在体跨尺度双光子显微成像技术 -

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在体跨尺度双光子显微成像技术

doi:10.37188/CO.2022-0086

陈帅^{1, 2,},
任林^{1, 2,},
周镇乔^2,,
李敏^2,,
贾宏博^{1, 2,,}

1.
广西大学物理科学与工程技术学院和脑与智能研究中心, 广西南宁 530004
2.
中国科学院苏州生物医学工程技术研究所, 江苏苏州 215163

基金项目:中国科学院院级重大科研仪器研制项目（No. GJJSTD2019003）；国家自然科学基金青年基金（No. 61705251）；苏州市基础研究试点项目（No. SJC2021021）；中国科学院科研仪器设备研制项目资助（No. YJKYYQ20200052）

详细信息

作者简介:
陈　帅（1994—），男，安徽淮南人，广西大学博士研究生，主要从事双光子显微镜系统的研究。E-mail：2007401031@st.gxu.edu.cn
贾宏博（1983—），吉林省吉林市，现任中国科学院苏州生物医学工程技术研究所技术总监、脑科学仪器创新中心主任研究员，中科院“百人计划——技术英才”入选者，广西大学“君武学者”特聘教授。2004年于北京大学获得学士学位，2006年于巴黎高等师范学校获得物理学硕士学位，2007年于巴黎高等师范学校获得生物学硕士学位，2011年于慕尼黑工业大学获得博士学位。主要专注于生物活体显微成像技术的创新及其在脑科学研究中应用的研究。E-mail：jiahb@sibet.ac.cn

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出版历程
- 收稿日期:2022-04-29
- 修回日期:2022-05-31
- 网络出版日期:2022-07-21

In-vivo across-scales two-photon microscopic imaging technique

CHEN Shuai^{1, 2,},
REN Lin^{1, 2,},
ZHOU Zhen-qiao^2,,
LI Min^2,,
JIA Hong-bo^{1, 2,,}

1.
School of Physical Science and Engineering Technology and Center for Brain and Intelligence Research, Guangxi University, Nanning 530004, China
2.
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China

Funds:Supported by Major Research Instrument Development Projects at the Chinese Academy of Sciences (No. GJJSTD2019003); National Natural Science Foundation of China Youth Fund (No. 61705251); Basic Research Pilot Project of Suzhou (No. SJC2021021); the Scientific Instrument Developing Project of the Chinese Academy of Sciences (No. YJKYYQ20200052)

More Information

Corresponding author:jiahb@sibet.ac.cn

摘要

摘要:
双光子显微镜在厚生物组织中依然可以保持良好的空间分辨率，这一优点使得其诞生不久就被应用于在体脑成像研究。而神经网络在时空多个维度均具有跨尺度的特点，为满足脑科学研究中在体跨尺度脑成像的需求，双光子显微镜近年来有了快速且显著的发展。本文首先介绍了双光子显微镜的工作原理，然后在成像视野、成像通量、成像深度、分辨率、微型化5个方面详细综述了双光子显微镜研究的新进展，并深入分析了跨尺度双光子在体显微成像技术的难点及未来挑战。
- 在体脑成像/
- 跨尺度/
- 双光子显微镜
Abstract:
Two-photon microscopy’s ability to maintain good spatial resolution in thick biological tissues has led to its application in in-vivo brain imaging studies soon after its conception. As neural networks have cross-scale multidimensional spatio-temporal properties, two-photon microscopy has developed rapidly and significantly in recent years to meet the demand for in-vivo cross-scale imaging of the brain. This paper firstly introduces the working principle of two-photon microscopy, then reviews the progress of two-photon microscopy from five perspectives: imaging field of view, imaging flux, imaging depth, resolution, miniaturization, and analyzes the difficulties and future challenges of cross-scale two-photon in-vivo microscopic imaging technology.
- in-vivo brain imaging/
- cross-scale/
- two-photon microscopy

