Signal subtraction using nanosecond optical pulses with two different pulse energies in two-photon photoacoustic microscopy (TP-PAM) with a ring-shaped acoustic transducer to evaluate precise thickness

We propose a signal subtraction method for two-photon photoacoustic microscopy (TP-PAM) with a ring-shaped acoustic transducer. This signal subtraction can reduce the contribution of one-photon photoacoustic (PA) signals based on the different pulse-energy dependence of one- and two-photon PA signals. The ring-shaped acoustic transducer extends the depth of focus in TP-PAM. The combination of the subtraction method and ring-shaped acoustic transducer in TP-PAM allows visualization of the edge of a target filled with a mixed solution of two-photon and one-photon absorbers and precise estimation of the thickness of the target.