Shao Wenjun, Kilic Kivilcim, Yin Wenqing, Wirak Gregory, Qin Xiaodan, Feng Hui, Boas David, Gabel Christopher V, Yi Ji
Department of Medicine, Boston University School of Medicine, Boston Medical Center, Boston, MA, USA.
Neurophotonics Center, Boston University, Boston, MA, USA.
Quant Imaging Med Surg. 2021 Mar;11(3):983-997. doi: 10.21037/qims-20-806.
Conventional light sheet fluorescence microscopy (LSFM), or selective plane illumination microscopy (SPIM), enables high-resolution 3D imaging over a large volume by using two orthogonally aligned objective lenses to decouple excitation and emission. The recent development of oblique plane microscopy (OPM) simplifies LSFM design with only one single objective lens, by using off-axis excitation and remote focusing. However, most reports on OPM have a limited microscopic field of view (FOV), typically within 1×1 mm. Our goal is to overcome the limitation with a new variant of OPM to achieve a mesoscopic FOV.
We implemented an optical design of mesoscopic scanning OPM to allow the use of low numerical aperture (NA) objective lenses. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. A telescope composed of cylindrical lenses was used to correct the distorted image caused by the demagnification design. We characterized the 3D resolutions and imaging volume by imaging fluorescent microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple ().
We demonstrate a mesoscopic FOV up to ~6×5×0.6 mm volumetric imaging, the largest reported FOV by OPM so far. The angle of the intermediate image plane is independent of the magnification as long as the size of the pupil aperture of the objectives is the same. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10×, NA 0.3) and median NA (20×, NA 0.5). Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. elegans are clearly visualized in 3D.
The proposed mesoscopic scanning OPM allows using low NA objectives such that centimeter-level FOV volumetric imaging can be achieved. With the extended FOV, simple sample mounting protocol, and the versatility of changeable FOVs/resolutions, our system will be ready for the varieties of applications requiring in vivo volumetric imaging over large length scales.
传统的光片荧光显微镜（LSFM），即选择性平面照明显微镜（SPIM），通过使用两个正交排列的物镜来分离激发光和发射光，从而能够在大体积范围内进行高分辨率三维成像。斜平面显微镜（OPM）的最新发展通过使用离轴激发和远程聚焦，仅用一个物镜简化了LSFM的设计。然而，大多数关于OPM的报道其微观视野（FOV）有限，通常在1×1毫米范围内。我们的目标是通过一种新型的OPM变体克服这一限制，以实现介观视野。
我们实施了介观扫描OPM的光学设计，以允许使用低数值孔径（NA）的物镜。在谢宾夫条件下，通过缩小倍率增加远程聚焦系统前中间图像的角度，从而显著提高远程聚焦系统中的光收集效率。使用由柱面透镜组成的望远镜来校正由缩小倍率设计引起的图像失真。我们通过对荧光微球成像来表征三维分辨率和成像体积，并在完整的斑马鱼幼虫、小鼠皮层和多个（此处原文似乎不完整）上展示了体积成像。
我们展示了高达约6×5×0.6毫米体积成像的介观视野，这是迄今为止OPM报道的最大视野。只要物镜的瞳孔孔径大小相同，中间图像平面的角度就与放大倍率无关。因此，该系统具有高度的通用性，允许在低（10×，NA 0.3）和中等NA（20×，NA 0.5）的不同物镜之间轻松切换。斑马鱼幼虫、小鼠皮层和秀丽隐杆线虫神经元中的详细微血管在三维中清晰可见。
所提出的介观扫描OPM允许使用低NA物镜，从而可以实现厘米级视野的体积成像。凭借扩展的视野、简单的样品安装协议以及可变视野/分辨率的通用性，我们的系统将适用于各种需要在大长度尺度上进行体内体积成像的应用。
