Two-photon light-sheet microscopy with isotropic near-diffraction-limited resolution in three dimensions.

Achieving isotropic high-resolution three-dimensional imaging across a large field of view (FOV) remains a key challenge in two-photon light-sheet microscopy (TPLSM). To address this limitation, we developed a TPLSM system that stitches multiple short Bessel beams into a large, ultrathin illumination light sheet and employs a dedicated control protocol to suppress stray light. The system delivers isotropic near-diffraction-limited resolution across a 332.8 μm FOV, enabling uniform visualization of fine structural details in biological samples and avoiding morphological misinterpretation. In vivo imaging of zebrafish embryos validates the system's performance, confirming its utility for high-fidelity biological observation. Furthermore, the proposed approach is broadly applicable to other microscope modalities using Bessel-beam illumination, extending its utility well beyond TPLSM.