Abstract

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

©2012 Optical Society of America

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  1. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
    [Crossref] [PubMed]
  2. J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express 12(11), 2478–2486 (2004).
    [Crossref] [PubMed]
  3. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
    [Crossref] [PubMed]
  4. H. Chen, H. Wang, M. N. Slipchenko, Y. Jung, Y. Shi, J. Zhu, K. K. Buhman, and J. X. Cheng, “A multimodal platform for nonlinear optical microscopy and microspectroscopy,” Opt. Express 17(3), 1282–1290 (2009).
    [Crossref] [PubMed]
  5. J. W. Jhan, W. T. Chang, H. C. Chen, M. F. Wu, Y. T. Lee, C. H. Chen, and I. Liau, “Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing structure-constituent correlation of tissues,” Opt. Express 16(21), 16431–16441 (2008).
    [Crossref] [PubMed]
  6. C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
    [Crossref] [PubMed]
  7. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5(8), 169–175 (1999).
    [Crossref] [PubMed]
  8. S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011).
    [Crossref]
  9. A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
    [Crossref] [PubMed]
  10. H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007).
    [Crossref] [PubMed]
  11. M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
    [Crossref] [PubMed]
  12. N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
    [Crossref] [PubMed]
  13. M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
    [Crossref] [PubMed]
  14. H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
    [Crossref]
  15. Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
    [Crossref] [PubMed]
  16. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1604–1610 (2002).
    [Crossref]
  17. S. Feng and H. G. Winful, “Physical origin of the Gouy phase shift,” Opt. Lett. 26(8), 485–487 (2001).
    [Crossref] [PubMed]
  18. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
    [Crossref] [PubMed]
  19. B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975).
    [PubMed]
  20. S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol. 20(1), 1–28 (2004).
    [Crossref] [PubMed]
  21. D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
    [Crossref] [PubMed]
  22. V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13(20), 8263–8276 (2005).
    [Crossref] [PubMed]

2011 (1)

S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011).
[Crossref]

2010 (1)

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

2009 (1)

2008 (4)

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

J. W. Jhan, W. T. Chang, H. C. Chen, M. F. Wu, Y. T. Lee, C. H. Chen, and I. Liau, “Integrated multiple multi-photon imaging and Raman spectroscopy for characterizing structure-constituent correlation of tissues,” Opt. Express 16(21), 16431–16441 (2008).
[Crossref] [PubMed]

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
[Crossref] [PubMed]

2007 (1)

H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007).
[Crossref] [PubMed]

2006 (2)

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
[Crossref] [PubMed]

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

2005 (2)

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
[Crossref] [PubMed]

V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13(20), 8263–8276 (2005).
[Crossref] [PubMed]

2004 (3)

J. Sun, T. Shilagard, B. Bell, M. Motamedi, and G. Vargas, “In vivo multimodal nonlinear optical imaging of mucosal tissue,” Opt. Express 12(11), 2478–2486 (2004).
[Crossref] [PubMed]

S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol. 20(1), 1–28 (2004).
[Crossref] [PubMed]

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

2003 (1)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

2002 (3)

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
[Crossref] [PubMed]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B 19(7), 1604–1610 (2002).
[Crossref]

2001 (1)

1999 (1)

1975 (1)

B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975).
[PubMed]

Aus der Au, J.

Barzda, V.

Beaurepaire, E.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Bell, B.

Bonn, M.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

Buhman, K. K.

Byers, B.

B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975).
[PubMed]

Campagnola, P. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

Chang, W. T.

Chen, C. H.

Chen, H.

Chen, H. C.

Cheng, J. X.

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

Combettes, L.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Couderc, V.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

Day, J. P. R.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

Débarre, D.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Elmore, S.

Fabre, A.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Feng, S.

Filippidis, G.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

Fotakis, C.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

Ganikhanov, F.

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
[Crossref] [PubMed]

Goetsch, L.

B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975).
[PubMed]

Greenhalgh, C.

Gualda, E. J.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

Hamaguchi, H. O.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007).
[Crossref] [PubMed]

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
[Crossref] [PubMed]

Hoppe, P. E.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

Huang, Y. S.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
[Crossref] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

Jaspersen, S. L.

S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol. 20(1), 1–28 (2004).
[Crossref] [PubMed]

Jhan, J. W.

Ji, N.

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
[Crossref] [PubMed]

Jung, Y.

Kano, H.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

H. Kano and H. O. Hamaguchi, “Supercontinuum dynamically visualizes a dividing single cell,” Anal. Chem. 79(23), 8967–8973 (2007).
[Crossref] [PubMed]

Karashima, T.

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
[Crossref] [PubMed]

Kraan, Y. M.

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

Lee, Y. T.

Légaré, F.

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
[Crossref] [PubMed]

Leproux, P.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

Liau, I.

