Abstract

Multimodal nonlinear optical microscopy is a valuable tool to study complex biological samples. We present an easy-to-operate approach to perform coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and third-harmonic generation (THG) imaging using a single laser source composed of an 80 MHz femtosecond (fs) laser, an optical parametric oscillator (OPO), and a PPLN crystal for frequency doubling. The platform allows vibrationally resonant CARS imaging of CH-rich myelin sheath in fresh spinal tissues and lipid bodies in live cells. Multimodal nonlinear optical imaging and microspectroscopy analysis of fresh liver tissues are demonstrated.

© 2009 Optical Society of America

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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  27. J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  31. T. W. Kee, and M. T. Cicerone, "Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy," Opt. Lett. 29, 2701-2703 (2004).
    [CrossRef] [PubMed]
  32. H. Kano, and H.-o. Hamaguchi, "Ultrabroadband (> 2500 cm-1) multiplex coherent anti-Stokes Raman scattering microspectroscopy using a supercontinuum generated from a photonic crystal fiber," Appl. Phys. Lett. 86, 121113-121113 (2005).
    [CrossRef]
  33. G. I. Petrov, and V. Yakovlev, "Enhancing red-shifted white-light continuum generation in optical fibers for applications in nonlinear Raman microscopy," Opt. Express 13, 1299-1306 (2005).
    [CrossRef] [PubMed]
  34. T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
    [CrossRef]
  35. Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
    [CrossRef] [PubMed]

2008 (3)

H.-W. Wang, T. T. Le, and J.-X. Cheng, "Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope," Opt. Commun. 281, 1813-1822 (2008).
[CrossRef]

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

J. Moger, B. D. Johnston, and C. R. Tyler, "Imaging metal oxide nanoparticles in biological structures with CARS microscopy," Opt. Express 16, 3408-3419 (2008).
[CrossRef] [PubMed]

2007 (3)

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

2006 (4)

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

F. Ganikhanov, S. Carrasco, X. S. Xie, M. Katz, W. Seitz, and D. Kopf, "Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy," Opt. Lett. 31, 1292-1294 (2006).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
[CrossRef] [PubMed]

2005 (5)

G. I. Petrov, and V. Yakovlev, "Enhancing red-shifted white-light continuum generation in optical fibers for applications in nonlinear Raman microscopy," Opt. Express 13, 1299-1306 (2005).
[CrossRef] [PubMed]

H. Kano, and H.-o. Hamaguchi, "Ultrabroadband (> 2500 cm-1) multiplex coherent anti-Stokes Raman scattering microspectroscopy using a supercontinuum generated from a photonic crystal fiber," Appl. Phys. Lett. 86, 121113-121113 (2005).
[CrossRef]

L. Li, H. Wang, and J.-X. Cheng, "Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains," Biophys. J. 89, 3480-3490 (2005).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

S.-H. Lim, A. G. Caster, and S. R. Leone, "Single-pulse phase-control interferometric coherent anti-Stokes Raman scattering spectroscopy," Phys. Rev. A 72, 041803 (2005).
[CrossRef]

2004 (3)

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
[CrossRef]

T. W. Kee, and M. T. Cicerone, "Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy," Opt. Lett. 29, 2701-2703 (2004).
[CrossRef] [PubMed]

2003 (4)

H. N. Paulsen, K. M. Hilligse, J. Thøgersen, S. R. Keiding, and J. J. Larsen, "Coherent anti-Stokes Raman scattering microscopy with a photonic crystal fiber based light source," Opt. Lett. 28, 1123-1125 (2003).
[CrossRef] [PubMed]

X. Nan, J.-X. Cheng, and X. S. Xie, "Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy," J. Lipid Res. 44, 2202-2208 (2003).
[CrossRef] [PubMed]

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotech. 21, 1356-1360 (2003).
[CrossRef]

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, 7075-7080 (2003).
[CrossRef] [PubMed]

2002 (6)

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

M. Muller, and J. M. Schins, "Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy," J. Phys. Chem. B 106, 3715-3723 (2002).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, "High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers," Opt. Lett. 27, 1168-1170 (2002).
[CrossRef]

J.-X. Cheng, and X. S. Xie, "Green's function formulation for third-harmonic generation microscopy," J. Opt. Soc. Am. B 19, 1604-1610 (2002).
[CrossRef]

2001 (1)

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

2000 (1)

1999 (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

1998 (1)

1997 (1)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

1982 (1)

Araki, T.

