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Nonresonant background suppression in wide-field Coherent anti-Stokes Raman scattering microscopy with transport of intensity equation based phase imaging

Zheng Juan-Juan Yao Bao-Li Shao Xiao-Peng

Nonresonant background suppression in wide-field Coherent anti-Stokes Raman scattering microscopy with transport of intensity equation based phase imaging

Zheng Juan-Juan, Yao Bao-Li, Shao Xiao-Peng
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  • Coherent anti-Stokes Raman scattering (CARS) microscopy is a valuable tool for label-free imaging of biological samples, since it enables providing contrast via vibrational resonances of a specific chemical bond. However, in a conventional CARS image the Raman resonant anti-Stokes radiation is often superimposed by a nonresonant contribution arising from the electronic part of the polarization. The situation becomes worse if a sample is composed of a significant amount of water, where a strong nonresonant background over the whole image is obtained.To date, various approaches including Epi, polarization sensitive, time-resolved, and CARS phase imaging have been implemented to suppress the undesirable nonresonant background in CARS microscopy. Notably, optical heterodyne based phase imaging schemes are of particular interest due to their intrinsic ability to retrieve Im(χ(3)), which is proportional to the Raman resonant signal. Nevertheless, all the reported phase imaging methods that require an independent reference wave lead to an increase in the setup complexity, thus making the measurement sensitive to external perturbations. In order to simplify the setup, single-beam scheme has also been utilized for vibrational CARS imaging by using wave-front sensors to acquire the phase of the complex anti-Stokes amplitude. However, this method demands highly accurate wave-front sensors.In this paper we present a reference-less CARS phase imaging technique to suppress nonresonant CARS background based on transport of intensity equation (TIE). Resonant CARS radiation ECARSR can be obtained when the frequency difference between the pump and Stokes beams is tuned to match a molecular vibration frequency (Raman resonant mode). In contrast, the nonresonant background ECARSNR can be obtained when the frequency difference between the pump and Stokes beams does not match a molecular vibration frequency (Raman resonant mode). Considering the fact that there is a phase shift of π/2 between the resonant and non-resonant CARS field, the phase imaging of both resonant and nonresonant CARS field can provide a background-free image. In implementation, three intensity images of the CARS field under resonant mode are recorded at three neighboring planes by moving the CCD camera along the axial direction. In the meantime, three images of the CARS field under non-resonant mode are also recorded. Considering the fact that the TIE links the intensity distributions in three neighboring planes (through which a beam transverses) with the phase distribution of the field, the phase images of the CARS field under both resonant and nonresonant modes are reconstructed from the recorded intensity images. The phase difference φχ between the resonant CARS field and the non-resonant CARS field is calculated. Eventually, the CARS background is efficiently suppressed by using the relation ICARSbf≅ICARSR·sin2φχ.Compared with conventional CARS background suppression techniques, the proposed method is robust against environmental disturbance, since it does not require an additional reference beam. Furthermore, the proposed method is easy to incorporate in a conventional CARS configuration. Therefore, the proposed method has the potential to become a versatile technique to image deep tissue with low background signal.
      Corresponding author: Yao Bao-Li, yaobl@opt.ac.cn
    • Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 61605150, 61475187, 61575154, 61377008), the Fundamental Research Funds for the Central Universities, China (Grant Nos. JB160511, XJS16005, JBG160502), the '
    [1]

    Chen T, Yu Z L, Zhang X N, Xie X S, Huang Y Y 2011 Sci. China: Chem. 41 1 (in Chinese) [陈涛, 虞之龙, 张先念, 谢晓亮, 黄岩谊 2011 中国科学: 化学 41 1]

    [2]

    Zhang S W, Chen D N, Liu S L, Liu W, Niu H B 2015 Acta Phys. Sin. 64 223301 (in Chinese) [张赛文, 陈丹妮, 刘双龙, 刘伟, 牛憨笨 2015 物理学报 64 223301]

    [3]

    Volkmer A, Cheng J X, Xie X S 2001 Phys. Rev. Lett. 87 023901

    [4]

