1364/OE.18.012807.
This study investigated theoretically and experimentally that two-photon excited fluorescence is enhanced and quenched via surface plasmons (SPs) excited by total internal reflection with a silver film. The fluorescence intensity is fundamentally affected by the local electromagnetic field enhancement and the quantum yield change according to the surrounding structure and materials. By utilizing the Fresnel equation and classical dipole radiation modeling, local electric field enhancement, fluorescence quantum yield, and fluorescence emission coupling yield via SPs were theoretically analyzed at different dielectric spacer thicknesses between the fluorescence dye and the metal film. The fluorescence lifetime was also decreased substantially via the quenching effect. A two-photon excited total internal reflection fluorescence (TIRF) microscopy with a time-correlated single photon counting device has been developed to measure the fluorescence lifetimes, photostabilities, and enhancements. The experimental results demonstrate that the fluorescence lifetimes and the trend of the enhancements are consistent with the theoretical analysis. The maximum fluorescence enhancement factor in the surface plasmon-total internal reflection fluorescence (SP-TIRF) configuration can be increased up to 30 fold with a suitable thickness SiO(2) spacer. Also, to compromise for the fluorescence enhancement and the fluorophore photostability, we find that the SP-TIRF configuration with a 10 nm SiO(2) spacer can provide an enhanced and less photobleached fluorescent signal via the assistance of enhanced local electromagnetic field and quenched fluorescence lifetime, respectively.
本研究通过理论和实验研究了双光子激发荧光如何通过银膜全内反射激发的表面等离子体激元（SPs）增强和猝灭。荧光强度从根本上受到局部电磁场增强以及根据周围结构和材料的量子产率变化的影响。通过利用菲涅耳方程和经典偶极辐射模型，在荧光染料与金属膜之间不同介电间隔层厚度下，对局部电场增强、荧光量子产率以及通过表面等离子体激元的荧光发射耦合产率进行了理论分析。荧光寿命也通过猝灭效应大幅降低。已开发出一种带有时间相关单光子计数装置的双光子激发全内反射荧光（TIRF）显微镜，用于测量荧光寿命、光稳定性和增强效果。实验结果表明，荧光寿命和增强趋势与理论分析一致。在表面等离子体激元 - 全内反射荧光（SP - TIRF）配置中，使用合适厚度的SiO₂间隔层时，最大荧光增强因子可提高至30倍。此外，为兼顾荧光增强和荧光团光稳定性，我们发现具有10 nm SiO₂间隔层的SP - TIRF配置可分别通过增强的局部电磁场和猝灭的荧光寿命，提供增强且光漂白较少的荧光信号。
