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Two-photon photoacoustic microscopy (TP-PAM) can visualize deep structures in living tissues with high spatial resolution determined by the volume of nonlinear absorption. Generally, the out-of-focus background fluorescence limits the imaging depth in nonlinear optical microscopies. In this study, to overcome this drawback that is also expected to exist in TP-PAM, we propose TP-PAM with spatial overlap modulation using femtosecond optical pulse train. Because the modulation depth of the spatial overlap in the focal region is much greater than those in out-of-focus regions, the out-of-focus background is effectively rejected by extracting the modulated photoacoustic signals.
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