Gratton E, Limkeman M, Lakowicz J R, Maliwal B P, Cherek H, Laczko G
Biophys J. 1984 Oct;46(4):479-86. doi: 10.1016/S0006-3495(84)84044-8.
We measured fluorescence phase shift and modulation data for one-, two- and, three-component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using the least-squares procedure described in the preceding paper (Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463-477). Using data obtained at a single emission bandpass, the lifetimes and preexponential factors of two-component mixtures could be easily resolved if the lifetimes differed by a factor of 2. With currently available instrumental stability, three-component mixtures could be resolved when the overall range of decay times was 10-fold, (e.g., 1.3, 4.4, and 12 ns). Measurement of phase and modulation data at several emission wavelengths, where the ratio of the preexponential factors varied, enhanced our ability to resolve closely spaced two and three-component decays. Two-component mixtures could then be resolved if the lifetimes differed by 30% (4.4 and 6.2 ns). Also, the multiple-wavelength data allowed the lifetimes and emission spectra of the three-components of a mixture to be resolved. These results demonstrated that resolution of multiexponential decay laws was possible using frequency-domain phase-modulation fluorometry.
我们测量了荧光团单组分、双组分和三组分混合物在1至140 MHz调制频率下的荧光相移和调制数据。这些数据使用前文（Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463 - 477）所述的最小二乘法进行分析。利用在单个发射带通获得的数据，如果双组分混合物的寿命相差2倍，其寿命和预指数因子可轻松解析。基于当前仪器的稳定性，当衰减时间的总范围为10倍（例如1.3、4.4和12 ns）时，三组分混合物可得到解析。在几个发射波长处测量相移和调制数据（此时预指数因子的比值会发生变化），增强了我们分辨间隔很近的双组分和三组分衰减的能力。如果双组分混合物的寿命相差30%（4.4和6.2 ns），则可以得到解析。此外，多波长数据还能分辨混合物中三组分的寿命和发射光谱。这些结果表明，使用频域相调制荧光法解析多指数衰减规律是可行的。
