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双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

侯国辉 罗腾 陈秉灵 刘杰 林子扬 陈丹妮 屈军乐

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双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

侯国辉, 罗腾, 陈秉灵, 刘杰, 林子扬, 陈丹妮, 屈军乐

Experimental study on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy

Hou Guo-Hui, Luo Teng, Chen Bing-Ling, Liu Jie, Lin Zi-Yang, Chen Dan-Ni, Qu Jun-Le
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  • 双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.
    Two-photon excitation fluorescence (2PEF) and coherent anti-Stokes Raman scattering (CARS) are both third-order nonlinear optical processes, but for a long time, the true relationship and differences between them are not clearly understood. For decades, the second harmonic generation has been studied in conjunction with two-photon excitation fluorescence, so it was thought that the latter was a second-order nonlinear optical process. In order to make the two nonlinear interaction processes clear enough, the two nonlinear interaction processes are worthy to study at the same time. In this paper, firstly, we give the relationships between the 2PEF, CARS signal and their third-order nonlinear susceptibility, respectively; secondly, we use our own near infrared super-continuum CARS microscopy system to study both processes. In doing so, we describe the relationship between their third-order nonlinear susceptibility and the signal. The reconstructed images derived from CARS and those derived from 2PEF differ significantly when imaging the same 1.01 $\muup$m fluorescence polystyrene beads. If the lateral spatial resolution of the CARS imaging system is larger than the fluorescence polystyrene beads, the measured size cannot be used to calculate the real spatial resolution of the CARS system. However, the resolution of the 2PEF microscopy system can be obtained through the de-convolution of the 2PEF image, which is approximately equivalent to the current resolution of the CARS imaging system, which is measured using 280 nm polystyrene beads. The images of 280 nm polystyrene beads and 190 nm fluorescent polystyrene beads also exhibit differences between the two samples and the environment around them, respectively. This means that although CARS and 2PEF are both third-order nonlinear optical processes, they have their own properties. In particular, CARS is a third-order nonlinear optical oscillation process which is caused by the phasing match condition, but 2PEF is not influenced by the phasing match condition. The phase matching condition is responsible for the differences around the sample in the images of the 280 nm pure polystyrene beads, but not for the 190 nm fluorescent polystyrene beads. The de-convolution results for the 1.01 $\muup$m fluorescence polystyrene beads and the 280 nm pure polystyrene beads are very similar, so we can use the de-convolution results for 2PEF by the 1.01 $\muup$m fluorescence polystyrene beads to approximate the current measure condition and the resolution of the CARS imaging system. If we want to gain a more accurate resolution from the CARS imaging system, the spherical sample should be smaller than the lateral spatial resolution of this system.
      通信作者: 陈丹妮, danny@szu.edu.cn;jlqu@szu.edu.cn ; 屈军乐, danny@szu.edu.cn;jlqu@szu.edu.cn
    • 基金项目: 国家重大科学仪器设备开发专项(批准号:2012YQ15009203)、国家自然科学基金(批准号:61235012)和国家重点基础研究发展计划(批准号:2015CB352005,2012CB825802)资助的课题.
      Corresponding author: Chen Dan-Ni, danny@szu.edu.cn;jlqu@szu.edu.cn ; Qu Jun-Le, danny@szu.edu.cn;jlqu@szu.edu.cn
    • Funds: Project supported by the Special Funds of the Major Scientific Instruments Equipment Development of China (Grant No. 2012YQ15009203), the National Natural Science Foundation of China (Grant No. 61235012), and the National Basic Research Program of China (Grant Nos. 2015CB352005, 2012CB825802).
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  • [1]

    Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp412-435

    [2]

    Potma E O, Xie X S N 2008 Handbook of Biomedical Nonlinear Optical Microscopy (New York: Oxford University Press) pp164-186

    [3]

    Zhang D, Slipchenko M N, Cheng J X 2011 Phys. Chem. Lett. 2 1248

    [4]

    Nestor J R 1978 Chem. Phys. 69 1778

    [5]

    Göeppert-Mayer M 1931 Ann. Phys. 9 273

    [6]

    So P T C, Dong C Y, Masters B R, Berland K M 2000 Ann. Rev. BioMed. Eng. 2 399

    [7]

    Song J J, Eesley G L, Levenson M D 1976 Appl. Phys. Lett. 29 567

    [8]

    Lotem H, Lynch R T J, Bloembergen N 1976 Phy. Rev. A 14 1748

    [9]

    Oudar J L, Smith R W, Shen Y R 1979 Appl. Phys. Lett. 34 758

    [10]

    Lee Y J, Cicerone M T 2008 Appl. Phys. Lett. 92 15

    [11]

    Isobe K, Kawano H, Takeda T, Suda A, Kumagai A, Mizuno H, Miyawaki A, Midorikawa K 2012 Biomed. Opt. Express 3 1594

    [12]

    Cheng J X, Volkmer A, Xie X S 2002 Opt. Soc. Am. B 19 1363

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  • 收稿日期:  2017-01-12
  • 修回日期:  2017-03-13
  • 刊出日期:  2017-05-05

双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

    基金项目: 国家重大科学仪器设备开发专项(批准号:2012YQ15009203)、国家自然科学基金(批准号:61235012)和国家重点基础研究发展计划(批准号:2015CB352005,2012CB825802)资助的课题.

摘要: 双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.

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