Deep Volumetric Super‐Resolution Imaging in Thick Biological Specimens With Sparse Scanning SIM

ABSTRACT High‐resolution fluorescence microscopy deep inside thick, live specimens remains a major challenge due to scattering and limited penetration depth. Structured illumination microscopy (SIM) is widely used for fast super‐resolution imaging, yet its sensitivity to scattering strongly restricts volumetric applications. Here we present sparse scanning SIM (SS‐SIM), a variant for deep imaging that integrates rapid laser beam scanning with pixel‐addressed intensity modulation and sCMOS detection. SS‐SIM operates with either one‐ or two‐photon excitation and achieves a spatial resolution of ∼150 nm, and maintains a 1.6/1.7‐fold (lateral/axial) resolution enhancement over wide‐field microscopy throughout the available imaging depth limited by the working distances of our water‐immersion objectives (300/600 µm), enabling volumetric visualization of dense biological samples. By combining simplicity, robustness, and super‐resolution performance, SS‐SIM expands the applicability of SIM to thick tissues, organoids, and small organisms, providing a practical route to high‐contrast volumetric imaging in complex biological environments.