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基于自相位调制光谱选择驱动的无标记自发荧光多倍频显微镜系统

王晓英 邢宇婷 陈润植 贾雪琦 吴继华 江进 李连勇 常国庆

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基于自相位调制光谱选择驱动的无标记自发荧光多倍频显微镜系统

王晓英, 邢宇婷, 陈润植, 贾雪琦, 吴继华, 江进, 李连勇, 常国庆

Simultaneous label-free autofluorescence-multiharmonic microscopy driven by femtosecond sources based on self-phase modulation enabled spectral selection

Wang Xiao-Ying, Xing Yu-Ting, Chen Run-Zhi, Jia Xue-Qi, Wu Ji-Hua, Jiang Jin, Li Lian-Yong, Chang Guo-Qing
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  • 由飞秒激光驱动的非线性光学显微镜技术在医学组织成像中具有很多独特的优势, 包括多种成像模态、高对比度、高分辨率和无标记的深层光学切片能力等. 由于缺乏波长可灵活调谐的飞秒激发光源, 导致在多模态成像的同时难以兼顾每种模态的对比度, 从而制约了非线性光学显微镜在医学诊断中的广泛应用. 本文采用基于自相位调制光谱选择的光纤激光光源, 获得了中心波长在990—1110 nm范围内可调谐的高能量飞秒脉冲, 并用于驱动非线性光学显微镜. 采用990 nm的飞秒脉冲, 通过双光子激发荧光和二倍频对胃组织成像, 进一步结合图像拼接技术成功获得了胃组织的双模态大视场图像; 利用1110 nm的飞秒脉冲, 实现了无标记自发荧光多倍频显微镜技术, 同时高效激发了胃组织的双光子激发荧光、三光子激发荧光、二倍频和三倍频信号, 获得了胃组织的多模态图像.
    Nonlinear optical microscopy technique has unique advantages in tissue imaging, such as enhanced contrast, high resolution, and label-free deep optical sectioning capabilities. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we propose a new wavelength conversion approach—self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. The SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using the suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this work, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with >5-nJ energy and ~84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain a large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources becomes a powerful tool in biomedical imaging.
      通信作者: 吴继华, jh_02821@126.com ; 常国庆, guoqing.chang@iphy.ac.cn
    • 基金项目: 战略支援部队特色医学中心医药卫生临床创新探索课题(批准号: 19ZX40)、国家自然科学基金(批准号: 11774234)和用于三维生物模型大深度和高精度成像的新型多光子显微镜研制项目(批准号: YJKYYQ20190034)资助的课题
      Corresponding author: Wu Ji-Hua, jh_02821@126.com ; Chang Guo-Qing, guoqing.chang@iphy.ac.cn
    • Funds: Project supported by the Strategic Support Force Medical Center Clinical Innovation Topics for Medicine and health, China (Grant No. 19ZX40), the National Natural Science Foundation of China (Grant No. 11774234), and the Program of Development of a New Multiphoton Microscope for Large Depth and High Precision Imaging of Three-dimensional Biological Models, China (Grant No. YJKYYQ20190034)
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    Masters B R, So P T C, Gratton E 1997 Biophys. J. 72 2405Google Scholar

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    König K, Riemann I 2003 J. Biomed. Opt. 8 432Google Scholar

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    König K, Ehlers A, Stracke F, Riemann I 2006 Skin Pharmacol. Physiol. 19 78Google Scholar

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    König K, Ehlers A, Riemann I, Schenkl S, Bückle R, Kaatz M 2007 Microsc. Res. Tech. 70 398Google Scholar

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    Paoli J, Smedh M, Wennberg A M, Ericson M B 2008 J. Invest. Dermatol. 128 1248Google Scholar

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    Breunig H G, Studier H, König K 2010 Opt. Express 18 7857Google Scholar

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    El Madani H A, Tancrède-Bohin E, Bensussan A, Colonna A, Dupuy A, Bagot M, Pena A M 2012 J. Biomed. Opt. 17 026009Google Scholar

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    Balu M, Mazhar A, Hayakawa C K, Mittal R, Krasieva T B, König K, Venugopalan V, Tromberg B J 2013 Biophys. J. 104 258Google Scholar

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    Cahill L C, Giacomelli M G, Tadayuki Y 2018 Lab. Invest. 98 150Google Scholar

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    Cahill L C, Fujimoto J G, Giacomelli M G 2019 Mod. Pathol. 32 1158Google Scholar

