Gryczynski Ignacy, Kuśba Józef, Lakowicz Joseph R
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, Maryland 21201.
Faculty of Applied Physics and Mathematics, Technical University of Gdansk, ul. Narutowicza 11/12, 80-952 Gdansk, Poland.
J Fluoresc. 1997 Sep;7(3):167-183. doi: 10.1007/BF02758217.
We examined a series of fluorophore mixtures to determine the wavelength selectivity of light quenching and the effects of light quenching on the emission spectra and intensity decays. Light quenching can be accomplished using a single excitation pulse train and quenching wavelength (one-beam) or with longer-wavelength quenching pulses time-delayed relative to the excitation pulses (two-beam). Both one-beam and two-beam light quenching were found to alter the intensity decays of the mixtures. The frequency-domain intensity decay data were analyzed to reveal the fractional intensity of each fluorophore in the mixture and the effects of light quenching on the fractional contribution of each fluorophore to the total intensity. Fluorophores were selected to provide a range of decay times and emission wavelengths. The extent of quenching in the mixtures was dependent on which fluorophore had the higher radiative decay rate and emission intensity at the quenching wavelength. A general theory is presented which describes the intensity decays in terms of the extent of quenching of each fluorophore and the time delay between excitation and quenching pulses. The effects of light quenching on the fractional intensities of each fluorophore in the mixture, recovered from the intensity decay analysis, were found to be in quantitative agreement with that predicted from steady-state measurements of light quenching and from the spectral properties of the fluorophores. The data on light quenching of mixtures demonstrate that light quenching may be used for selective quenching of fluorophores and thus of potential value for studies of multichromophoric systems.
我们研究了一系列荧光团混合物，以确定光猝灭的波长选择性以及光猝灭对发射光谱和强度衰减的影响。光猝灭可以使用单个激发脉冲序列和猝灭波长（单光束）来实现，也可以使用相对于激发脉冲有时间延迟的较长波长猝灭脉冲（双光束）来实现。发现单光束和双光束光猝灭都会改变混合物的强度衰减。对频域强度衰减数据进行分析，以揭示混合物中每个荧光团的分数强度以及光猝灭对每个荧光团对总强度的分数贡献的影响。选择荧光团以提供一系列衰减时间和发射波长。混合物中的猝灭程度取决于在猝灭波长处哪个荧光团具有更高的辐射衰减率和发射强度。提出了一种通用理论，该理论根据每个荧光团的猝灭程度以及激发和猝灭脉冲之间的时间延迟来描述强度衰减。从强度衰减分析中恢复的光猝灭对混合物中每个荧光团分数强度的影响，与从光猝灭的稳态测量和荧光团的光谱特性预测的结果在定量上一致。关于混合物光猝灭的数据表明，光猝灭可用于荧光团的选择性猝灭，因此对于多发色团系统的研究具有潜在价值。