Axäng, C.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Beaurepaire, E.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Bonn, M.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

Book, L. D.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

Brackmann, C.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Brakenhoff, G.

Burger, K. N. J.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

Campagnola, P. J.

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotech. 21, 1356-1360 (2003).
[CrossRef]

Carrasco, S.

Caster, A. G.

S.-H. Lim, A. G. Caster, and S. R. Leone, "Single-pulse phase-control interferometric coherent anti-Stokes Raman scattering spectroscopy," Phys. Rev. A 72, 041803 (2005).
[CrossRef]

Chen, Z.

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

Cheng, J. X.

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

Cheng, J.-X.

H.-W. Wang, T. T. Le, and J.-X. Cheng, "Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope," Opt. Commun. 281, 1813-1822 (2008).
[CrossRef]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
[CrossRef] [PubMed]

L. Li, H. Wang, and J.-X. Cheng, "Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains," Biophys. J. 89, 3480-3490 (2005).
[CrossRef] [PubMed]

X. Nan, J.-X. Cheng, and X. S. Xie, "Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy," J. Lipid Res. 44, 2202-2208 (2003).
[CrossRef] [PubMed]

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, "High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers," Opt. Lett. 27, 1168-1170 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

J.-X. Cheng, and X. S. Xie, "Green's function formulation for third-harmonic generation microscopy," J. Opt. Soc. Am. B 19, 1604-1610 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

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, 7075-7080 (2003).
[CrossRef] [PubMed]

Cicerone, M. T.

Combettes, L.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Cote, D.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

Debarre, D.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Dudovich, N.

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Duncan, M. D.

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Enejder, A.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Enejder, A. M. K.

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
[CrossRef]

Evans, C. L.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

Fabre, A.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Fu, Y.

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
[CrossRef] [PubMed]

Ganikhanov, F.

Hamaguchi, H.-o.

H. Kano, and H.-o. Hamaguchi, "Ultrabroadband (> 2500 cm-1) multiplex coherent anti-Stokes Raman scattering microspectroscopy using a supercontinuum generated from a photonic crystal fiber," Appl. Phys. Lett. 86, 121113-121113 (2005).
[CrossRef]

Hashimoto, M.

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

Hellerer, T.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
[CrossRef]

Hillertz, P.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Hilligse, K. M.

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

Huff, T. B.

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[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, 7075-7080 (2003).
[CrossRef] [PubMed]

Johnston, B. D.

Jones, D. J.

Kano, H.

H. Kano, and H.-o. Hamaguchi, "Ultrabroadband (> 2500 cm-1) multiplex coherent anti-Stokes Raman scattering microspectroscopy using a supercontinuum generated from a photonic crystal fiber," Appl. Phys. Lett. 86, 121113-121113 (2005).
[CrossRef]

Katz, M.

Kawata, S.

Kee, T. W.

Keiding, S. R.

Kopf, D.

Krasieva, T. B.

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

Larsen, J. J.

Le, T. T.

H.-W. Wang, T. T. Le, and J.-X. Cheng, "Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope," Opt. Commun. 281, 1813-1822 (2008).
[CrossRef]

Leone, S. R.

S.-H. Lim, A. G. Caster, and S. R. Leone, "Single-pulse phase-control interferometric coherent anti-Stokes Raman scattering spectroscopy," Phys. Rev. A 72, 041803 (2005).
[CrossRef]

Li, L.

L. Li, H. Wang, and J.-X. Cheng, "Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains," Biophys. J. 89, 3480-3490 (2005).
[CrossRef] [PubMed]

Lim, S.-H.

S.-H. Lim, A. G. Caster, and S. R. Leone, "Single-pulse phase-control interferometric coherent anti-Stokes Raman scattering spectroscopy," Phys. Rev. A 72, 041803 (2005).
[CrossRef]

Lin, C. P.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

Loew, L. M.

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotech. 21, 1356-1360 (2003).
[CrossRef]

Manuccia, T. J.

Moger, J.

Muller, M.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

M. Muller, and J. M. Schins, "Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy," J. Phys. Chem. B 106, 3715-3723 (2002).
[CrossRef]

J. Squier, M. Muller, G. Brakenhoff, and K. R. Wilson, "Third harmonic generation microscopy," Opt. Express 3, 315-324 (1998).
[CrossRef] [PubMed]

Nan, X.

