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用于多尺度高分辨率三维成像的双环光片荧光显微技术

王鹏 周瑶 赵宇轩 费鹏

王鹏, 周瑶, 赵宇轩, 费鹏. 用于多尺度高分辨率三维成像的双环光片荧光显微技术[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0093
引用本文: 王鹏, 周瑶, 赵宇轩, 费鹏. 用于多尺度高分辨率三维成像的双环光片荧光显微技术[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0093
WANG Peng, ZHOU Yao, ZHAO Yu-xuan, FEI Peng. Double-ring-modulated light sheet fluorescence microscopic technique for multi-scale high-resolution 3D imaging[J]. Chinese Optics. doi: 10.37188/CO.2022-0093
Citation: WANG Peng, ZHOU Yao, ZHAO Yu-xuan, FEI Peng. Double-ring-modulated light sheet fluorescence microscopic technique for multi-scale high-resolution 3D imaging[J]. Chinese Optics. doi: 10.37188/CO.2022-0093

用于多尺度高分辨率三维成像的双环光片荧光显微技术

doi: 10.37188/CO.2022-0093
基金项目: 国家重点研发计划 (No. 2017YFA0700501);国家自然科学基金 (No. 21874052, No. 21927802)
详细信息
    作者简介:

    王 鹏 (1997—),男,河南漯河人,2019年于中山大学获得学士学位,现为华中科技大学光学与电子信息学院光电工程系硕士研究生,主要从事光片荧光显微成像技术的研究。E-mail:wp24@hust.edu.cn

    周 瑶(1998—),女,四川成都人,2020年于华中科技大学获得学士学位,现为华中科技大学光学与电子信息学院光电工程系博士研究生,主要从事新型荧光显微成像技术的研究。E-mail:zhouyao2021@hust.edu.cn

    赵宇轩(1993—),男,河南洛阳人,华中科技大学博士研究生,2017年于华南理工大学获得学士学位,目前聚焦于超分辨荧光显微成像新技术的研究及其在活细胞显微成像中的应用。在Nature Methods , Optica , Journal of Biophotonics等期刊发表论文5篇,授权发明专利/软件著作权5项。E-mail:yxzh@hust.edu.cn

    费 鹏(1982—),男,江西九江人,北京大学博士,UCLA博士后,国家海外高层次青年人才,现任华中科技大学光电学院/武汉光电国家研究中心教授。目前聚焦于荧光显微成像新技术的研究,并致力开拓他们在生物医学中的应用。在Nature Methods (2), Nature Materials, Nature Communications 等期刊发表论文70余篇,授权发明专利/软件著作权20余项,并实现国际转化。任SPIE Journal of Biomedical Optics期刊Associate Editor。E-mail:feipeng@hust.edu.cn

Double-ring-modulated light sheet fluorescence microscopic technique for multi-scale high-resolution 3D imaging

Funds: Supported by National Key Research and Development Program of China (No. 2017YFA0700501); National Natural Science Foundation of China (No. 21874052, No. 21927802)
More Information
  • 摘要:

    本文提出了一种无衍射光片荧光显微成像技术,可以方便地对从微米到厘米各种尺寸的多种生物样本进行多尺度三维荧光成像。为了解决传统贝塞尔光片旁瓣过重的问题,提出了一种双环调控的方法能够可调节地产生0.4微米到5微米之间不同厚度类型的无衍射光片,同时其旁瓣占比被压低到30%以下。基于这种崭新的调控手段设计搭建了一套多尺度光片荧光显微成像系统并展示了其适用性极强的多尺度成像能力;例如活细胞双色三维动态成像、膨胀细胞三维超分辨成像以及介观尺度下全器官的高通量三维成像。证明了该多尺度成像模式能够显著地提升光片荧光显微镜的成像效率并以此推进细胞生物学、组织病理学、神经科学等多种生物医学相关研究。

     

  • 图 1  光束整形原理和光片性能表征:(a)双环掩膜调控生成低旁瓣贝塞尔光片;(b)不同尺度类型光片性能表征及对比

    Figure 1.  Principle of double-ring beam reshaping and its comparative performance: (a) The generation of low-side lobe Bessel light sheet based on the modulation by a double-ring mask; (b) The comparative performance of our multi-scale low-side lobe light sheet

    图 2  系统光路图与时序控制图:(a)多尺度光片显微成像系统光路图;(b)不同应用场景的时序控制图

    Figure 2.  Schematic of our system and the control flow charts: (a) The optical design of our multi-scale light sheet fluorescence microscope; (b) The control charts for different imaging applications

