Abstract

We developed a multi-focus excitation coherent anti-Stokes Raman scattering (CARS) microscope using a microlens array scanner for real-time molecular imaging. Parallel exposure of a specimen with light from two highly controlled picosecond mode-locked lasers (jitter of 30 fs through an electronic low-pass filter with 150 Hz bandwidth, point-by-point wavelength scan within 300 ms) and parallel detection with an image sensor enabled real-time imaging. We demonstrated real-time CARS imaging of polystyrene beads (frame rate of 30 fps), a giant multi-lamellar vesicle of dipalmitoylphosphatidylcholine (frame rate of 10 fps), and living HeLa cells (frame rate of 10 fps).

© 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. M. Hashimoto, T. Araki, and S. Kawata, "Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration," Opt. Lett. 25, 1768-1770 (2000).
    [CrossRef]
  3. X. Nan, J. 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]
  4. X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
    [CrossRef] [PubMed]
  5. X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (2006).
    [CrossRef] [PubMed]
  6. X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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. H. Kano and H. Hamaguchi, "Supercontinuum dynamically visualizes a dividing single cell," Anal. Chem. 79, 8967-8973 (2007).
    [CrossRef] [PubMed]
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    [CrossRef]
  9. 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]
  10. C. L. Evans, X. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, "Chemically-selective imaging of brain structures with CARS microscopy," Opt. Express 15, 12076-12087 (2007).
    [CrossRef] [PubMed]
  11. 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. Aca. Sci. 102, 16807-16812 (2005).
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    [CrossRef]
  14. Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
    [CrossRef] [PubMed]
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  16. T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
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    [CrossRef]
  19. M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
    [CrossRef]
  20. T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
    [CrossRef]
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    [CrossRef]
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  24. T. O’Leary and I. Levin, "Raman spectroscopic study of the melting behavior of anhydrous dipalmitoylphosphatidylcholine bilayers," J. Phys. Chem. 88, 1790-1796 (1984).
    [CrossRef]

2007 (2)

2006 (4)

X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (2006).
[CrossRef] [PubMed]

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

2005 (3)

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

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. Aca. Sci. 102, 16807-16812 (2005).
[CrossRef]

2003 (4)

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[CrossRef]

M. Hashimoto, T. Araki, and S. Kawata, "Multi-focus coherent anti-Stokes Raman scattering microscopy," Microsc. Microanal. 9(Suppl 2), 1090-1091 (2003).

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

X. Nan, J. 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]

2002 (2)

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (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]

2001 (1)

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

2000 (1)

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]

K. Konig, T. W. Becker, P. Fisher, I. Riemann, and K. -J. Halbhuber, "Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes," Opt. Lett. 24, 113-115 (1999).
[CrossRef]

1998 (3)

1995 (1)

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

1984 (1)

T. O’Leary and I. Levin, "Raman spectroscopic study of the melting behavior of anhydrous dipalmitoylphosphatidylcholine bilayers," J. Phys. Chem. 88, 1790-1796 (1984).
[CrossRef]

Ajito, K.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Araki, T.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

M. Hashimoto, T. Araki, and S. Kawata, "Multi-focus coherent anti-Stokes Raman scattering microscopy," Microsc. Microanal. 9(Suppl 2), 1090-1091 (2003).

M. Hashimoto, T. Araki, and S. Kawata, "Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration," Opt. Lett. 25, 1768-1770 (2000).
[CrossRef]

Asada, T.

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

Barbosa, L. C.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Becker, T. W.

Berns, M. W.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Cesar, C. L.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Chapman, C. F.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Cheng, D. K.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Cheng, J.

X. Nan, J. 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]

de Paula, A. M.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

de Thomaz, A. A.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Evans, C. L.

C. L. Evans, X. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, "Chemically-selective imaging of brain structures with CARS microscopy," Opt. Express 15, 12076-12087 (2007).
[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. Aca. Sci. 102, 16807-16812 (2005).
[CrossRef]

Fisher, P.

Fontes, A.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Fujita, K.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Halbhuber, K. -J.

Hamaguchi, H.

H. Kano and H. Hamaguchi, "Supercontinuum dynamically visualizes a dividing single cell," Anal. Chem. 79, 8967-8973 (2007).
[CrossRef] [PubMed]

Hashimoto, M.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

M. Hashimoto, T. Araki, and S. Kawata, "Multi-focus coherent anti-Stokes Raman scattering microscopy," Microsc. Microanal. 9(Suppl 2), 1090-1091 (2003).

M. Hashimoto, T. Araki, and S. Kawata, "Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration," Opt. Lett. 25, 1768-1770 (2000).
[CrossRef]

Hayazawa, N.