Malone, C. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

Mari, M.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

Millard, A. C.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

Mohler, W. A.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

Motamedi, M.

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

Okuno, M.

M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
[Crossref] [PubMed]

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[Crossref] [PubMed]

Olsen, B. R.

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
[Crossref] [PubMed]

Otto, C.

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

Pena, A. M.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Pfeffer, C. P.

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
[Crossref] [PubMed]

Roos, D.

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

Schanne-Klein, M. C.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Shen, Y. R.

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
[Crossref] [PubMed]

Shi, Y.

Shilagard, T.

Silberberg, Y.

Slipchenko, M. N.

Squier, J.

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Supatto, W.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Tavernarakis, N.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

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P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

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[Crossref] [PubMed]

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A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
[Crossref] [PubMed]

van Beek, J.

van Manen, H. J.

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

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E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
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W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
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W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
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N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
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A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
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S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011).
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N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
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Zhu, J.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

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A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
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M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. O. Hamaguchi, “Quantitative CARS molecular fingerprinting of single living cells with the use of the maximum entropy method,” Angew. Chem. Int. Ed. Engl. 49(38), 6773–6777 (2010).
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S. L. Jaspersen and M. Winey, “The budding yeast spindle pole body: structure, duplication, and function,” Annu. Rev. Cell Dev. Biol. 20(1), 1–28 (2004).
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Biochemistry (1)

Y. S. Huang, T. Karashima, M. Yamamoto, and H. O. Hamaguchi, “Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy,” Biochemistry 44(30), 10009–10019 (2005).
[Crossref] [PubMed]

Biophys. J. (1)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 82(1), 493–508 (2002).
[Crossref] [PubMed]

J. Am. Chem. Soc. (1)

N. Ji, K. Zhang, H. Yang, and Y. R. Shen, “Three-dimensional chiral imaging by sum-frequency generation,” J. Am. Chem. Soc. 128(11), 3482–3483 (2006).
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B. Byers and L. Goetsch, “Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae,” J. Bacteriol. 124(1), 511–523 (1975).
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J. Microsc. (1)

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229(1), 141–150 (2008).
[Crossref] [PubMed]

J. Opt. Soc. Am. B (1)

J. Phys. Chem. B (1)

H. J. van Manen, Y. M. Kraan, D. Roos, and C. Otto, “Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy,” J. Phys. Chem. B 108(48), 18762–18771 (2004).
[Crossref]

J. Struct. Biol. (1)

C. P. Pfeffer, B. R. Olsen, F. Ganikhanov, and F. Légaré, “Multimodal nonlinear optical imaging of collagen arrays,” J. Struct. Biol. 164(1), 140–145 (2008).
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Laser Photon. Rev. (1)

S. Yue, M. N. Slipchenko, and J. X. Cheng, “Multimodal nonlinear optical microscopy,” Laser Photon. Rev. 5(4), 496–512 (2011).
[Crossref]

Nat. Methods (1)

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3(1), 47–53 (2006).
[Crossref] [PubMed]

Opt. Express (5)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (2)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[Crossref] [PubMed]

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. U.S.A. 99(17), 11014–11019 (2002).
[Crossref] [PubMed]

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Figures (4)

Fig. 1
Fig. 1 Diagrams of (a) the THG process, (b) the TSFG process and (c) the experimental setup for multiplex CARS, SHG, THG and multiplex TSFG multimodal imaging. PCF stands for photonic crystal fiber, SF short-pass filter, LF long-pass filter, NF notch filter, and DM dichroic mirror.

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Fig. 2
Fig. 2 (a) Spectral profiles of the visible region and the NIR region; the others are the images of yeast cells corresponding to (b) optical; (c) SH; (d) TH; (e) TSF; (f) 1440 cm−1(CH2 bend mode) ; (g) 1003 cm−1(phenylalanine residue) image. The white bar in (b) corresponds to 5 μm. Blue and orange crosses are the position where each spectrum was obtained.

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Fig. 3
Fig. 3 (a) Optical image, (b) corresponding SH image. On the left side image, two cells have a bright spot and one cell (upper one) do not. On the right side image, all cell have a bright spot. (c) The averaged Im[χ(3)] spectrum at the 4 SH bright spots indicated in the right side image of (b).

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Fig. 4
Fig. 4 (a) TSF image; (b) CARS image at 1440 cm−1 (CH2 bend); (c) correlation image between (a) and (b); (d) high correlation area (yellow) and low correlation area (red) of TSF image (a); (e) The integrated spectrum over the peripheral red area of (d).

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Equations (1)

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C ij  =  a ij ( a ij ) Max ⋅ b ij ( b ij ) Max     ( a ij ( a ij ) Max ⋅ b ij ( b ij ) Max ) Max ,

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