Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering

Photoacoustic microscopy usually uses high-frequency photoacoustic waves, which provide not only high spatial resolution but also limitation of the penetration depth. In this study, we developed two-photon absorption-induced photoacoustic microscopy (TP-PAM) to improve the depth resolution without use of high-frequency photoacoustic waves. The spatial resolution in TP-PAM is determined by two-photon absorption. TP-PAM with a 20X objective lens (numerical aperture: 0.4) provides an optically-determined depth resolution of 44.9 ± 2.0 μm, which is estimated by the full width at half maximum of the photoacoustic signal from an infinitely small target, using low-frequency bandpass filtering of photoacoustic waves. The combination of TP-PAM and frequency filtering provides high spatial resolution.