Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon 791-0295, Ehime, Japan.
Translational Research Center, Ehime University Hospital, Toon 791-0295, Ehime, Japan.
Int J Mol Sci. 2023 Jun 15;24(12):10186. doi: 10.3390/ijms241210186.
Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.
双光子激发在光片显微镜中的应用推进了对多细胞生物的活体成像。在之前的研究中，我们使用低放大倍数（10×）、中数值孔径（NA0.5）检测物镜，开发了一种具有近 1mm 视场和小于 4μm 轴向分辨率的双光子贝塞尔光束光片显微镜。在这项研究中，我们旨在构建一种具有更高分辨率成像的光片显微镜，同时保持大视场，使用低放大倍数（16×）和高数值孔径（NA0.8）物镜。为了解决潜在的照明和检测不匹配问题，我们研究了使用景深（DOF）扩展方法。具体来说，我们使用了由五层环形区域组成的阶梯式装置，将 DOF 扩展了两倍，足以覆盖光片厚度。使用荧光珠进行分辨率测量表明分辨率的降低很小。然后，我们将该系统应用于斑马鱼的活体成像，并发现可以补偿光束注入远端的图像质量下降。这表明，扩展 DOF 系统与宽场双光子光片显微镜相结合，为具有亚细胞分辨率的大型多细胞生物标本的活体成像应用提供了一种简单易用的设置。