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图 1（a）单光子和（b）双光子荧光激发原理

Figure 1.Excitation principles of (a) single photon and (b) two-photon fluorescence

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图 2双光子显微镜结构示意图

Figure 2.Schematic diagram of two-photon microscope structure

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图 3大尺度成像视野双光子显微镜。（a）多区域实时双光子成像技术^[25]；（b）双扫描系统双光子显微镜^[26]；（c）双扫描区域同步成像双光子显微镜^[28]；（d）多区域随机扫描双光子显微镜^[31]。

Figure 3.Two-photon microscope with large-scale imaging field of view. (a) Multi-area real-time two-photon imaging technology^[25]; (b) dual scanning system two-photon microscope^[26]; (c) two-photon microscope with dual scanning area simultaneous imaging^[28]; (d) two-photon microscope with multi-region follow-on scanning^[31]

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图 4快速扫描双光子成像技术。（a）光珠双光子显微镜^[37]；（b）多焦点快速扫描双光子显微镜^[38]；（c）快速线扫描双光子显微镜^[39]

Figure 4.Fast scanning two-photon imaging technology. (a) Light beads two-photon microscope^[37]; (b) multi-focus fast scanning two-photon microscope^[38]; (c) fast line scanning two-photon microscope^[39]

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图 5超深度探测显微成像技术。（a）梯度折射率透镜双光子显微镜^[44]；（b）三光子小鼠神经元成像^[49]；（c）三光子小鼠脑血管成像^[51]

Figure 5.Ultra depth detection microscopic imaging. (a) Gradient refractive index lens two-photon microscope^[44]; (b) three-photon neuron imaging in mice^[49]; (c) three-photon cerebral vascular imaging in mice^[51]

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图 6超分辨率双光子显微镜。（a）STEM成像原理和实验数据^[58]；（b）SIM结构光生成方法和结构光与均匀光照射探测精度对比^[60]

Figure 6.Super-resolution two-photon microscope. (a) STEM imaging principles and experimental data^[58]; (b) SIM structured light generation method and comparison of structured light and uniform light irradiation detection accuracy^[60]

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图 7超分辨率双光子显微镜实验结果^[65-66]

Figure 7.Experimental results of super-resolution two-photon microscope^[65-66]

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表 1双光子显微镜在多个尺度方向的性能提升进展现状

Table 1.Progress in performance improvement of two-photon microscopy in multiple scale directions

提升的尺度方向	提升方法	结果	相关文献
成像视野	1. 使用两套或多套独立扫描探测系统； 2. 自制大视野介观物镜，双焦点扫描，脉冲延时与时分复用； 3. 自制大口径介观物镜，共振镜串联大孔径振镜，随机扫描； 4. 使用多层阵列复合物镜结构，二次聚焦放大，多光轴耦合。	将传统显微镜的成像视野直径由不到1 mm提升至12 mm。	[25−33]
成像通量	1. 光珠双光子荧光显微镜，轴向多焦点扫描； 2. 微透镜阵列将光束分束，平面多焦点扫描； 3. 线扫描，通过压缩传感算法反解出二维荧光图像。	成像通量由百万量级提高至亿量级。图像帧率达到kHz量级。	[35−39]
成像深度	1. 使用更低能量（波长1300 nm）的光子，结合自适应光学，三光子激发； 2. 配合使用梯度折射率透镜，任意深度探测。	成像深度由传统双光子0.7 mm提升至2.1 mm（三光子，无外源装置侵入）或任意深度（配合植入器件）。	[44−46]、[49−51]
成像分辨率	1. 受激辐射耗尽双光子荧光显微镜； 2. 结构光双光子荧光显微镜。	将双光子的成像分辨率由~500 nm提升至80 nm。	[54]、[57−59]、[60−62]
微型化	1. 光纤传导激发光，MEMS、微型物镜等器件。	重量由数十 kg减少到 ~3 g；被观测动物在实验过程中可以自由移动。	[65−68]

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