Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis
Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis
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DOI：
10.1073/pnas.180317197
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发表时间：
2000-09-12
影响因子：
11.1
通讯作者：
Schwille, P
中科院分区：
文献类型：
--
作者：
Heinze, KG;Koltermann, A;Schwille, P
共聚焦荧光相关光谱作为一种时间平均波动分析，结合了最高灵敏度和高统计置信度，已被证明是一种非常通用和强大的工具，用于检测和时间研究表面、溶液和活细胞中超低浓度的生物分子。为了探索不同分子物种的相互作用，以详细了解生物学相关机制，具有不同光谱激发和发射的双重或多重荧光团测定的相互关联研究特别有前景。尽管荧光互相关分析提供的检测特异性有了相当大的提高，但迄今为止很少有应用报道，可能是因为正确对准和控制实验装置稳定性的实际挑战。在这项工作中，我们证明了双光子激发结合双色荧光相关光谱可以成为在单分子尺度上显著简化多个荧光物种同时研究的关键。双光子激发允许通过相同的激发波长加入具有很大差异发射的共同荧光团，因为选择规则和振动耦合的差异可以引起单光子和双光子激发光谱之间的相当大的位移。介绍了双色双光子荧光互相关分析的概念，并通过限制性内切酶EcoRI检测双标记DNA底物的选择性切割实验进行了实验证明。
Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very Versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.