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 $250\mathrm{\mu m}\times 250\mathrm{\mu m}$ 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.

中文翻译：

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灵活的、视频速率和像差补偿的轴向双线扫描成像，具有视场连接和阶梯式远程聚焦

具有大轴向间隔的平行双平面成像能够在不同的兴趣视图中同时观察生物结构和活动。然而，三维视场（FOV）位置的调整和光学切片效应的不灵活性，以及​​受球面像差限制的有效轴向范围相对较小，阻碍了平行双平面成像的应用。在此，我们提出了一种灵活的、视频速率和散焦像差补偿的轴向双线扫描成像方法。我们使用阶梯镜远程生成和检测双轴线，补偿球面像差和 FOV 连接，重新排列成一条头对头线，用于高速光学切片采集。两个视场的横向和轴向位置可以分别在成像前和成像过程中灵活调整。该方法还允许根据特定的实验要求调整光学切片效果。我们通过实验验证了 300 μm 轴向范围内一致的成像性能。我们通过同时成像两个布朗运动展示了高吞吐量$250\mathrm{微米}\times 250\mathrm{微米}$FOV 的轴向和横向间隔分别为 150 μm 和 240 μm，频率为 24.5 Hz。我们还通过同时获取斑马鱼大脑视顶盖和后脑中的神经活动，展示了在功能成像中的潜在应用。因此，与现有的平行双平面成像相比，所提出的方法具有优势，并且有可能促进大轴向范围内的活体生物研究。