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在体跨尺度双光子显微成像技术

陈帅 任林 周镇乔 李敏 贾宏博

陈帅, 任林, 周镇乔, 李敏, 贾宏博. 在体跨尺度双光子显微成像技术[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0086
引用本文: 陈帅, 任林, 周镇乔, 李敏, 贾宏博. 在体跨尺度双光子显微成像技术[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0086
CHEN Shuai, REN Lin, ZHOU Zhen-qiao, LI Min, JIA Hong-bo. In-vivo across-scales two-photon microscopy[J]. Chinese Optics. doi: 10.37188/CO.2022-0086
Citation: CHEN Shuai, REN Lin, ZHOU Zhen-qiao, LI Min, JIA Hong-bo. In-vivo across-scales two-photon microscopy[J]. Chinese Optics. doi: 10.37188/CO.2022-0086

在体跨尺度双光子显微成像技术

doi: 10.37188/CO.2022-0086
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,在中科院苏州医工所参加新一代跨尺度双光子显微镜研制工作,主要从事双光子显微镜系统的研究。Email:2007401031.st.gxu.edu.cn

    任 林(1995—),男,四川宜宾人,广西大学博士研究生,2021年于长春大学获得硕士学位,在中科院苏州医工所参加新一代跨尺度双光子显微镜研制工作,主要从事光学设计、图像处理方面的研究。E-mail:815559030@qq.com

    周镇乔(1987—),男,广东省江门人,中国科学院苏州生物医生工程技术研究所副研究员,2015年于华中科技大学获得博士学位,作为核心骨干成员参加新一代跨尺度双光子显微镜研制工作,主要从事新型双光子显微成像技术研究,提出多光轴耦合、二次聚焦放大的双光子成像新方法,使双光子成像视野跨度达到12 mm,达到国际领先水平。Email:zhouzq@sibet.ac.cn

    李 敏(1981—),女,山东博兴县人,中国科学院苏州生物医生工程技术研究所副研究员,2010年于中国科学院长春光学精密机械与物理研究所获得博士学位,主要从事新型显微光学成像技术和内窥显微成像技术研究。E-mail:limin@sibet.ac.cn

    贾宏博(1983—),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获得北京大学学士学位,2006获得巴黎高等师范学校物理学硕士学位,2007年获得巴黎高等师范学校生物学硕士学位,2011年获得慕尼黑工业大学博士学位。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

  • 中图分类号: Q6

In-vivo across-scales two-photon microscopy

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);Supported by Basic Research Pilot Project of Suzhou, Grant No. SJC2021021;Supported by the Scientific Instrument Developing Project of the Chinese Academy of Sciences, Grant No. YJKYYQ20200052。
More Information
  • 摘要:

    双光子显微镜在厚生物组织中保持良好空间分辨率的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、微型化五个方面详细综述了双光子显微镜的新进展,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。

     

  • 图 1  单光子、双光子荧光激发(a)单光子激发原理;(b)双光子激发原理

    Figure 1.  Single photon and two-photon fluorescence excitation. (a)Single photon excitation principle; (b)Two-photon excitation principle

    图 2  双光子显微镜结构

    Figure 2.  Two-photon microscope structure

    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MATRIEX[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.  Large-scale imaging field of view two-photon microscope. (a) Multi-area real-time two-photon imaging technology MATRIEX; (b) Dual scanning system two-photon microscope; (c) Dual scanning area simultaneous imaging two-photon microscope; (d) Multi-region follow-on scanning two-photon microscope.

    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; (b) Multi-focus fast scanning two-photon microscope; (c) Fast line scanning two-photon microscope.

    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; (b) Three-photon neuron imaging in mice; (c) Three-photon cerebral vascular imaging in mice

    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; (b) SIM structured light generation method and comparison of structured light and uniform light irradiation detection accuracy

    图 7  超分辨率双光子显微镜[65-66]

    Figure 7.  Super-resolution two-photon microscope

    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.   Multi-dimensional enhancement across scales in two-photon microscopy

    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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