High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy

Super-resolution imaging has revolutionized our ability to visualize biological structures at subcellular scales. However, deep-tissue super-resolution imaging remains constrained by background interference, which leads to limited depth penetration and compromised imaging fidelity. To overcome these challenges, we propose a novel imaging system, confocal² spinning-disk image scanning microscopy (C²SD-ISM). It integrates a spinning-disk (SD) confocal microscope, which physically eliminates out-of-focus signals, forming the first confocal level. A digital micromirror device (DMD) is employed for sparse multifocal illumination, combined with a dynamic pinhole array pixel reassignment (DPA-PR) algorithm for ISM super-resolution reconstruction, forming the second confocal level. The dual confocal configuration enhances system resolution, while effectively mitigating scattering background interference. Compared to computational out-of-focus signal removal, SD preserves the original intensity distribution as the penetration depth increases, achieving an imaging depth of up to 180 μm. Additionally, the DPA-PR algorithm effectively corrects Stokes shifts, optical aberrations, and other non-ideal conditions, achieving a lateral resolution of 144 nm and an axial resolution of 351 nm, and a linear correlation of up to 92% between the original confocal and the reconstructed image, thereby enabling high-fidelity super-resolution imaging. Moreover, the system's programmable illumination via the DMD allows for seamless realization with structured illumination microscopy modality, offering excellent scalability and ease of use. Altogether, these capabilities make the C²SD-ISM system a versatile tool, advancing cellular imaging and tissue-scale exploration for modern bioimaging needs.