Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

中文翻译：

---

使用新型单物镜平面照明双光子显微镜对脑类器官进行快速 3D 成像。

人类诱导多能干细胞 (hiPSC) 在疾病建模方面具有巨大潜力。hiPSC 衍生的人类星形胶质细胞和神经元培养物允许以亚细胞分辨率研究神经信号通路。组合培养和三维 (3-D) 胚胎体 (EB) 扩大了对多细胞现象的研究范围。大脑类器官具有最高水平的复杂性，在许多方面概括了发育中大脑的解剖和功能特征，使得研究人类疾病的发育和形态学方面成为可能。用于这些不同尺度的 3D 组织成像的理想显微镜将结合共焦激光扫描和光片显微镜的功能：微米光学切片能力和亚微米空间分辨率、大视场和高帧速率，侵入性低，即理想情况下，比共焦显微镜具有更好的光子效率。在目前的工作中，我们描述了这样一种使用平面双光子（2P）激发的仪器。其特殊性在于，与两镜头或三镜头光片显微镜不同，它使用单个低放大倍率、高数值孔径物镜来生成和扫描虚拟光片。该显微镜基于改进的 Nipkow-Petráň 转盘方案构建，可实现宽视场激发。然而，与使用串联盘的横河设计不同，我们的概念将微透镜、二向色镜和检测针孔结合在单个盘上。这种新设计有利于 2P 激发，避免了串联盘因红外激发光和可见荧光之间的波长差异较大而出现的问题。使用相同的物镜收集由光片激发的 2P 荧光，并将其成像到快速 sCMOS 相机上。我们展示了 TO-PRO3 染色的 EB 和脑类器官的 3D 成像，未透明且使用三乙醇胺甲酰胺 (RTF) 快速部分透明化后，我们将我们的仪器与共焦激光扫描显微镜 (CLSM) 的性能进行了比较具有相似的数值孔径。与具有相似空间分辨率的共焦显微镜相比，我们的大视场 2P 旋转圆盘显微镜的成像速度提高了一个数量级，光漂白更少，并且具有更好的深度穿透能力。