Flexible, video-rate, and aberration-compensated axial dual-line scanning imaging with field-of-view jointing and stepped remote focusing

Parallel dual-plane imaging with a large axial interval enables the simultaneous observation of biological structures and activities in different views of interest. However, the inflexibility in adjusting the field-of-view (FOV) positions in three dimensions and optical sectioning effects, as well as the relatively small effective axial range limited by spherical aberration, have hindered the application of parallel dual-plane imaging. Herein, we propose a flexible, video-rate, and defocus-aberration-compensated axial dual-line scanning imaging method. We used a stepped mirror to remotely generate and detect dual axial lines with compensation for spherical aberration and FOV-jointing to rearrange into a head-to-head line for high-speed optical sectioning acquisition. The lateral and axial positions of the two FOVs could be flexibly adjusted before and during imaging, respectively. The method also allows the adjustment of optical sectioning effects according to specific experimental requirements. We experimentally verified the consistent imaging performance over an axial range of 300 μm. We demonstrated high throughput by simultaneously imaging Brownian motions in two 250 μm×250  μm FOVs with axial and lateral intervals of 150 μm and 240 μm, respectively, at 24.5 Hz. We also showed potential application in functional imaging by simultaneously acquiring neural activities in the optic tectum and hindbrain of a zebrafish brain. The proposed method is, thus, advantageous compared to existing parallel dual-plane imaging and potentially facilitates intravital biological study in large axial range.