One aspect of the present disclosure relates to systems and methods that can be used for super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM). One or more laser light sources can deliver excitation light with an intensity modulated by a periodic function to a sample, which includes a fluorophore concentration. A receiver can receive a fluorescence signal emitted from the fluorophore concentration over a time period. A computing device can transform the fluorescence signal to the frequency domain and measure a fluorescence lifetime associated with the sample in the frequency domain based on a DC component and at least one higher harmonic component generated by non-linear optical mixing of the modulated excitation light. Using the DC component and the at least one higher harmonic component can increase the sensitivity of an image created by the MPM-FD-FLIM system.
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