Line-scanning microscopy with laterally symmetric imaging using simultaneous cross-line-illumination

• Dan Shen, Yafeng Li, Meng Wang, Yutong Han, Bolin Lu, Hui Gong, Qingming Luo, and Jing Yuan
• received 02/23/2024; accepted 05/01/2024; posted 05/02/2024; Doc. ID 521819
• Abstract: Using an on-the-fly scanning scheme, line confocal microscopy can obtain complex structures of large biological tissues with high throughput. Yet, it suffers from lateral imaging asymmetry and thus sacrifices the detection accuracy of the observation results. Here, we propose cross-line illumination microscopy (cLIM) that acquires the imaging data of two perpendicular directions simultaneously through the same objective lens in a line scanning and utilizes two-direction deconvolution (De2dir) fusion to achieve lateral symmetric imaging performance. Imaging fluorescence beads indicates that cLIM reduces lateral resolution asymmetry from 46.1% to 2.5% and improves lateral resolution by 31.0%, compared with traditional line-scanning imaging. Compared with commercial point-confocal microscopy, the cLIM has a 25.84× increase in imaging speed and 1.93× better background-suppressing ability by imaging an 11,306 × 7,783 × 100 μm3 mouse kidney slice. We also show the advantages of the cLIM in observing direction-sensitive texture features by imaging a muscular tissue slice. cLIM offers a novel solution to achieve laterally symmetric line scanning imaging with simple modifications while maintaining high throughput and accuracy for imaging large-scale samples.