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

Abstract 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‐OB 3 ) 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‐OB 3 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‐OB 3 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‐OB 3 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.