Keating-Nakamoto S M, Cherek H, Lakowicz J R
Anal Chem. 1987 Jan 15;59(2):271-8. doi: 10.1021/ac00129a012.
Phase sensitive emission spectra recorded at multiple frequencies were used to determine the lifetimes and steady-state spectra of the fluorophores In two-component mixtures. This analysis does not require any previous knowledge of the lifetimes, fractional Intensities, or the emission spectra and is thus an Improvement over single frequency phase sensitive detection which requires known emission spectra or known lifetimes. Phase sensitive emission spectra are recorded at several frequencies and arbitrarily chosen detector phase angles. The data are analyzed by use of nonlinear least squares to recover the lifetimes and wavelength-dependent fractional Intensities. The latter values determine the emission spectrum and relative intensity of each component in the mixture. Using this technique, we resolved two-component mixtures of fluorescein and 9-amlnoacrldlne, 2-(-toluidinyl)-naphthalene-6-sulfonlc acid and 6-proplonyl-2-(dlmethylamlno)naphthalene, and -acetyl-l-tyrosinamide and -acetyl-l-tryptophanamide. In these mixtures the lifetimes differ by about 2-fold. Analysis of simulated data is presented to Illustrate the requirements for a satisfactory resolution. For simulated two-component mixtures, the components can be resolved If the lifetimes differ by 2-fold or greater, even with extensive overlap of the emission spectra.
在多频率下记录的相敏发射光谱用于确定双组分混合物中荧光团的寿命和稳态光谱。该分析不需要事先了解寿命、分数强度或发射光谱，因此是对单频相敏检测的改进，单频相敏检测需要已知的发射光谱或已知的寿命。在几个频率和任意选择的探测器相角下记录相敏发射光谱。通过使用非线性最小二乘法分析数据，以恢复寿命和波长相关的分数强度。后一个值决定了混合物中各组分的发射光谱和相对强度。使用该技术，我们解析了荧光素和9-氨基吖啶、2-（对甲苯胺基）-萘-6-磺酸和6-丙酰基-2-（二甲基氨基）萘、以及N-乙酰-L-酪氨酰胺和N-乙酰-L-色氨酰胺的双组分混合物。在这些混合物中，寿命相差约2倍。给出了模拟数据的分析，以说明获得满意分辨率的要求。对于模拟的双组分混合物，即使发射光谱有大量重叠，只要寿命相差2倍或更大，各组分就能被解析。
