As a non-invasive technique, optical imaging has become a widely used tool in both biological research and clinical diagnostics to investigate biological tissues. A key parameter to consider is the penetration depth of optical imaging in the tissues. Several techniques have been developed to enhance the penetration depth of optical imaging within scattering biological tissues, such as optical coherence microscopy (OCM) and multi-photon microscopy (MPM). Recently, focal modulation microscopy (FMM) has been developed and an imaging depth comparable to these techniques has been achieved. Here, combined with focal modulation techniques, two-photon focal modulation microscopy (TPFMM) is demonstrated theoretically and experimentally. First, TPFMM in turbid media using a novel spatiotemporal phase modulator (STPM) is theoretically investigated using the vector diffraction theory. At the destructive stage during the excitation beam modulation, this STPM is equivalent to a strip-shaped pupil filter with a sinusoidal phase distribution. Compared to the previous filter patterns with sharp phase transitions, the contribution of out-of-focus ballistic excitation to the background is largely reduced using the continuous phase filters. In addition, this new STPM has been designed and integrated into TPFMM to achieve high performance imaging of the biological tissues. It is found that TPFMM using this new STPM can significantly suppress scattered excitation and reduce out-of-focus ballistic excitation with acceptable modulation depth and resolution. Therefore, TPFMM with some new STPMs has the great potential to further extend the penetration depth in imaging the scattering biological tissues.
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