Quantifying uncertainty in phasor-based time-domain fluorescence lifetime imaging microscopy.

The phasor approach to time-domain fluorescence lifetime imaging microscopy (FLIM) offers a powerful, fit-free method for analyzing complex fluorescence decay signals. However, its quantitative accuracy is fundamentally limited by noise-particularly photon shot noise-which introduces variability and deviations in lifetime estimation and fluorophore unmixing. In this study, we present a theoretical uncertainty model for phasor-based time-domain FLIM that analytically captures the propagation of shot noise and quantifies its impact on phasor coordinates and fluorophore weight estimation. We validate the model using Monte Carlo simulations and experimental data acquired from standard fluorescent dyes and biological tissue samples. Our model improves the overall reliability and efficiency of phasor-based time-domain FLIM, particularly in photon-limited imaging applications.