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    Sun C K, Chien T K, Ming L W 2019 J. Biophotonics 12 1

    [13]

    You S, Tu H, Chaney E J, Sun Y, Zhao Y, Bower A J, Liu Y Z, Marjanovic M, Sinha S, Pu Y, Boppart S A 2018 Nat. Commun. 9 2125Google Scholar

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    Brown E, McKee T, diTomaso E, Pluen A, Seed B, Boucher Y, Jain R K 2003 Nat. Med. 9 796Google Scholar

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    Sun C K, Chen C C, Chu S W, Tsai T H, Chen Y C, Lin B L 2003 Opt. Lett. 28 2488Google Scholar

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    Chen S Y, Wu H Y, Sun C K 2009 J. Biomed. Opt. 14 060505Google Scholar

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    Yasui T, Takahashi Y, Ito M, Fukushima S, Araki T 2009 Appl. Opt. 48 D88Google Scholar

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    Chen S Y, Chen S U, Wu H Y, Lee W J, Liao Y H, Sun C K 2010 IEEE J. Sel. Top. Quantum Electron. 16 478Google Scholar

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    Tsai N R, Chen S Y, Shieh D B, Lou P J, Sun C K 2011 Biomed. Opt. Express 2 2317Google Scholar

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    Zumbusch A, Holtom G R, Xie X S 1999 Phys. Rev. Lett. 82 4142Google Scholar

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    Hellerer T, Enejder A M, Zumbusch A 2004 Appl. Phys. Lett. 85 25Google Scholar

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    Legesse F B, Medyukhina A, Heuke S, Popp J 2015 Comput. Med. Imaging Graph. 43 36Google Scholar

    [24]

    Freudiger C W, Min W, Saar B G, Lu S, Holtom G R, He C, Tsai J C, Kang J X, Xie X S 2008 Science 322 1857Google Scholar

    [25]

    Ji M, Lewis S, Camelo-Piragua S, Ramkissoon S H, Snuderl M, Venneti S, Fisher-Hubbard A, Garrard M, Fu D, Wang A C, Heth J A, Maher C O, Sanai N, Johnson T D, Freudiger C W, Sagher O, Xie X S, Orringer D A 2015 Sci. Transl. Med. 7 309ra163Google Scholar

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    Georgakoudi I, Quinn K P 2012 Annu. Rev. Biomed. Eng. 14 351Google Scholar

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    Chang T, Zimmerley S, Quinn K P, Jouenne I L, Kaplan D L, Beaurepaire E, Georgakoudi I 2013 Biomaterials 34 8607Google Scholar

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    Hou J, Wright H J, Chan N S K, Tran R D H, Razorenova O V, Potma E O, Tromberg B J 2016 J Biomed Opt. 21 060503Google Scholar

    [29]

    Huang S H, Heikal A A, Webb W W 2002 Biophys. J. 82 2811Google Scholar

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    Zoumi A, Yeh A, Tromberg B J 2002 Proc. Natl. Acad. Sci. U. S. A. 99 11014Google Scholar

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    Zipfel W R, Williams R M, Christie R, Nikitin A Y, Hyman B T, Webb W W 2003 Proc. Natl. Acad. Sci. U. S. A. 100 7075Google Scholar

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    Chu S W, Chen I H, Liu T M, Chen P C, Sun C K, Lin B L 2001 Opt. Lett. 26 1909Google Scholar

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    Xu C, Wise F W 2013 Nat. Photonics 7 875Google Scholar

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    Lim H, Ilday F O, Buckley J, Chong A, Wise F W 2004 Electron. Lett. 40 1523Google Scholar

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    Takayanagi J, Sugiura T, Yoshida M, Nishizawa N 2006 IEEE Photonics Technol. Lett. 18 2284Google Scholar

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    Li K C, Huang L L H, Liang J H, Chang M C 2016 Biomed. Opt. Express 7 4803Google Scholar

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    Chen H W, Haider Z, Lim J, Xu S, Yang Z, Kärtner F X, Chang G, 2013 Opt. Lett. 38 4927Google Scholar

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    Chan M C, Lien C H, Lu J Y, Lyu B H 2014 Opt. Express 22 9498Google Scholar

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    Tauser F, Adler F, Leitenstorfer A 2004 Opt. Lett. 29 516Google Scholar

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    Tu H, Lægsgaard J, Zhang R, Tong S, Liu Y, Boppart S A 2013 Opt. Express 21 23188Google Scholar

