Flow cytometry is being applied more extensively because of the outstanding advantages of multicolor fluorescence analysis. However, the intensity measurement is susceptible to the nonlinearity of the detection method. Moreover, in multicolor analysis, it is impossible to discriminate between fluorophores that spectrally overlap; this influences the accuracy of the fluorescence pulse signal representation. Here, we focus on spectral overlap in two-color analysis, and assume that the fluorescence follows the single exponential decay model. We overcome these problems by analyzing the influence of the spectral overlap quantitatively, which enables us to propose a method of fluorescence pulse signal representation based on time-delay estimation (between fluorescence and scattered pulse signals). First, the time delays are estimated using a modified chirp Z-transform (MCZT) algorithm and a fine interpolation of the correlation peak (FICP) algorithm. Second, the influence of hardware is removed via calibration, in order to acquire the original fluorescence lifetimes. Finally, modulated signals containing phase shifts associated with these lifetimes are created artificially, using a digital signal processing method, and reference signals are introduced in order to eliminate the influence of spectral overlap. Time-delay estimation simulation and fluorescence signal representation experiments are conducted on fluorescently labeled cells. With taking the potentially overlap of autofluorescence as part of the observed fluorescence spectrum, rather than distinguishing the individual influence, the results show that the calculated lifetimes with spectral overlap can be rectified from 8.28 and 4.86 ns to 8.51 and 4.63 ns, respectively, using the comprehensive approach presented in this work. These values agree well with the lifetimes (8.48 and 4.67 ns) acquired for cells stained with single-color fluorochrome. Further, these results indicate that the influence of spectral overlap can be eliminated effectively. Moreover, modulation, mixing with reference signals, and low-pass filtering are performed with a digital signal processing method, thereby obviating the need for a high-speed analog device and complex circuit system. Finally, the flexibility of the comprehensive method presented in this work is significantly higher than that of existing methods.
Keywords: digital signal processing; flow cytometry; fluorescence lifetime; spectrally overlapping signals; time-delay estimation.