Adapting phasor analysis for nonlinear pump-probe microscopy

Phasor analysis has become a powerful tool for examining signals in fluorescence life-time microscopy (FLIM), where the analysis provides a fast, robust and intuitive means of separating different fluorescent species and mixtures thereof. In this work we adapt this analysis for pump-probe microscopy, a method that provides molecular contrast of pigmented samples by probing their excited state dynamic properties. The bipolar nature of the pump-probe signals presents important differences in the resulting phasors compared to FLIM—here, we discuss these differences and describe the behavior of bipolar signals in phasor analysis. Results show that this method is indeed able to separate multiple molecular species of interests and allows facile assessment of pigment chemistry and its distribution in samples.