    Cheng J X, Book L D, Xie X S 2001 Opt. Lett. 26 1341

    [5]

    Volkmer A, Book L D, Xie X S 2002 Appl. Phys. Lett. 80 1505

    [6]

    Krishnamachari V V, Potma E O 2007 J. Opt. Soc. Am. A 24 1138

    [7]

    Potma E O, Evans C L, Xie X S 2006 Opt. Lett. 31 241

    [8]

    Jurna M, Korterik J P, Otto C, Offerhaus H L 2007 Opt. Express 15 15207

    [9]

    Jurna M, Korterik J P, Otto C, Herek J L, Offerhaus H L 2008 Opt. Express 16 15863

    [10]

    Jurna M, Korterik J P, Otto C, Herek J L, Offerhaus H L 2009 Phys. Rev. Lett. 103 043905

    [11]

    Evans C L, Potma E O, Xie X S 2004 Opt. Lett. 29 2923

    [12]

    Akimov D, Chatzipapadopoulos S, Meyer T, Tarcea N, Dietzek B, Schmitt M, Popp J 2009 J. Raman Spectrosc. 40 941

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    Berto P, Gachet D, Bon P, Monneret S, Rigneault H 2012 Phys. Rev. Lett. 109 093902

    [14]

    Berto P, Jesacher A, Roider C, Monneret S, Rigneault H, Ritsch-Marte M 2013 Opt. Lett. 38 709

    [15]

    Zheng J, Akimov D, Heuke S, Schmitt M, Yao B, Ye T, Lei M, Gao P, Popp J 2015 Opt. Express 23 10756

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    Teague M R 1983 J. Opt. Soc. Am. 73 1434

    [17]

    Roddier F 1988 Appl. Opt. 27 1223

    [18]

    Nugent K A, Gureyev T E, Cookson D F, Paganin D, Barnea Z 1996 Phys. Rev. Lett. 77 2961

    [19]

    McMahon P J, Allman B E, Jacobson D L, Arif M, Werner S A, Nugent K A 2003 Phys. Rev. Lett. 91 145502

    [20]

    Bajt S, Barty A, Nugent K A, McCartney M, Wall M, Paganin D 2000 Ultramicroscopy 83 67

    [21]

    Kou S S, Waller L, Barbastathis G, Sheppard C J R 2010 Opt. Lett. 35 447

    [22]

    Gorthi S S, Schonbrun E 2012 Opt. Lett. 37 707

    [23]

    Zuo C, Chen Q, Sun J S, Asund A 2016 Chin. J. Lasers 43 0609002 (in Chinese) [左超, 陈钱, 孙佳嵩, Asund A 2016 中国激光 43 0609002]

    [24]

    Teague M R 1982 J. Opt. Soc. Am. 72 1199

    [25]

    Frank J, Altmeyer S, Wernicke G 2010 J. Opt. Soc. Am. A 27 2244

    [26]

    Zuo C, Chen Q, Yu Y, Asundi A 2013 Opt. Express 21 5346

    [27]

    Frankot R T, Chellappa Z 1988 IEEE Trans. Patt. Anal. Mach. Intell. 10 439

    [28]

    Gao P, Pedrini G, Osten W 2013 Opt. Lett. 38 5204

    [29]

    Gao P, Pedrini G, Zuo C, Osten W 2014 Opt. Lett. 39 3615

    [30]

    Shi K, Li H, Xu Q, Psaltis D, Liu Z 2010 Phys. Rev. Lett. 104 093902

    [31]

    Gao P, Pedrini G, Osten W 2013 Opt. Lett. 38 1328

    [32]

    Popescu G, Ikeda T, Goda K, Best-Popescu C A, Laposata M, Manley S, Dasari R R, Badizadegan K, Feld M S 2006 Phys. Rev. Lett. 97 218101

    [33]

    Alexandrov S A, Hillman T R, Gutzler T, Sampson D D 2006 Phys. Rev. Lett. 97 168102

    [34]

    Barty A, Nugent K A, Paganin D, Roberts A 1998 Opt. Lett. 23 817

  • [1]

