Abstract

We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy of live cells and tissues with user-variable spectral resolution and broad Raman tunability (2500 - 4100 cm-1), using a femtosecond Ti:Sapphire pump and photonic crystal fiber output for the broadband synchronized Stokes pulse. Spectral chirp of the fs laser pulses was a user-variable parameter for optimization in a spectral focussing implementation of multimodal CARS microscopy. High signal-to-noise, high contrast multimodal imaging of live cells and tissues was achieved with pixel dwell times of 2-8 μs and low laser powers (< 30 mW total).

© 2009 Optical Society of America

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  1. A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
    [CrossRef]
  2. J.-X. Cheng and X. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004). URL http://pubs3.acs.org/acs/journals/doilookup?in doi=10.1021/jp035693v.
    [CrossRef]
  3. W. Denk, J. Strickler, and W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  4. P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
    [CrossRef]
  5. D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-5-8-169.
    [CrossRef] [PubMed]
  6. X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
    [CrossRef] [PubMed]
  7. T. T. Le, I. M. Langohr, M. J. Locker, M. Sturek, and J.-X. Cheng, "Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy," J. Biomed. Opt. 12, 054007 (2007). URL http://link.aip.org/link/?JBO/12/054007/1.
    [CrossRef] [PubMed]
  8. D. J. Jones, E. O. Potma, J. xin Cheng, B. Burfeindt, Y. Pang, J. Ye, and X. S. Xie, "Synchronization of two passively mode-locked, picosecond lasers within 20 fs for coherent anti-Stokes Raman scattering microscopy," Rev. Sci. Instrum. 73, 2843-2848 (2002). URL http://link.aip.org/link/?RSI/73/2843/1.
    [CrossRef]
  9. 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). URLhttp://ol.osa.org/abstract.cfm?URI=ol-31-9-1292.
    [CrossRef] [PubMed]
  10. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, "Selective imaging of saturated andunsaturated lipids by wide-fieldCARS-microscopy," Opt. Express 16, 2699-2708 (2008). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-16-4-2699.
    [CrossRef] [PubMed]
  11. J.-x. Cheng, A. Volkmer, L. Book, and X. Xie, "An Epi-Detected Coherent Anti-Stokes Raman Scattering (ECARS) Microscope with High Spectral Resolution and High Sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001). URL http://pubs3.acs.org/acs/journals/doilookup?in doi=10.1021/jp003774a.
    [CrossRef]
  12. N. Dudovich, D. Oron, and Y. Silberberg, "Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy," Nature 418, 512-514 (2002).
    [CrossRef] [PubMed]
  13. E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
    [CrossRef] [PubMed]
  14. A. M. Zheltikov and A. N. Naumov, "High-resolution four-photon spectroscopy with chirped pulses," Quantum Electron. 30, 606-610 (2000). URL http://stacks.iop.org/1063-7818/30/606.
    [CrossRef]
  15. T. Hellerer, A. M. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004). URL http://link.aip.org/link/?APL/85/25/1.
    [CrossRef]
  16. I. Rocha-Mendoza, W. Langbein, and P. Borri, "Coherent anti-Stokes Raman microspectroscopy using spectral focusing with glass dispersion," Appl. Phys. Lett. 93, 201103 (2008). URL http://link.aip.org/link/?APL/93/201103/1.
    [CrossRef]
  17. R. M. Onorato, N. Muraki, K. P. Knutsen, and R. J. Saykally, "Chirped coherent anti-Stokes Raman scattering as a high-spectral- and spatial-resolution microscopy," Opt. Lett. 32, 2858-2860 (2007). URL http://ol.osa.org/abstract.cfm?URI=ol-32-19-2858.
    [CrossRef] [PubMed]
  18. H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).
  19. K. M. Hilligsøe, T. Andersen, H. Paulsen, C. Nielsen, K. Mølmer, S. Keiding, R. Kristiansen, K. Hansen, and J. Larsen, "Supercontinuum generation in a photonic crystal fiber with two zero dispersion wavelengths," Opt. Express 12, 1045-1054 (2004). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-12-6-1045.
    [CrossRef] [PubMed]
  20. M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
    [CrossRef] [PubMed]
  21. B. Schrader and W. Meier, Raman/Infrared Altas of Organic Compounds (Verlag Chemie, Weinheim, 1978).
  22. A. M.-A. Martin Schwartz and W. H. Koehler, "Fermi resonance in aqueous methanol," J. Mol. Struct. 63, 279-285 (1980).
    [CrossRef]
  23. H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, J. Insull, William, M. E.  Rosenfeld, C. J. Schwartz, W. D. Wagner, and R. W. Wissler, "A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis : A Report From the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association," Arterioscler Thromb Vasc Biol 15, 1512-1531 (1995). URL http://atvb.ahajournals.org/cgi/content/abstract/15/9/1512.
    [CrossRef] [PubMed]
  24. S. Murugkar, C. Brideau, A. Ridsdale, M. Naji, P. K. Stys, and H. Anis, "Coherent anti-Stokes Raman scattering microscopy using photonic crystal fiber with two closely lying zero dispersion wavelengths," Opt. Express 15, 14,028-14,037 (2007). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-15-21-14028
    [CrossRef]

