Beyond conventional wisdom: unveiling quantitative insights in fluorescence lifetime imaging via realistic simulation of biological systems

Fluorescence lifetime imaging microscopy (FLIM) and photometry (FLiP) are illuminating the dynamics of biological signals. Because fluorescence lifetime is an intensive property of a fluorophore that is insensitive to sensor expression levels, it excels over fluorescence intensity measurements by allowing comparison across animals, over chronic time periods, and quantitation of the absolute levels of biological signals. However, the insensitivity of lifetime to sensor expression level does not always hold true in biological experiments where autofluorescence, ambient light, dark currents and afterpulses of the detectors are present. To quantitatively evaluate the potential and limitations of fluorescence lifetime measurements, we introduce FLiSimBA, a flexible platform enabling realistic Fluorescence Lifetime Simulation for Biological Applications. FLiSimBA accurately recapitulates experimental data and provides quantitative analyses. Using FLiSimBA, we determine the photons required for minimum detectable differences in lifetime and quantify the impact of hardware innovation. Furthermore, we challenge the conventional view that fluorescence lifetime is insensitive to sensor expression levels and define the conditions in which sensor express levels do not result in statistically significant difference in biological experiments. Thus, we introduce an adaptable simulation tool that allows systematic exploration of parameters to define experimental advantages and limitations in biological applications. Moreover, we provide a statistical framework and quantitative insights into the impact of key experimental parameters on signal-to-noise ratio and fluorescence lifetime responses. Our tool and results will enable the growing community of FLIM users and developers to optimize FLIM experiments, expose limitations, and identify opportunities for future innovation of fluorescence lifetime technologies. ### Competing Interest Statement The authors have declared no competing interest.