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    Liu Y, Tu H, Benalcazar W A, Chaney E J, Boppart S A 2012 IEEE J. Sel. Top. Quantum Electron. 18 1209Google Scholar

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    Gottschall T, Meyer T, Schmitt M, Popp J, Limpert J, Tünnermann A 2015 Opt. Express 23 23968Google Scholar

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    Liu W, Li C, Zhang Z, Kärtner F X, Chang G Q 2016 Opt. Express 24 15328Google Scholar

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    Liu W, Chia S H, Chung H Y, Greinert R, Kärtner F X, Chang G Q 2017 Opt. Express 25 6822Google Scholar

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    Chung H Y, Liu W, Cao Q, Kärtner F X, Chang G Q 2017 Opt. Express 25 15760Google Scholar

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    Chung H Y, Greinert R, Kärtner F X, Chang G Q 2019 Biomed. Opt. Express 10 514Google Scholar

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    Herz J, Siffrin V, Hauser A E, Brandt A U, Leuenberger T, Radbruch H, Zipp F, Niesne R A 2010 Biophys. J. 98 715Google Scholar

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  • 图 1  基于SESS的非线性光学显微镜实验装置. ISO: 隔离器, LD: 二极管泵浦激光源, WDM: 波分复用器, Amp: 光纤放大器, HWP: 半波片, PBS: 偏振分束器, L: 透镜, LPF: 长通滤波器, NLOM: 非线性光学显微镜

    Fig. 1.  Schematic setup of the nonlinear optical microscopy driven by SESS. ISO: isolator, LD: laser diode, WDM: wavelength-division multiplexing, Amp: amplifier, HWP: half-wave plate, PBS: polarization beam splitter, L: lens, LPF: long pass filter, NLOM: nonlinear optical microscopy.

    图 2  放大脉冲的光谱和自相关轨迹 (a)光谱; (b)自相关轨迹. 红色曲线: 测量得到的自相关轨迹, 黑色曲线: 通过光谱计算得到的变换极限脉冲的自相关轨迹

    Fig. 2.  Spectrum and autocorrelation trace of the amplified pulse: (a) Spectrum; (b) autocorrelation trace. Red curve: measured autocorrelation trace. Black curve: calculated from the transform-limited pulses allowed by the amplified pulse spectrum.

    图 3  当耦合到光纤里的能量为44 nJ时输出的展宽光谱以及990 nm处滤波得到的脉冲光谱和自相关轨迹 (a)展宽光谱; (b)自相关轨迹. 红色曲线: 测量得到的自相关轨迹, 黑色曲线: 通过光谱计算得到的变换极限脉冲的自相关轨迹. 插图: 990 nm处滤波得到的脉冲光谱

    Fig. 3.  Spectrum broadening with coupled energy of 44 nJ, spectrum and autocorrelation trace of the filtered pulses at 990 nm: (a) Broadened spectrum; (b) autocorrelation trace. Red curve: measured autocorrelation trace. Black curve: calculated from the transform-limited pulses allowed by the spectrum at 990 nm. Inset: filtered spectrum at 990 nm.

    图 4  当耦合到光纤里的能量为50 nJ时输出的展宽光谱以及1110 nm处滤波得到的脉冲光谱和自相关曲线 (a)展宽光谱; (b)自相关轨迹. 红色曲线: 测量得到的自相关轨迹, 黑色曲线: 通过光谱计算得到的变换极限脉冲的自相关轨迹, 插图: 1110 nm处滤波得到的脉冲光谱

    Fig. 4.  Spectrum broadening with coupled energy of 50 nJ, spectrum and autocorrelation trace of the filtered pulses at 1110 nm: (a) Broadened spectrum; (b) autocorrelation trace. Red curve: measured autocorrelation trace. Black curve: calculated from the transform-limited pulses allowed by the spectrum at 1110 nm. Inset: filtered spectrum at 1110 nm.

    图 5  离体人体胃组织的2PEF/SHG图像 (a) 2PEF图像; (b) SHG图像; (c) 2PEF和SHG的叠加图像. 白色箭头: 上皮细胞; 黄色箭头: 胶原蛋白. 比例尺: 100 μm

    Fig. 5.  2PEF/SHG images of ex vivo human gastric tissue: (a) 2PEF image; (b) SHG image; (c) merging of 2PEF and SHG images. White arrow: epithelial cells; yellow arrow: collagen. Scale bar: 100 μm.