2006 (1)

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

2003 (1)

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

2002 (1)

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

1999 (2)

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

1992 (1)

E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
[CrossRef] [PubMed]

1990 (1)

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

1980 (1)

A. M.-A. Martin Schwartz and W. H. Koehler, "Fermi resonance in aqueous methanol," J. Mol. Struct. 63, 279-285 (1980).
[CrossRef]

1976 (1)

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).

Bloembergen, N.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).

Campagnola, P. J.

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

Denk, W.

W. Denk, J. Strickler, and 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]

Duppen, K.

E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
[CrossRef] [PubMed]

Fan, J.

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

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, 4142-4145 (1999).
[CrossRef]

Ito, T.

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

Kawashima, S.

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

Koehler, W. H.

A. M.-A. Martin Schwartz and W. H. Koehler, "Fermi resonance in aqueous methanol," J. Mol. Struct. 63, 279-285 (1980).
[CrossRef]

Lewis, A.

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

Loew, L. M.

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

Lotem, H.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).

Lynch, R. T.

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).

Martin Schwartz, A. M.-A.

A. M.-A. Martin Schwartz and W. H. Koehler, "Fermi resonance in aqueous methanol," J. Mol. Struct. 63, 279-285 (1980).
[CrossRef]

Nan, X.

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

Nibbering, E. T. J.

E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
[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]

Pezacki, J. P.

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

Shiomi, M.

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[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]

Stolow, A.

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

Strickler, J.

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

Tonary, A. M.

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

Webb, W.

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

Wei, M.-d.

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

Wiersma, D. A.

E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
[CrossRef] [PubMed]

Xie, X. S.

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Yamada, S.

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

Zumbusch, A.

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Arterioscler Thromb Vasc Biol (1)

M. Shiomi, T. Ito, S. Yamada, S. Kawashima, and J. Fan, "Development of an Animal Model for Spontaneous Myocardial Infarction (WHHLMI Rabbit)," Arterioscler Thromb Vasc Biol 23, 1239-1244 (2003).
[CrossRef] [PubMed]

Biophys. J. (1)

P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, "High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation," Biophys. J. 77, 3341-3349 (1999).
[CrossRef]

ChemBioChem (1)

X. Nan, A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezacki, "Intracellular Imaging of HCV RNA and Cellular Lipids by Using Simultaneous Two-Photon Fluorescence and Coherent Anti-Stokes Raman Scattering Microscopies," ChemBioChem 7, 1895-1897 (2006).
[CrossRef] [PubMed]

J. Mol. Struct. (1)

A. M.-A. Martin Schwartz and W. H. Koehler, "Fermi resonance in aqueous methanol," J. Mol. Struct. 63, 279-285 (1980).
[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]

Phys. Rev. A (1)

H. Lotem, R. T. Lynch, and N. Bloembergen, "Interference between Raman resonances in four-wave difference mixing," Phys. Rev. A 14, 1748-1755 (1976).

Phys. Rev. Lett. (2)

E. T. J. Nibbering, D. A. Wiersma, and K. Duppen, "Ultrafast nonlinear spectroscopy with chirped optical pulses," Phys. Rev. Lett. 68, 514-517 (1992).
[CrossRef] [PubMed]

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Science (1)

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

Other (15)

H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, J. Insull, William, M. E.  Rosenfeld, C. J. Schwartz, W. D. Wagner, and R. W. Wissler, "A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis : A Report From the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association," Arterioscler Thromb Vasc Biol 15, 1512-1531 (1995). URL http://atvb.ahajournals.org/cgi/content/abstract/15/9/1512.
[CrossRef] [PubMed]

S. Murugkar, C. Brideau, A. Ridsdale, M. Naji, P. K. Stys, and H. Anis, "Coherent anti-Stokes Raman scattering microscopy using photonic crystal fiber with two closely lying zero dispersion wavelengths," Opt. Express 15, 14,028-14,037 (2007). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-15-21-14028
[CrossRef]

J.-X. Cheng and X. Xie, "Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications," J. Phys. Chem. B 108, 827-840 (2004). URL http://pubs3.acs.org/acs/journals/doilookup?in doi=10.1021/jp035693v.
[CrossRef]

