Analysis of Flow Cytometric Fluorescence Lifetime with Time-Delay Estimation of Pulse Signals

The measurement of fluorescence lifetimes emerged in flow cytometry because it is not impacted by the non-linearity, which occurs in fluorescence intensity measurements. However, this significantly increases the cost and complexity of a traditional flow cytometer. This work reports a simple method of fluorescence lifetime measurement of a flow cytometer based on the cytometric fluorescence pulse time-delay estimation and hardware time-delay calibration. The modified chirp Z-transform (MCZT) algorithm, combined with the algorithm of fine interpolation of correlation peak (FICP), is applied to improve the temporal resolution of the cross-correlation function of the scattering and fluorescence signals, which in turn improves the time-delay estimation accuracy. The estimation accuracy is verified by Gauss fitting. Cells that were labeled simultaneously with three-color reagents are measured; the statistical results of 5000 cells are compared with reference values and are verified with the pulse width variation. The results show the potential of fluorescence lifetime measurements in the traditional flow cytometer.