Differential synthetic illumination based on multi-line detection for resolution and contrast enhancement of line confocal microscopy

Line confocal (LC) microscopy is a fast 3D imaging technique, but its asymmetric detection slit limits resolution and optical sectioning. To address this, we propose the differential synthetic illumination (DSI) method based on multi-line detection to enhance the spatial resolution and optical sectioning capability of the LC system. The DSI method allows the imaging process to simultaneously accomplish on a single camera, which ensures the rapidity and stability of the imaging process. DSI-LC improves X- and Z-axis resolution by 1.28 and 1.26 times, respectively, and optical sectioning by 2.6 times compared to LC. Furthermore, the spatially resolved power and contrast are also demonstrated by imaging pollen, microtubule, and the fiber of the GFP fluorescence-labeled mouse brain. Finally, Video-rate imaging of zebrafish larval heart beating in a 665.6×332.8μm² field-of-view is achieved. DSI-LC provides a promising approach for 3D large-scale and functional imaging in vivo with improved resolution, contrast, and robustness.