    图 6  离体人体胃组织的2PEF/SHG/3PEF/THG图像 (a) 2PEF图像; (b) SHG图像; (c) 3PEF图像; (d) THG图像; (e) 2PEF/SHG/3PEF/THG的叠加图像. 粉色箭头: 弹力纤维; 黄色箭头: 胶原纤维; 白色箭头: 脂肪细胞. 比例尺: 100 μm.

    Fig. 6.  2PEF/SHG/3PEF/THG images of ex vivo human gastric tissue: (a) 2PEF image; (b) SHG image; (c) 3PEF image; (d) THG image; (e) merging of 2PEF/SHG/3PEF/THG images. Pink arrow: elastic; yellow arrow: collagen; white arrow: adipocyte. Scale bar: 100 μm.

  • [1]

    Hanson K M, Bardeen C J 2009 Photochem. Photobiol. 85 33Google Scholar

    [2]

    Masters B R, So P T C, Gratton E 1997 Biophys. J. 72 2405Google Scholar

    [3]

    König K, Riemann I 2003 J. Biomed. Opt. 8 432Google Scholar

    [4]

    König K, Ehlers A, Stracke F, Riemann I 2006 Skin Pharmacol. Physiol. 19 78Google Scholar

    [5]

    König K, Ehlers A, Riemann I, Schenkl S, Bückle R, Kaatz M 2007 Microsc. Res. Tech. 70 398Google Scholar

    [6]

    Paoli J, Smedh M, Wennberg A M, Ericson M B 2008 J. Invest. Dermatol. 128 1248Google Scholar

    [7]

    Breunig H G, Studier H, König K 2010 Opt. Express 18 7857Google Scholar

    [8]

    El Madani H A, Tancrède-Bohin E, Bensussan A, Colonna A, Dupuy A, Bagot M, Pena A M 2012 J. Biomed. Opt. 17 026009Google Scholar

    [9]

    Balu M, Mazhar A, Hayakawa C K, Mittal R, Krasieva T B, König K, Venugopalan V, Tromberg B J 2013 Biophys. J. 104 258Google Scholar

    [10]

    Cahill L C, Giacomelli M G, Tadayuki Y 2018 Lab. Invest. 98 150Google Scholar

    [11]

    Cahill L C, Fujimoto J G, Giacomelli M G 2019 Mod. Pathol. 32 1158Google Scholar

    [12]

    Sun C K, Chien T K, Ming L W 2019 J. Biophotonics 12 1

    [13]

    You S, Tu H, Chaney E J, Sun Y, Zhao Y, Bower A J, Liu Y Z, Marjanovic M, Sinha S, Pu Y, Boppart S A 2018 Nat. Commun. 9 2125Google Scholar

    [14]

    Brown E, McKee T, diTomaso E, Pluen A, Seed B, Boucher Y, Jain R K 2003 Nat. Med. 9 796Google Scholar

    [15]

    Sun C K, Chen C C, Chu S W, Tsai T H, Chen Y C, Lin B L 2003 Opt. Lett. 28 2488Google Scholar

    [16]

    Tai S P, Tsai T H, Lee W J, Shieh D B, Liao Y H, Huang H Y, Zhang K, Liu H L, Sun C K 2005 Opt. Express 13 8231Google Scholar

    [17]

    Chen S Y, Wu H Y, Sun C K 2009 J. Biomed. Opt. 14 060505Google Scholar

    [18]

    Yasui T, Takahashi Y, Ito M, Fukushima S, Araki T 2009 Appl. Opt. 48 D88Google Scholar

    [19]

    Chen S Y, Chen S U, Wu H Y, Lee W J, Liao Y H, Sun C K 2010 IEEE J. Sel. Top. Quantum Electron. 16 478Google Scholar

    [20]

    Tsai N R, Chen S Y, Shieh D B, Lou P J, Sun C K 2011 Biomed. Opt. Express 2 2317Google Scholar

    [21]

    Zumbusch A, Holtom G R, Xie X S 1999 Phys. Rev. Lett. 82 4142Google Scholar

    [22]

    Hellerer T, Enejder A M, Zumbusch A 2004 Appl. Phys. Lett. 85 25Google Scholar

    [23]

    Legesse F B, Medyukhina A, Heuke S, Popp J 2015 Comput. Med. Imaging Graph. 43 36Google Scholar

    [24]

    Freudiger C W, Min W, Saar B G, Lu S, Holtom G R, He C, Tsai J C, Kang J X, Xie X S 2008 Science 322 1857Google Scholar

    [25]