    Chen T, Yu Z L, Zhang X N, Xie X S, Huang Y Y 2011 Sci. China: Chem. 41 1 (in Chinese) [陈涛, 虞之龙, 张先念, 谢晓亮, 黄岩谊 2011 中国科学: 化学 41 1]

    [2]

    Zhang S W, Chen D N, Liu S L, Liu W, Niu H B 2015 Acta Phys. Sin. 64 223301 (in Chinese) [张赛文, 陈丹妮, 刘双龙, 刘伟, 牛憨笨 2015 物理学报 64 223301]

    [3]

    Volkmer A, Cheng J X, Xie X S 2001 Phys. Rev. Lett. 87 023901

    [4]

    Cheng J X, Book L D, Xie X S 2001 Opt. Lett. 26 1341

    [5]

    Volkmer A, Book L D, Xie X S 2002 Appl. Phys. Lett. 80 1505

    [6]

    Krishnamachari V V, Potma E O 2007 J. Opt. Soc. Am. A 24 1138

    [7]

    Potma E O, Evans C L, Xie X S 2006 Opt. Lett. 31 241

    [8]

    Jurna M, Korterik J P, Otto C, Offerhaus H L 2007 Opt. Express 15 15207

    [9]

    Jurna M, Korterik J P, Otto C, Herek J L, Offerhaus H L 2008 Opt. Express 16 15863

    [10]

    Jurna M, Korterik J P, Otto C, Herek J L, Offerhaus H L 2009 Phys. Rev. Lett. 103 043905

    [11]

    Evans C L, Potma E O, Xie X S 2004 Opt. Lett. 29 2923

    [12]

    Akimov D, Chatzipapadopoulos S, Meyer T, Tarcea N, Dietzek B, Schmitt M, Popp J 2009 J. Raman Spectrosc. 40 941

    [13]

    Berto P, Gachet D, Bon P, Monneret S, Rigneault H 2012 Phys. Rev. Lett. 109 093902

    [14]

    Berto P, Jesacher A, Roider C, Monneret S, Rigneault H, Ritsch-Marte M 2013 Opt. Lett. 38 709

    [15]

    Zheng J, Akimov D, Heuke S, Schmitt M, Yao B, Ye T, Lei M, Gao P, Popp J 2015 Opt. Express 23 10756

    [16]

    Teague M R 1983 J. Opt. Soc. Am. 73 1434

    [17]

    Roddier F 1988 Appl. Opt. 27 1223

    [18]

    Nugent K A, Gureyev T E, Cookson D F, Paganin D, Barnea Z 1996 Phys. Rev. Lett. 77 2961

    [19]

    McMahon P J, Allman B E, Jacobson D L, Arif M, Werner S A, Nugent K A 2003 Phys. Rev. Lett. 91 145502

    [20]

    Bajt S, Barty A, Nugent K A, McCartney M, Wall M, Paganin D 2000 Ultramicroscopy 83 67

    [21]

    Kou S S, Waller L, Barbastathis G, Sheppard C J R 2010 Opt. Lett. 35 447

    [22]

    Gorthi S S, Schonbrun E 2012 Opt. Lett. 37 707

    [23]

    Zuo C, Chen Q, Sun J S, Asund A 2016 Chin. J. Lasers 43 0609002 (in Chinese) [左超, 陈钱, 孙佳嵩, Asund A 2016 中国激光 43 0609002]

    [24]

    Teague M R 1982 J. Opt. Soc. Am. 72 1199

    [25]

    Frank J, Altmeyer S, Wernicke G 2010 J. Opt. Soc. Am. A 27 2244

    [26]

    Zuo C, Chen Q, Yu Y, Asundi A 2013 Opt. Express 21 5346

    [27]

    Frankot R T, Chellappa Z 1988 IEEE Trans. Patt. Anal. Mach. Intell. 10 439

    [28]

    Gao P, Pedrini G, Osten W 2013 Opt. Lett. 38 5204

    [29]

    Gao P, Pedrini G, Zuo C, Osten W 2014 Opt. Lett. 39 3615

    [30]

    Shi K, Li H, Xu Q, Psaltis D, Liu Z 2010 Phys. Rev. Lett. 104 093902

    [31]

