Single-objective two-photon oblique light sheet microscopy with a low repetition rate for 3D high-SNR imaging

Two-photon light sheet microscopy is a powerful technology for rapid three-dimensional (3D) fluorescence imaging with low phototoxicity, which has led to its wide adoption in biomedical and life science applications. However, conventional multiobjective lens configurations limit compatibility with large or irregularly shaped biological samples, restricting broader applicability. Moreover, image quality is often compromised due to the inherent trade-off between the low numerical aperture required for selective planar illumination and the reduced efficiency of two-photon excitation. In this study, we present a single-objective two-photon oblique light sheet microscopy (TP-ob-LSM) system employing a low-repetition-rate light source. This single-objective lens design enables simultaneous excitation and detection, thereby significantly enhancing compatibility with diverse biological sample geometries. Furthermore, by adjusting the repetition rate while maintaining constant average power, we increased pulse energy and achieved a 22.9-fold improvement in signal-to-noise ratio compared to conventional high-repetition-rate systems through system-level optimization of the excitation regime. We demonstrate large-volume reconstructions (9 mm ×6 mm ×0.18 mm ) and deep-tissue imaging in 1-mm-thick mouse brain slices with clear dendritic morphology. The proposed TP-ob-LSM with low-repetition-rate excitation offers a promising solution for high-quality 3D imaging across a wide range of biomedical applications.