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 ...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(C^(2)SD-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 180μm.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 C^(2)SD-ISM system a versatile tool,advancing cellular imaging and tissue-scale exploration for modern bioimaging needs.展开更多
基金supported by the National Key R&D Program of China(2022YFC3401100)the National Natural Science Foundation of China(62025501,92150301,62335008,62405010,and 62305004)the Postdoctoral Fellowship Program of CPSF(GZB20250669)。
文摘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(C^(2)SD-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 180μm.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 C^(2)SD-ISM system a versatile tool,advancing cellular imaging and tissue-scale exploration for modern bioimaging needs.