X. Nan, J.-X. Cheng, and X. S. Xie, "Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy," J. Lipid Res. 44, 2202-2208 (2003).
[CrossRef] [PubMed]

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, 7075-7080 (2003).
[CrossRef] [PubMed]

Oron, D.

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Paulsen, H. N.

Pena, A.-M.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Petrov, G. I.

Pilon, M.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, "Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy," Proc. Natl. Acad. Sci. U. S. A. 104, 14658-14663 (2007).
[CrossRef] [PubMed]

Potma, E. O.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, "High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers," Opt. Lett. 27, 1168-1170 (2002).
[CrossRef]

Puoris'haag, M.

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

Reintjes, J.

Rinia, H. A.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

Schanne-Klein, M.-C.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Schins, J. M.

M. Muller, and J. M. Schins, "Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy," J. Phys. Chem. B 106, 3715-3723 (2002).
[CrossRef]

Seitz, W.

Shi, R.

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
[CrossRef] [PubMed]

Silberberg, Y.

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Squier, J.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Supatto, W.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Tang, S.

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

Tempea, G.

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

Thøgersen, J.

Tordjmann, T.

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Tromberg, B. J.

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

Tyler, C. R.

Volkmer, A.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

Wang, H.

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy," Opt. Express 14, 3942-3951 (2006).
[CrossRef] [PubMed]

L. Li, H. Wang, and J.-X. Cheng, "Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains," Biophys. J. 89, 3480-3490 (2005).
[CrossRef] [PubMed]

Wang, H.-W.

H.-W. Wang, T. T. Le, and J.-X. Cheng, "Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope," Opt. Commun. 281, 1813-1822 (2008).
[CrossRef]

Webb, W. W.

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, 7075-7080 (2003).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

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, 7075-7080 (2003).
[CrossRef] [PubMed]

Wilson, K. R.

Xie, X. S.

F. Ganikhanov, S. Carrasco, X. S. Xie, M. Katz, W. Seitz, and D. Kopf, "Broadly tunable dual-wavelength light source for coherent anti-Stokes Raman scattering microscopy," Opt. Lett. 31, 1292-1294 (2006).
[CrossRef] [PubMed]

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

X. Nan, J.-X. Cheng, and X. S. Xie, "Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy," J. Lipid Res. 44, 2202-2208 (2003).
[CrossRef] [PubMed]

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, "High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers," Opt. Lett. 27, 1168-1170 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

J.-X. Cheng, and X. S. Xie, "Green's function formulation for third-harmonic generation microscopy," J. Opt. Soc. Am. B 19, 1604-1610 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Yakovlev, V.

Ye, 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, 7075-7080 (2003).
[CrossRef] [PubMed]

Zumbusch, A.

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Appl. Phys. Lett. (3)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

H. Kano, and H.-o. Hamaguchi, "Ultrabroadband (> 2500 cm-1) multiplex coherent anti-Stokes Raman scattering microspectroscopy using a supercontinuum generated from a photonic crystal fiber," Appl. Phys. Lett. 86, 121113-121113 (2005).
[CrossRef]

T. Hellerer, A. M. K. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004).
[CrossRef]

Biophys. J. (4)

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Muller, "Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy," Biophys. J. 95, 4908-4914 (2008).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

Y. Fu, H. Wang, R. Shi, and J.-X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys. J. 92, 3251-3259 (2007).
[CrossRef] [PubMed]

L. Li, H. Wang, and J.-X. Cheng, "Quantitative Coherent Anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains," Biophys. J. 89, 3480-3490 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

S. Tang, T. B. Krasieva, Z. Chen, G. Tempea, and B. J. Tromberg, "Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy," J. Biomed. Opt. 11, 020501-020503 (2006).
[CrossRef] [PubMed]

J. Lipid Res. (1)

X. Nan, J.-X. Cheng, and X. S. Xie, "Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy," J. Lipid Res. 44, 2202-2208 (2003).
[CrossRef] [PubMed]

J. Neurosci. Res. (1)

Y. Fu, H. Wang, T. B. Huff, R. Shi, and J.-X. Cheng, "Coherent anti-stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination," J. Neurosci. Res. 85, 2870-2881 (2007).
[CrossRef] [PubMed]

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

J. Phys. Chem. B (4)

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "Multiplex coherent anti-Stokes Raman scattering microspectroscopy and study of lipid vesicles," J. Phys. Chem. B 106, 8493-8498 (2002).
[CrossRef]

