Super-Resolution Two-Photon Fluorescence Tomography Through the Phase-Shifted Optical Beatings of Bessel Beams for High-Resolution Deeper Tissue 3D Imaging

A novel z-scanning-free epi-detected super-resolution two-photon fluorescence tomography (TPFT) technique enabling super-resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase-shifted optical beatings of Bessel beams (PS-OB³) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super-resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth-resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth-resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth-scanning. PS-OB³ TPFT provides ≈1.3- and 2-fold improvements in lateral and axial resolutions in comparison with conventional point-scan TPF imaging. It is also illustrated that the epi-detected PS-OB³ TPFT imaging with inherent scattering-resilient properties of the Bessel beams employed gives over 2-fold improvement in imaging depth in porcine brain tissue compared to conventional point-scan Gaussian beam TPF imaging. The z-scanning-free optical sectioning ability of PS-OB³ method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super-resolution deeper 3D imaging in biological and biomedical tissues.