Three-dimensional resolution enhancement of two-photon microscopy by combining point spread function engineering and...

Two-photon microscopy (TPM) enables deep, minimally invasive volumetric imaging of biological tissues, but its three-dimensional (3D) spatial resolution is fundamentally limited by diffraction, particularly along the axial direction. Here, we present a 3D resolution enhancement strategy for TPM by combining point spread function (PSF) engineering with multi-image deconvolution. By redistributing the objective pupil, we generate a modulated excitation PSF with enhanced axial high-frequency components. Two complementary volumetric datasets acquired under conventional Gaussian and modulated illumination are then jointly reconstructed using a Hessian-regularized deconvolution framework. Experiments on 200-nm fluorescent beads demonstrate lateral and axial resolution improvements of 1.56× and 1.67×, respectively, over conventional TPM, with a further 1.26× axial improvement relative to deconvolution using only the Gaussian-PSF image. Imaging of actin filaments in HeLa cells confirms improved contrast and visualization of fine 3D structures. Brain-slice experiments further show that aberrations can severely compromise performance, whereas aberration correction restores the axial-resolution enhancement. These results establish PSF engineering combined with multi-image deconvolution as an effective route for enhancing 3D resolution in TPM.