Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

We describe the use of phase-sensitive detection of fluorescence to resolve the lifetimes and fractional intensities from multi-component fluorescence samples, using data obtained at a single modulation frequency. Phase-sensitive spectra of the mixture are recorded at arbitrarily chosen detector phase angles. The steady-state spectrum of each component must be known. The phase-sensitive spectra are fitted, using a nonlinear least-squares algorithm, to obtain the lifetimes and fractional intensities of each fluorophore in the mixture. Simulations for two- and three-component mixtures are presented to illustrate how the resolution is affected by spectral overlap and lifetime separation. Experimentally, we resolved two- and three-component mixtures of protein-like fluorophores (N-acetyl-L-tyrosinamide, N-acetyl- L-tryptophanamide, indole and 2,3-dimethylindole) using data collected at 30 MHz. These fluorophores have closely spaced lifetimes of 1.5, 2.9, 4.5 and 4.3 ns, respectively, and display extensive spectral overlap. These results demonstrate that phase-sensitive spectra, recorded at only one modulation frequency with a standard phase fluorometer, can be used to resolve multi-component emissions.