D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-5-8-169.
[CrossRef] [PubMed]

T. T. Le, I. M. Langohr, M. J. Locker, M. Sturek, and J.-X. Cheng, "Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy," J. Biomed. Opt. 12, 054007 (2007). URL http://link.aip.org/link/?JBO/12/054007/1.
[CrossRef] [PubMed]

D. J. Jones, E. O. Potma, J. xin Cheng, B. Burfeindt, Y. Pang, J. Ye, and X. S. Xie, "Synchronization of two passively mode-locked, picosecond lasers within 20 fs for coherent anti-Stokes Raman scattering microscopy," Rev. Sci. Instrum. 73, 2843-2848 (2002). URL http://link.aip.org/link/?RSI/73/2843/1.
[CrossRef]

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). URLhttp://ol.osa.org/abstract.cfm?URI=ol-31-9-1292.
[CrossRef] [PubMed]

C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, "Selective imaging of saturated andunsaturated lipids by wide-fieldCARS-microscopy," Opt. Express 16, 2699-2708 (2008). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-16-4-2699.
[CrossRef] [PubMed]

J.-x. Cheng, A. Volkmer, L. Book, and X. Xie, "An Epi-Detected Coherent Anti-Stokes Raman Scattering (ECARS) Microscope with High Spectral Resolution and High Sensitivity," J. Phys. Chem. B 105, 1277-1280 (2001). URL http://pubs3.acs.org/acs/journals/doilookup?in doi=10.1021/jp003774a.
[CrossRef]

A. M. Zheltikov and A. N. Naumov, "High-resolution four-photon spectroscopy with chirped pulses," Quantum Electron. 30, 606-610 (2000). URL http://stacks.iop.org/1063-7818/30/606.
[CrossRef]

T. Hellerer, A. M. Enejder, and A. Zumbusch, "Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 25-27 (2004). URL http://link.aip.org/link/?APL/85/25/1.
[CrossRef]

I. Rocha-Mendoza, W. Langbein, and P. Borri, "Coherent anti-Stokes Raman microspectroscopy using spectral focusing with glass dispersion," Appl. Phys. Lett. 93, 201103 (2008). URL http://link.aip.org/link/?APL/93/201103/1.
[CrossRef]

R. M. Onorato, N. Muraki, K. P. Knutsen, and R. J. Saykally, "Chirped coherent anti-Stokes Raman scattering as a high-spectral- and spatial-resolution microscopy," Opt. Lett. 32, 2858-2860 (2007). URL http://ol.osa.org/abstract.cfm?URI=ol-32-19-2858.
[CrossRef] [PubMed]

K. M. Hilligsøe, T. Andersen, H. Paulsen, C. Nielsen, K. Mølmer, S. Keiding, R. Kristiansen, K. Hansen, and J. Larsen, "Supercontinuum generation in a photonic crystal fiber with two zero dispersion wavelengths," Opt. Express 12, 1045-1054 (2004). URL http://www.opticsexpress.org/abstract.cfm?URI=oe-12-6-1045.
[CrossRef] [PubMed]

B. Schrader and W. Meier, Raman/Infrared Altas of Organic Compounds (Verlag Chemie, Weinheim, 1978).

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Figures (8)

Fig. 1.
Fig. 1.

(a) Theoretically predicted resonant and nonresonant CARS signal levels are plotted as a function of pump and Stokes spectral width. The three resonant curves correspond to the Raman linewidth of polystyrene beads 9.2 cm-1, lipids 100 cm-1 and water 400 cm-1. The nonresonant signal (NR) is plotted for comparison. (b) The performance ℙ f , the contrast ratio Ir /Inr multiplied by the absolute signal level Ir , is shown as a function of pulse spectral width. It can be seen that ℙ f peaks when the spectral width of the pump and Stokes matches the linewidth of the Raman mode being probed.

Fig. 2.
Fig. 2.

Determination of resonant versus nonresonant CARS signals in the lipid region (effective pulse spectral widths of the pump and Stokes were ~40 cm-1). (a) The intensity profile of the laser focus was scanned along the axial direction across an interface from glass (purely nonresonant CARS signal) to octadecene (resonant CARS signal). The resonant signal was much larger. (b) A spectral scan of octadecene showing the line shape and the contrast between on and off resonant signal levels. For comparison, the spontaneous Raman spectrum of octadecene is included. These two scans yield a ratio of resonant to non-resonant signal exceeding 40:1, indicating that the resonant signal was not overwhelmed by the nonresonant background.

Fig. 3.
Fig. 3.

