Time-domain fluorescence lifetime imaging by nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses.

We propose a time-domain approach for fluorescence lifetime measurements using nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses. Nonlinear fluorescence signals generated by fluorescence reduction due to stimulated emission were detectable through a lock-in technique. Changing the time delay between the two-wavelength pulses enables acquisition of a time-resolved nonlinear fluorescence signal, which directly reflects the fluorescence lifetime of the sample and is thus applicable to fluorescence lifetime imaging. We also quantitatively demonstrate that nonlinear fluorescence microscopy possesses better optical resolution than conventional laser-scanning fluorescence microscopy. Experimental trials indicate that straightforward fluorescence lifetime imaging with high optical resolution is readily available.