    Gao P, Pedrini G, Osten W 2013 Opt. Lett. 38 1328

    [32]

    Popescu G, Ikeda T, Goda K, Best-Popescu C A, Laposata M, Manley S, Dasari R R, Badizadegan K, Feld M S 2006 Phys. Rev. Lett. 97 218101

    [33]

    Alexandrov S A, Hillman T R, Gutzler T, Sampson D D 2006 Phys. Rev. Lett. 97 168102

    [34]

    Barty A, Nugent K A, Paganin D, Roberts A 1998 Opt. Lett. 23 817

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  • Received Date:  17 December 2016
  • Accepted Date:  31 March 2017
  • Published Online:  05 June 2017

Nonresonant background suppression in wide-field Coherent anti-Stokes Raman scattering microscopy with transport of intensity equation based phase imaging

    Corresponding author: Yao Bao-Li, yaobl@opt.ac.cn
  • 1. School of Physics and Optoelectronic Engineering, Xidian University, Xi'an 710071, China;
  • 2. State Key Laboratory of Transient Optics and Photonics, Xi'an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, Xi'an 710119, China
Fund Project:  Project supported by the National Natural Science Foundation of China (Grant Nos. 61605150, 61475187, 61575154, 61377008), the Fundamental Research Funds for the Central Universities, China (Grant Nos. JB160511, XJS16005, JBG160502), the '

Abstract: Coherent anti-Stokes Raman scattering (CARS) microscopy is a valuable tool for label-free imaging of biological samples, since it enables providing contrast via vibrational resonances of a specific chemical bond. However, in a conventional CARS image the Raman resonant anti-Stokes radiation is often superimposed by a nonresonant contribution arising from the electronic part of the polarization. The situation becomes worse if a sample is composed of a significant amount of water, where a strong nonresonant background over the whole image is obtained.To date, various approaches including Epi, polarization sensitive, time-resolved, and CARS phase imaging have been implemented to suppress the undesirable nonresonant background in CARS microscopy. Notably, optical heterodyne based phase imaging schemes are of particular interest due to their intrinsic ability to retrieve Im(χ(3)), which is proportional to the Raman resonant signal. Nevertheless, all the reported phase imaging methods that require an independent reference wave lead to an increase in the setup complexity, thus making the measurement sensitive to external perturbations. In order to simplify the setup, single-beam scheme has also been utilized for vibrational CARS imaging by using wave-front sensors to acquire the phase of the complex anti-Stokes amplitude. However, this method demands highly accurate wave-front sensors.In this paper we present a reference-less CARS phase imaging technique to suppress nonresonant CARS background based on transport of intensity equation (TIE). Resonant CARS radiation ECARSR can be obtained when the frequency difference between the pump and Stokes beams is tuned to match a molecular vibration frequency (Raman resonant mode). In contrast, the nonresonant background ECARSNR can be obtained when the frequency difference between the pump and Stokes beams does not match a molecular vibration frequency (Raman resonant mode). Considering the fact that there is a phase shift of π/2 between the resonant and non-resonant CARS field, the phase imaging of both resonant and nonresonant CARS field can provide a background-free image. In implementation, three intensity images of the CARS field under resonant mode are recorded at three neighboring planes by moving the CCD camera along the axial direction. In the meantime, three images of the CARS field under non-resonant mode are also recorded. Considering the fact that the TIE links the intensity distributions in three neighboring planes (through which a beam transverses) with the phase distribution of the field, the phase images of the CARS field under both resonant and nonresonant modes are reconstructed from the recorded intensity images. The phase difference φχ between the resonant CARS field and the non-resonant CARS field is calculated. Eventually, the CARS background is efficiently suppressed by using the relation ICARSbf≅ICARSR·sin2φχ.Compared with conventional CARS background suppression techniques, the proposed method is robust against environmental disturbance, since it does not require an additional reference beam. Furthermore, the proposed method is easy to incorporate in a conventional CARS configuration. Therefore, the proposed method has the potential to become a versatile technique to image deep tissue with low background signal.

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