Multimodal CARS microscopy optical arrangement. Pulses from the Ti:Sa oscillator are sent through a Faraday isolator (FI) followed by a prism compressor. A 50:50 beam splitter (BS) divided the incoming light. One arm was sent through a photonic crystal fiber (PCF) and bandpass filter (BP) before being recombined on a dichroic mirror (DM). The other half was sent through a time delay arm that included a neutral density filter (ND). In both the pump and Stokes paths, glass (SF6) could be added to control the chirp. The recombined beam was sent into the FV300 microscope. Inside the FV300, there were more dichroic mirrors and filters to separate the signals from the excitation light. Note that the epi-detected fluorescence signals were collected inside the microscope.

Fig. 4.
Fig. 4.

Time-frequency plots showing the pump and Stokes pulses as ellipses in (ω,t) space. In this representation, transform limited pulses have vertical major axes whereas those of chirped pulses are tilted. The instantaneous bandwidth is determined by measuring the height of an ellipse at any instant of time. In CARS, the spectral resolution is set by the first two photon interactions and, in this representation, the spectral resolution ∆Ω is determined by the total height of the ellipse ωp - ωs . (a) Pulses having unmatched chirps and large instantaneous bandwidths. Here, the spectral resolution ∆Ω is poor because the instantaneous bandwidth is large and the difference between the pump and Stokes is changing as a function of time. (b) Chirp matched pulses with narrow instantaneous bandwidths. Here the spectral resolution ∆Ω is improved because the instantaneous bandwidths of both the pump and Stokes are narrower and the frequency difference between the pump and Stokes is nearly constant. It is seen that changing the time delay between pump and Stokes scans the instantaneous difference frequency, corresponding to different Raman modes being probed (Ω1, Ω2).

Fig. 5.
Fig. 5.

FV300 CARS spectrum of methanol obtained by scanning the time delay between the pump and Stokes pulses. One curve (dashed) was obtained with near transform limited pulses having large instantaneous bandwidths and unmatched chirps (corresponding to Fig. 4(a)). The second curve (solid) was obtained with pulses that were nearly chirp matched and narrow instantaneous bandwidths (corresponding to Fig. 4(b)). As expected, varying the chirp and ensuring matched chirp rates improves the spectral resolution. In this case, the spectral resolution varied from >160 cm-1 to <60 cm-1.

Fig. 6.
Fig. 6.

Forward detected FV300 CARS imaging of fixed rat dorsal root nerves. The CARS resonance was set to 2850 cm-1. The lipid-rich myelin sheath surrounds the neuronal axon and generates a strong CARS signal. The pixel intensity profile of the indicated line is shown, revealing high contrast. The scale bar is 50 μm. The pixel dwell time was 8 μs.

Fig. 7.
Fig. 7.

Multimodal CARS microscopy of rabbit aorta. A 50 micron thick section of aorta was tangentially cut to the lumenal side and imaged for lipids (CARS - red) collagen (second harmonic - blue) and smooth muscle elastin (fluorescence - green). All three signals are endogenous to the sample. The CARS and SHG were collected in the forward direction whereas the TPF was collected in the epi-direction. This image is a z-projection of a 50 image data set of a 3D scan through the sample (images set 1 μm apart). The scale bar is 50 μm. The pixel dwell time was 8 μs.

Fig. 8.
Fig. 8.

(Media 1) CARS video of lipid trafficking in live human liver cells. Here we show the first and last frames from a hour long time course of live HuH 7 cells, using label-free CARS imaging of lipids. The full video is available online. The high stability of this multimodal CARS microscope allows near continuous imaging of live cells.

Equations (6)

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E p ( t ) E s * ( t ) = E p E s π 2 Δ p Δ s ( 2 ln 2 ) 2 exp ( π 2 ( Δ p 2 + Δ s 2 ) t 2 2 ln 2 ) exp ( i ( ω p ω s ) t )
E p ( t ) E s * ( t ) = E p E s π 2 Δ p Δ s ( 2 ln 2 + ia ) ( 2 ln 2 ib )
exp [ π 2 2 ln 2 t 2 ( Δ p 2 ( 2 ln 2 ) 2 + a 2 + Δ s 2 ( 2 ln 2 ) 2 + b 2 ) ]
exp [ i ( ω p ω s ) t + i π 2 t 2 ( Δ p 2 a ( 2 ln 2 ) 2 + a 2 Δ s 2 b ( 2 ln 2 ) 2 + b 2 ) ]
E p ( t ) E s * ( t ) = E p E s π 2 Δ 2 ( 2 ln 2 ) 2 + a 2 exp ( π 2 4 ln 2 Δ 2 t 2 ( 2 ln 2 ) 2 + a 2 ) exp ( i ( ω p ω s ) t )
Δ effective = Δ 2 1 + ( a 2 ln 2 ) 2

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