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大视场高分辨HiLo光切片显微成像系统

郎松 张艳微 郑汉青 徐林钰 汪路涵 巩岩

郎松, 张艳微, 郑汉青, 徐林钰, 汪路涵, 巩岩. 大视场高分辨HiLo光切片显微成像系统[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0087
引用本文: 郎松, 张艳微, 郑汉青, 徐林钰, 汪路涵, 巩岩. 大视场高分辨HiLo光切片显微成像系统[J]. 中国光学(中英文). doi: 10.37188/CO.2022-0087
LANG Song, ZHANG Yan-wei, ZHENG Han-qing, XU Lin-yu, WANG Lu-han, GONG Yan. Wide-field-of-view and high-resolution HiLo optical sectioning microscopy system[J]. Chinese Optics. doi: 10.37188/CO.2022-0087
Citation: LANG Song, ZHANG Yan-wei, ZHENG Han-qing, XU Lin-yu, WANG Lu-han, GONG Yan. Wide-field-of-view and high-resolution HiLo optical sectioning microscopy system[J]. Chinese Optics. doi: 10.37188/CO.2022-0087

大视场高分辨HiLo光切片显微成像系统

doi: 10.37188/CO.2022-0087
基金项目: 国家自然科学基金(No.61975228&62005307);中国科学院科研仪器设备研制项目(No.YJKYYQ20190048);济南高校“20条”(No.2019GXRC042)
详细信息
    作者简介:

    郎 松(1991—),男,安徽阜阳人,中科院苏州医工所助理研究员,中国科学技术大学在读博士研究生,2013年于西北工业大学获得学士学位,2015年于哈尔滨工业大学获得硕士学位,主要从事光机结构设计、生物医用光学成像技术等方面的研究。E-mail:langs@sibet.ac.cn

    巩 岩(1968—),男,吉林梅河口人,中科院苏州医工所研究员,博士生导师,1990年于浙江大学获得学士学位,1998年于中科院研究生院所获得硕士学位,2002年于中科院研究生院获得博士学位,主要从事光学系统设计、先进计算显微光学和成像技术等方面的研究。E-mail:gongy@sibet.ac.cn

  • 中图分类号: O439

Wide-field-of-view and high-resolution HiLo optical sectioning microscopy system

Funds: Supported by the National Natural Science Foundation of China (No.61975228 and 62005307); Scientific Instrument Developing Project of the Chinese Academy of Sciences (No.YJKYYQ20190048); The Science and Technology Department of Jinan City (No.2019GXRC042)
More Information
  • 摘要:

    现代生物学和生物医学领域迫切需要兼顾大视场、高分辨率的显微成像技术和仪器对生物样品跨尺度观测,以满足重大科学问题的研究需求。受限于系统的空间带宽积,传统商业显微镜无法满足这一需求,且现有高空间带宽积显微成像系统存在体积庞大、实施成本高昂等问题。本文基于HiLo光切片技术和自主设计的大视场高分辨显微物镜,研发了具有高空间带宽积特点的大视场高分辨HiLo光切片显微成像系统,测试了系统的成像视场和分辨率,应用该系统对小鼠脑切片开展了白光照明明场成像实验,并与OLYMPUS商业显微镜成像结果做了对比,同时,应用该系统对小麦种子荧光切片开展了光切片成像和宽场荧光成像对比实验。实验结果表明, 大视场高分辨HiLo光切片显微成像系统成像视场达到4.8 mm×3.6 mm (对角视场6.0 mm),横向分辨率达到0.74 μm,轴向分辨率达到4.16 μm。大视场高分辨HiLo光切片显微成像系统具有兼顾大视场和高分辨率成像的优势和快速光切片成像的能力,能够对大体积生物样本开展快速三维成像,将为胚胎发育、脑成像、数字病理诊断等研究提供有力的技术支撑。

     

  • 图 1  大视场高分辨显微物镜光学设计评价图

    Figure 1.  Design evaluation graphs of the wide-field-of-view and high-resolution objective

    图 2  大视场高分辨HiLo光切片显微成像系统光学原理图

    Figure 2.  Optical schematic diagram of wide-field-of-view and high-resolution HiLo optical sectioning microscopy system

    图 3  系统实物照片

    Figure 3.  Photo of the system

    图 4  系统成像视场测试结果图

    Figure 4.  The test results of FOV of the system

    图 5  系统横向分辨率测试结果图

    Figure 5.  The test results of lateral resolution of the system

    图 6  系统轴向分辨率测试结果图

    Figure 6.  The test results of the axial resolution of the system

    图 7  HE染色的小鼠脑切片白光照明明场成像结果对比图

    Figure 7.  Comparison of brightfield imaging results of a HE-stained mouse brain slice illuminated by white light

    图 8  FITC-WGA染色的小麦种子荧光切片三维光切片成像和三维宽场荧光成像结果对比图(50层图像切片,单层图像切片厚2 μm)

    Figure 8.  Comparison of 3D optical sectioning and wide field fluorescence imaging results of a FITC-WGA-stained wheat seed fluorescence slice (50 slices, 2μm thick of every slice)

    表  1  大视场高分辨显微物镜设计参数

    Table  1.   Design parameters of the wide-field-of-view and high-resolution objective

    参数指标
    物方视场(Field of View, FOV)Φ6.0 mm
    数值孔径(Numerical Aperture, NA)0.5
    工作距(Working Distance, WD)2 mm
    焦距(Focus, F)40 mm
    工作波段(Working Waveband)436 nm ~ 800 nm
    浸没介质(Immersion Medium)空气
    下载: 导出CSV

    表  2  系统参数

    Table  2.   Parameters of the system

    参数指标
    照明光源白光、488 nm/561 nm激光
    成像视场4.8 mm×3.6 mm(对角6.0 mm)
    分辨率横向0.73 μm,轴向4.80 μm
    放大倍率11.1 X
    系统空间带宽积151 M
    工作模式明场成像、宽场荧光成像、HiLo光切片成像
    外形尺寸565 mm(长)×890 mm(宽)×830 mm(高)
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-04-29
  • 录用日期:  2022-07-14
  • 修回日期:  2022-06-30
  • 网络出版日期:  2022-08-03

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