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摘要:
光学显微成像技术具有实时性、高分辨率和非侵入性等特点，其成像尺度可跨越细胞、组织乃至生命体，极大地拓展了人们对生命本质的认识边界。然而，受限于光学显微成像系统有限的空间带宽积（Space-Bandwidth Product，SBP），常规的光学显微镜难以同时兼具大视场和高分辨率，使得显微成像在大视场生物成像应用中受到较大的限制，例如，对脑神经网络以突触为单位的神经回路成像。近年来，大视场光学显微成像技术得到不断的发展，其SBP的视场相较于传统的光学显微镜有了十倍甚至百倍的提升，在保持高分辨率的基础上拓展了成像视场，从而可以满足生物医学领域重大问题的研究需求。本文介绍了近年来几种典型的大视场光学显微成像技术及其生物医学应用，并对其未来发展做了展望。
Abstract:With the characteristics of real-time, high-resolution and non-invasive, optical microscopy can scale from cells, tissues to whole living organisms, which has greatly expanded our understanding to the nature of life. However, due to the limited Space-Bandwidth Product (SBP), it is hard for a conventional optical microscope to achieve a large field of view with a high resolution. This makes it very difficult for microscopic imaging in large field of view biological imaging applications, such as imaging of neural circuits between the synapse of the brain neural networks. Recently, large field-of-view imaging technology has received increasing attention and experienced rapid development. The SBP has been improved ten times or even a hundred times as compared to a traditional optical microscope and the field-of-view has been expanded without sacrificing resolution, which, in turn, has resolved some major problems in biomedical research. This review introduces the progress, characteristics and corresponding biological applications of several typical trans-scale optical imaging techniques in recent years, and gives an outlook on their future development.
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图 1 大视场物镜双光子脑成像[12]。(a)成像系统光路图和物镜实物图（左上）；(b) 活体鼠脑神经细胞的双光子成像，深度为150 μm；(c)图(b)中虚线框内的细节放大图
Figure 1. Large FOV objective two-photon brain imaging[12]. (a) Optical path diagram of the imaging system and objective lens (upper left); (b) two-photon imaging of neuronal cells activity of mice brain in vivo, the depth of imaging is 150 μm; (c) magnification of some details in white box of (b)
图 2 基于散斑光片照明的大视场成像系统[25]。(a) 系统光路示意图，激光经磨砂玻璃片形成的散斑图案投影在体积为4.4 mm×3 mm×3 mm的样品内，形成厚度约为3 μm的散斑光片照明；(b) 用光片照明模式实现斑马鱼全身成像；(c)用共聚焦模式实现的斑马鱼全身成像
Figure 2. Speckle light sheet illumination-based large FOV imaging system[25]. (a) System setting, the speckle pattern formed by the laser through the ground glass disk is projected into the sample with a volume of 4.4 mm×3 mm×3 mm, forming a speckle light sheet with a thickness of about 3 μm for illumination; (b) Zebrafish whole body imaging with light sheet illumination; (c) Zebrafish whole body imaging with confocal microscopy
图 3 微透镜的排列与扫描方向示意图[37]
Figure 3. Schematic diagram of microlenses arrangement and scanning direction
图 5 无透镜分辨率增强成像系统[21]。(a) 系统结构和其使用的无序表面示意图；(b) 系统对小鼠肾脏切片的高通量成像，并选取三个子区域b1, b2, b3（图中红色框内）的成像结果与使用20 X, 0.75 NA物镜的荧光显微镜成像结果做对比，两者的相似度约为0.75
Figure 5. Resolution enhanced lensless imaging system[21]. (a) Schematic diagram of system design and the disordered surface; (b) high-throughput imaging of mouse kidney slices. Imaging results of three sub-regions b1, b2, b3 (red boxes) are compared with that of the fluorescence microscope using an objective with 20 X and 0.75 NA. The similarity is about 0.75
表 1 4类典型的大视场光学显微成像技术参数对比
Table 1. Comparison of four representative optical microscopy imaging techniques with large FOV
技术类别 成像方式 分辨率/μm 帧率（frame·s−1） 视场 优缺点 适用场景 大视场物镜成像 宽场[27] 0.7 92 20 mm2 可兼容多种成像方式；
像质分布不均匀活体、细胞、切片观察 双光子[17,27] 0.6 5×10−3 20 mm2 光片[29] 0.7 0.15 20 mm2 曲面探测成像 串行成像[37] 1.5 0.7 1256 mm2 整体像质更好；成像方式
多局限于宽场活体、细胞、切片观察 并行成像[19] 1.2 30 113 mm2 阵列显微 宽场+扫描[41] 1.7 6 60 mm2 简单；焦深浅 切片观测 无透镜显微 多波长复用[53] 0.69 0.5 29 mm2 简单，低成本；成像保真度有限 细胞、切片观测 倾斜成像[55] 0.69 5.6×10−2 120 mm2 编码叠层成像[26] 0.3 6.7×10−2 240 mm2 
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