M. Muller, and J. M. Schins, "Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy," J. Phys. Chem. B 106, 3715-3723 (2002).
[CrossRef]

J. X. Cheng and X. S. Xie, "Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications," J. Phys. Chem. B 108, 827-840 (2004).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, "An epi-detected coherent anti-Stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

Nat. Biotech. (1)

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotech. 21, 1356-1360 (2003).
[CrossRef]

Nat. Meth. (1)

D. Debarre, 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. Meth. 3, 47-53 (2006).
[CrossRef]

Nature (1)

N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
[CrossRef] [PubMed]

Opt. Commun. (1)

H.-W. Wang, T. T. Le, and J.-X. Cheng, "Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope," Opt. Commun. 281, 1813-1822 (2008).
[CrossRef]

Opt. Express (4)

Opt. Lett. (6)

Phys. Rev. A (1)

S.-H. Lim, A. G. Caster, and S. R. Leone, "Single-pulse phase-control interferometric coherent anti-Stokes Raman scattering spectroscopy," Phys. Rev. A 72, 041803 (2005).
[CrossRef]

Phys. Rev. Lett. (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Proc. Natl. Acad. Sci. U. S. A. (3)

C. L. Evans, E. O. Potma, M. Puoris'haag, D. Cote, C. P. Lin, and X. S. Xie, "Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy," Proc. Natl. Acad. Sci. U. S. A. 102, 16807-16812 (2005).
[CrossRef] [PubMed]

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, 7075-7080 (2003).
[CrossRef] [PubMed]

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[CrossRef] [PubMed]

Science (1)

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[CrossRef] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Schematic diagram of a multimodal NLO microscope using a fs laser source. The Mai Tai output at 790 nm (ω1) is split into two beams, with 80% of power being used to pump the Opal-BB. The idler output at 2036 nm from Opal-BB is doubled using a PPLN crystal. The rest of Mai Tai output is sent through a delay line and collinearly combined with the frequency-doubled idler beam at 1018 nm (ω2) for CARS imaging on a FV1000 laser-scanning inverted microscope. The 790 nm beam is also used for TPF and SHG imaging. The signal beam at 1290 nm (ω3) is used for SHG and THG imaging. Two internal spectral detectors are used for the microspectroscopy analysis. External detectors (not shown) are added for simultaneous forward and backward detection.

Fig. 2.
Fig. 2.

Spatial resolution. (a) Epi-detected CARS image of TiO2 nanoparicles, excited at 790 and 1018 nm. (b-c) Lateral and axial intensity profiles along the circled TiO2 nanoparticle in (a). (d) Epi-detected THG image of TiO2 nanoparicles, excited at 1290 nm. (e-f) Lateral and axial intensity profiles along the circled TiO2 nanoparticle in (d). Scale bars = 5 μm.

Fig. 3.
Fig. 3.

Vibrational contrast of C-H rich objects. (a-d) The forward-detected CARS images of a subcutaneous fat tissue by the fs (a-b) and ps (c-d) lasers, respectively. The Raman shift is marked in each image. Scale bars = 20 μm. (e) The CARS spectra of subcutaneous fat recorded with the fs (grey) and ps (black) lasers. (f-g) The backward-detected CARS images of myelin sheath surrounding parallel axons in a fresh spinal tissue (f) and paranodal myelin at a node of Ranvier (g). Scale bars = 10 μm.

Fig. 4.
Fig. 4.

Comparison of forward-detected THG (a) and CARS (b) images of lipid droplets inside live KB cells. Scale bars = 10 μm.

Fig. 5.
Fig. 5.

Multimodal nonlinear optical imaging of fresh liver tissues from high fat fed mice. (a) TPF image excited at 790 nm and detected in the 450-550 nm region. Fibrous structure on the liver surface produced strong intrinsic fluorescence. (b) SHG image of collagen fibers at 1 μm below the liver surface, excited by the 1290 nm beam. (c) Forward-detected CARS image of lipid droplets in liver cells. (d) TPF image of the same cells as in (c) excited by the 790 nm fs laser. (e) Microspectroscopy analysis of the circled area shown in (c) using a λ-scanner detector and the same lasers. The CARS signal peaked at 645 nm and the autofluorescence peaked around 490 nm are shown. (f) TPF spectra of the entire image shown in (d) by different excitation wavelengths. Scale bars = 10 μm.

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