物理化学学报 >> 2016, Vol. 32 >> Issue (5): 1123-1128.doi: 10.3866/PKU.WHXB201603234

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基于散射信号的超分辨光学相减显微镜

周前1,2,3,于建强4,赵立波3,李德胜5,吴魁6,朱建华1,2,袁景和3,*(),方晓红3   

  1. 1 四川大学物理学系,成都610064
    2 高能量密度物理及技术教育部重点实验室,成都610064
    3 中国科学院化学研究所分子纳米结构与纳米技术实验室,北京100190
    4 北方工业大学理学院,北京100144
    5 中国科学院化学研究所高分子物理与化学实验室,北京100190
    6 中国科学院化学研究所活体分析化学实验室,北京100190
  • 收稿日期:2016-02-25 发布日期:2016-05-07
  • 通讯作者: 袁景和 E-mail:jhyuan@iccas.ac.cn
  • 基金资助:
    国家重点基础研究发展规划项目(973)(2013CB933701);国家自然科学基金(21127901);国家自然科学基金(91413119);中国科学院“关键技术人才”项目

Super-Resolution Optical Subtraction Microscopy Using Optical Scattering Imaging

Qian ZHOU1,2,3,Jian-Qiang YU4,Li-Bo ZHAO3,De-Sheng LI5,Kui WU6,Jian-Hua ZHU1,2,Jing-He YUAN3,*(),Xiao-Hong FANG3   

  1. 1 Department of Physics, Sichuan University, Chengdu 610064, P. R. China
    2 Key Laboratory of High Energy Density Physics and Technology of Ministry of Education, Chengdu 610064, P. R. China
    3 Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China
    4 College of Science, North China University of Technology, Beijing 100144, P. R. China
    5 State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China
    6 Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China
  • Received:2016-02-25 Published:2016-05-07
  • Contact: Jing-He YUAN E-mail:jhyuan@iccas.ac.cn
  • Supported by:
    the National Key Basic Research Program of China (973)(2013CB933701);National Natural Science Foundation of China(21127901);National Natural Science Foundation of China(91413119);Key Technology Talent Program of Chinese Academy of Sciences

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摘要：

现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力。因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率。不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响。随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ, 1λ = 650 nm),并且通过探测散射信号得到更多的样品细节信息。

关键词: 超分辨显微镜, 图像相减, 非荧光成像, 光散射成像

Abstract:

The existing form of super-resolution microscopy based on specific fluorescent tagging is unable to obtain super-resolution images of non-fluorescent samples. Hence, we have developed optical subtraction microscopy for obtaining super-resolution imaging in such cases. This method is based on image subtraction between the two optical scattering images from general confocal excitation and doughnut-shaped excitation, respectively. Unlike super-resolution fluorescence microscopy, subtraction microscopy requires no preprocessing of the sample, and the excitation power can be kept low to avoid sample damage. The non-fluorescent imaging of gold nanobeads and polymer nanofibers has been realized to demonstrate the feasibility of super-resolution subtraction microscopy. The lateral resolution decreases to 215 nm (0.33λ, 1λ = 650 nm) in subtraction imaging, and greater imaging detail of the sample is achieved via optical scattering.

Key words: Super-resolution microscopy, Subtraction imaging, Non-fluorescence imaging, Optical scattering imaging

MSC2000: 

  • O644