Structured illumination microscopy for high SNR 3D imaging from millimeter-thick tissues to cellular dynamics

Three-dimensional (3D) optical imaging of intact tissues with high spatiotemporal resolution is a highly desirable goal, as it offers visualization of biological structures and processes in their physiological context. Optical sectioning structured illumination microscopy (OS-SIM) stands out among various 3D imaging techniques for its comprehensive superiority, as it offers high spatial resolution, rapid imaging speed, and low phototoxicity. However, under the challenging conditions of dominant background and increased fluorescence scattering in thick tissues, which leads to a decrease in modulation contrast, OS-SIM faces the problem of low signal-to-noise ratio (SNR) and limited imaging depth. We have developed a high SNR OS-SIM strategy for 3D imaging of biological samples ranging from millimeter-thick tissues to cellular dynamics. By enhancing the SNR by ∼10 dB with rapid processing, our method enables imaging of tissue-cleared sample with thickness >2 mm and low light dose for live-cell recording, while also doubling the imaging penetration depth. With our OS-SIM system, we achieve high-quality 3D images of various biological samples, including mouse brains, Drosophila clock neurons, organoids, and live cells. We anticipate that this approach will render OS-SIM a powerful technique for the research of cellular organelles or thick tissues in 3D morphology.