    图 3  单环贝塞尔光片与双环贝塞尔光片成像性能对比:(a)使用旁瓣占比73%的2.5 μm单环贝塞尔光片获得的点扩散函数x-z面最大强度投影,比例尺:20 μm;(b)使用旁瓣占比18%的2.5 μm双环贝塞尔光片获得的点扩散函数x-z面最大强度投影,比例尺:20 μm

    Figure 3.  Comparison of imaging results between single-ring Bessel light sheet and DR-SPIM: (a) X-z plane max intensity projection of point spread function by 2.5 μm single-ring Bessel light sheet with 73% side lobes, scale bar: 20 μm; (b) X-z plane max intensity projection of point spread function by 2.5 μm DR-SPIM with 18% side lobes, scale bar: 20 μm

    图 4  U2OS细胞中线粒体外膜的分布及其运动变化:(a)整个细胞中线粒体外膜的三维分布图,比例尺:10 μm;(b)局部线粒体外膜的运动变化过程,比例尺:2 μm

    Figure 4.  3D dynamics of mitochondrial outer membrane in U2OS cell: (a) Three-dimensional distribution of mitochondrial outer membrane in a whole live cell, scale bar: 10 μm; (b) The movement of local mitochondrial outer membrane, scale bar: 2 μm

    图 5  U2OS细胞中线粒体外膜与Drp1的分布与相互作用:(a)线粒体外膜与Drp1的三维分布图,比例尺:3 μm;(b)感兴趣区域内线粒体外膜与Drp1相互作用过程,比例尺:1 μm

    Figure 5.  3D interactions between mitochondrial outer membrane and Drp1 in U2OS cell: (a) Three-dimensional co-localization of mitochondrial outer membrane and Drp1, scale bar: 3 μm; (b) Interactions between mitochondrial outer membrane and Drp1 in a region of interest, scale bar: 1 μm

    图 6  多尺度成像应用结果可视化:(a)4倍膨胀核孔可视化结果,比例尺:10 μm;(b)4倍膨胀核孔的轴向视角,比例尺:10 μm;(c)区域三维可视化,比例尺:2 μm;(d)区域x-y面最大强度投影,比例尺:1 μm;(e)区域y-z面最大强度投影,比例尺:1 μm;(f)鼠脑全脑可视化结果,比例尺:1 mm;(g)位于皮层的感兴趣区域三维可视化结果,比例尺:100 μm;(h)位于丘脑的感兴趣区域三维可视化结果,比例尺:100 μm

    Figure 6.  3D Visualization of multi-scale imaging results: (a) 3D visualization of an expanded nuclear hole (expansion factor ~4), scale bar: 10 μm; (b) axial 3D view of this nuclear hole, scale bar: 10 μm; (c) 3D visualization of a selected small region, scale bar: 2 μm; (d) Max intensity projection of the x-y plane of the region, scale bar: 1 μm; (e) Max intensity projection of the y-z plane of the region, scale bar: 1 μm; (f) 3D Visualization of a whole mouse brain, scale bar: 1 mm; (g) 3D visualization of a region of interest in cortex, scale bar: 100 μm; (h) 3D visualization of a region of interest in thalamus, scale bar: 100 μm

    表  1  不同应用场景下使用的掩膜参数与物镜

    Table  1.   Mask parameters and objectives in different application scenarios

    应用场景线粒体外膜的高
    速、低光毒性成像
    线粒体外膜与
    Drp1相互作用的
    双色成像
    4倍膨胀核孔
    三维体成像
    透明化鼠脑
    三维体成像
    单环贝塞尔光片与双环贝塞尔
    光片成像性能对比
    光片类型500 nm双环贝塞尔光片3.5 μm双环贝塞尔
    光片
    2.5 μm单环贝塞尔
    光片
    2.5 μm双环贝塞尔光片
    掩膜参数(mm)Router_max1.8571.6521.952.395
    Router_min0.9820.8731.031.266
    Rinner_max0.7890.70101.016
    Rinner_min0.3860.2700.392
    照明物镜28.6 × SO Microscope Objective, 0.66 NA, 3.74mm Water, Special OpticsXLFLUOR4X/340, Olympus
    探测物镜LUMFLN60XW,OlympusUMPLFLN10XW, OlympusUMPLFLN20XW,Olympus
    下载: 导出CSV
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  • 收稿日期:  2022-05-09
  • 录用日期:  2022-07-07
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