Polygon-synchronized line-confocal microscopy with offset-slit detection for high-speed volumetric fluorescence imaging

Fast volumetric fluorescence imaging requires both optical sectioning and high acquisition speed. We present a polygon-synchronized line-confocal microscope (Poly-LSCM) that combines a 16-facet polygon scanner, direct sCMOS detection, and offset-slit image reconstruction. The system eliminates image stitching and enables high-speed line-confocal imaging with improved scan uniformity. A subtraction-based reconstruction strategy further suppresses out-of-focus background and enhances image contrast. Poly-LSCM achieves a lateral resolution of 510 nm, an axial full width at half maximum of 738 nm, and imaging rates exceeding 200 fps. We demonstrate volumetric imaging of thick Thy1-EGFP mouse brain tissue and high-speed imaging of fluorescent microspheres in microfluidic channels. These results establish Poly-LSCM as a practical approach for high-throughput fluorescence imaging with millisecond-scale temporal resolution.