A spatiotemporal phase modulator (STPM) is theoretically investigated using the vectorial diffraction theory. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and a stripe-shaped filter with a sinusoidal phase distribution. It is found that two-photon focal modulation microscopy (TPFMM) using this STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as two-photon microscopy. The modulation depth is also evaluated and a compromise exists between the signal-to-background ratio and signal-to-noise ratio. The theoretical investigations provide important insights into future implementations of TPFMM and its potential to further extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues.
Line-scan focal modulation microscopy (LSFMM) is an emerging imaging technique that affords high imaging speed and good optical sectioning at the same time. We present a systematic investigation into optimal design of the pupil filter for LSFMM in an attempt to achieve the best performance in terms of spatial resolutions, optical sectioning, and modulation depth. Scalar diffraction theory was used to compute light propagation and distribution in the system and theoretical predictions on system performance, which were then compared with experimental results.
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.