Meng Qi, Xu Tianqi, Smith Zachary J, Chu Kaiqin
University of Science and Technology of China, Department of Precision Machinery and Precision Instrumentation, Anhui, Hefei, China.
Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Anhui, Hefei, China.
Biomed Opt Express. 2020 Jun 16;11(7):3769-3782. doi: 10.1364/BOE.393494. eCollection 2020 Jul 1.
One critical challenge in studying neural circuits of freely behaving model organisms is to record neural signals distributed within the whole brain, yet simultaneously maintaining cellular resolution. However, due to the dense packing of neuron cells in animal brains, high numerical aperture (NA) objectives are often required to differentiate neighboring neurons with the consequent need for axial scanning for whole brain imaging. Extending the depth of focus (EDoF) will be beneficial for fast 3D imaging of those neurons. However, current EDoF-enabled microscopes are primarily based on objectives with small NAs (≤0.3 ) such that the paraxial approximation can be applied. In this paper, we started from a nonparaxial approximation of the defocus aberration and derived a new phase mask that was appropriate for large NA microscopic systems. We validated the performance experimentally with a spatial light modulator (SLM) to create the designed phase mask. The performance was tested on different samples such as multilayered fluorescence beads and thick brain tissues, as well as with different objectives. Results confirmed that our design has extended the depth of focus about 10 fold and the image quality is much higher than those based on the most common EDoF method, the cubic phase method, popularly used to generate Airy beams. Meanwhile, our phase mask is rotationally symmetric and easy to fabricate. We fabricated one such phase plate and tested it on the pan-neuronal labeled (). The imaging performance demonstrated that we can capture all neurons in the whole brain with one snapshot and with cellular resolution, while the imaging speed is increased about 3 fold compared to the system using SLM. Thus we have shown that our method can not only provide the required imaging speed and resolution for studying neural activities in model animals, but also can be implemented as a low-cost, add-on module that can immediately augment existing fluorescence microscopes with only minor system modifications, and yielding substantially higher photon efficiency than SLM-based methods.
在研究自由活动模式生物的神经回路时，一个关键挑战是记录分布在整个大脑中的神经信号，同时保持细胞分辨率。然而，由于动物大脑中神经元细胞的密集排列，通常需要高数值孔径（NA）物镜来区分相邻神经元，因此需要进行轴向扫描以实现全脑成像。扩展焦深（EDoF）将有利于对这些神经元进行快速三维成像。然而，目前具备EDoF功能的显微镜主要基于小NA（≤0.3）的物镜，以便可以应用傍轴近似。在本文中，我们从离焦像差的非傍轴近似出发，推导了一种适用于大NA显微系统的新型相位掩膜。我们通过空间光调制器（SLM）实验验证了该设计的性能，以创建设计的相位掩膜。在多层荧光珠和厚脑组织等不同样本以及不同物镜上测试了该性能。结果证实，我们的设计将焦深扩展了约10倍，图像质量远高于基于最常用的EDoF方法（用于生成艾里光束的立方相位法）的图像质量。同时，我们的相位掩膜具有旋转对称性且易于制造。我们制作了一个这样的相位板，并在全神经元标记的（）上进行了测试。成像性能表明，我们可以通过一次快照以细胞分辨率捕获全脑中的所有神经元，而与使用SLM的系统相比，成像速度提高了约3倍。因此，我们已经表明，我们的方法不仅可以为研究模式动物的神经活动提供所需的成像速度和分辨率，而且可以作为一种低成本的附加模块来实现，只需对现有荧光显微镜进行少量系统修改，就能立即增强其功能，并且产生比基于SLM的方法高得多的光子效率。