    Ji M, Lewis S, Camelo-Piragua S, Ramkissoon S H, Snuderl M, Venneti S, Fisher-Hubbard A, Garrard M, Fu D, Wang A C, Heth J A, Maher C O, Sanai N, Johnson T D, Freudiger C W, Sagher O, Xie X S, Orringer D A 2015 Sci. Transl. Med. 7 309ra163Google Scholar

    [26]

    Georgakoudi I, Quinn K P 2012 Annu. Rev. Biomed. Eng. 14 351Google Scholar

    [27]

    Chang T, Zimmerley S, Quinn K P, Jouenne I L, Kaplan D L, Beaurepaire E, Georgakoudi I 2013 Biomaterials 34 8607Google Scholar

    [28]

    Hou J, Wright H J, Chan N S K, Tran R D H, Razorenova O V, Potma E O, Tromberg B J 2016 J Biomed Opt. 21 060503Google Scholar

    [29]

    Huang S H, Heikal A A, Webb W W 2002 Biophys. J. 82 2811Google Scholar

    [30]

    Zoumi A, Yeh A, Tromberg B J 2002 Proc. Natl. Acad. Sci. U. S. A. 99 11014Google Scholar

    [31]

    Zipfel W R, Williams R M, Christie R, Nikitin A Y, Hyman B T, Webb W W 2003 Proc. Natl. Acad. Sci. U. S. A. 100 7075Google Scholar

    [32]

    Chu S W, Chen I H, Liu T M, Chen P C, Sun C K, Lin B L 2001 Opt. Lett. 26 1909Google Scholar

    [33]

    Xu C, Wise F W 2013 Nat. Photonics 7 875Google Scholar

    [34]

    Lim H, Ilday F O, Buckley J, Chong A, Wise F W 2004 Electron. Lett. 40 1523Google Scholar

    [35]

    Takayanagi J, Sugiura T, Yoshida M, Nishizawa N 2006 IEEE Photonics Technol. Lett. 18 2284Google Scholar

    [36]

    Li K C, Huang L L H, Liang J H, Chang M C 2016 Biomed. Opt. Express 7 4803Google Scholar

    [37]

    Chen H W, Haider Z, Lim J, Xu S, Yang Z, Kärtner F X, Chang G, 2013 Opt. Lett. 38 4927Google Scholar

    [38]

    Chan M C, Lien C H, Lu J Y, Lyu B H 2014 Opt. Express 22 9498Google Scholar

    [39]

    Tauser F, Adler F, Leitenstorfer A 2004 Opt. Lett. 29 516Google Scholar

    [40]

    Tu H, Lægsgaard J, Zhang R, Tong S, Liu Y, Boppart S A 2013 Opt. Express 21 23188Google Scholar

    [41]

    Liu Y, Tu H, Benalcazar W A, Chaney E J, Boppart S A 2012 IEEE J. Sel. Top. Quantum Electron. 18 1209Google Scholar

    [42]

    Gottschall T, Meyer T, Schmitt M, Popp J, Limpert J, Tünnermann A 2015 Opt. Express 23 23968Google Scholar

    [43]

    Liu W, Li C, Zhang Z, Kärtner F X, Chang G Q 2016 Opt. Express 24 15328Google Scholar

    [44]

    Liu W, Chia S H, Chung H Y, Greinert R, Kärtner F X, Chang G Q 2017 Opt. Express 25 6822Google Scholar

    [45]

    Chung H Y, Liu W, Cao Q, Kärtner F X, Chang G Q 2017 Opt. Express 25 15760Google Scholar

    [46]

    Chung H Y, Liu W, Cao Q, Song L W, Kärtner F X, Chang G Q 2018 Opt. Express 26 3684Google Scholar

    [47]

    Chung H Y, Liu W, Cao Q, Greinert R, Kärtner F X, Chang G Q 2019 IEEE J. Sel. Top. Quantum Electron 25 6800708Google Scholar

    [48]

    Chung H Y, Greinert R, Kärtner F X, Chang G Q 2019 Biomed. Opt. Express 10 514Google Scholar

    [49]

    Herz J, Siffrin V, Hauser A E, Brandt A U, Leuenberger T, Radbruch H, Zipp F, Niesne R A 2010 Biophys. J. 98 715Google Scholar

    [50]

    Bissell M J, Radisky D 2001 Nat. Rev. Cancer 1 46Google Scholar

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出版历程
  • 收稿日期:  2021-12-09
  • 修回日期:  2022-01-12
  • 上网日期:  2022-02-10
  • 刊出日期:  2022-05-20

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