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

Hell, S. W.

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]

Ichimura, T.

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

Inouye, Y.

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

Kaneko, T.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Kano, H.

H. Kano and H. Hamaguchi, "Supercontinuum dynamically visualizes a dividing single cell," Anal. Chem. 79, 8967-8973 (2007).
[CrossRef] [PubMed]

Kawata, S.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

M. Hashimoto, T. Araki, and S. Kawata, "Multi-focus coherent anti-Stokes Raman scattering microscopy," Microsc. Microanal. 9(Suppl 2), 1090-1091 (2003).

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

M. Hashimoto, T. Araki, and S. Kawata, "Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration," Opt. Lett. 25, 1768-1770 (2000).
[CrossRef]

Kesari, S.

Kobayashi, M.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

Konig, K.

Levin, I.

T. O’Leary and I. Levin, "Raman spectroscopic study of the melting behavior of anhydrous dipalmitoylphosphatidylcholine bilayers," J. Phys. Chem. 88, 1790-1796 (1984).
[CrossRef]

Liu, Y.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Minamikawa, T.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

Moreira, W. L.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

Muller, 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]

Nakamura, O.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Nan, X.

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (2006).
[CrossRef] [PubMed]

X. Nan, J. 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]

Neves, A. A. R.

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (2005).
[CrossRef]

O’Leary, T.

T. O’Leary and I. Levin, "Raman spectroscopic study of the melting behavior of anhydrous dipalmitoylphosphatidylcholine bilayers," J. Phys. Chem. 88, 1790-1796 (1984).
[CrossRef]

Oyamada, M.

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Pezzacki, J. P.

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

Potma, E. O.

X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (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. Aca. Sci. 102, 16807-16812 (2005).
[CrossRef]

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[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. Aca. Sci. 102, 16807-16812 (2005).
[CrossRef]

Riemann, I.

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]

Schonle, A.

Sonek, G. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Stolow, A.

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

Straub, M.

Straub, S.

S. Straub and S. W. Hell, "Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging," Bioimaging 88, 177-185 (1998).
[CrossRef]

Takamatsu, T.

M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, "Second-harmonic-generation microscope with a microlens array scanner," Opt. Lett. 27, 1324-1326 (2002).
[CrossRef]

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Tanaka, H.

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Tanimoto, N.

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

Tonary, E. A. M.

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

Tromberg, B. J.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Wong, S. T. C.

Xie, X. S.

C. L. Evans, X. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, "Chemically-selective imaging of brain structures with CARS microscopy," Opt. Express 15, 12076-12087 (2007).
[CrossRef] [PubMed]

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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]

X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (2006).
[CrossRef] [PubMed]

X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

X. Nan, J. 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 and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[CrossRef]

A. Zumbusch, G. R. Holtom, and X. S. Xie, "Three-Dimensional vibrational imaging by coherent anti-Stokes Raman scattering," Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Xu, X.

Yang, W. Y.

X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

Young, G. S.

Yu, J.

X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
[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]

Acta Histochem. Cytochem. (1)

T. Kaneko, K. Fujita, H. Tanaka, M. Oyamada, O. Nakamura, S. Kawata, and T. Takamatsu, "Real-time twophoton microscopy and its application for in situ imaging," Acta Histochem. Cytochem. 34, 399-403 (2001).
[CrossRef]

Anal. Chem. (1)

H. Kano and H. Hamaguchi, "Supercontinuum dynamically visualizes a dividing single cell," Anal. Chem. 79, 8967-8973 (2007).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

T. Minamikawa, N. Tanimoto, M. Hashimoto, T. Araki, M. Kobayashi, K. Fujita, and S. Kawata, "Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors," Appl. Phys. Lett. 89, 191101 (2006).
[CrossRef]

Bioimaging (1)

S. Straub and S. W. Hell, "Multifocal multiphoton microscopy: a fast and efficient tool for 3-D fluorescence imaging," Bioimaging 88, 177-185 (1998).
[CrossRef]

Biophys. J. (2)

X. Nan, E. O. Potma, and X. S. Xie, "Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy," Biophys. J. 91, 728-735 (2006).
[CrossRef] [PubMed]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence of localized cell heating induced by infrared optical tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

ChemBioChem (1)

X. Nan, E. A. M. Tonary, A. Stolow, X. S. Xie, and J. P. Pezzacki, "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. Lipid Res. (1)

X. Nan, J. 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. Phys. Chem. (1)

T. O’Leary and I. Levin, "Raman spectroscopic study of the melting behavior of anhydrous dipalmitoylphosphatidylcholine bilayers," J. Phys. Chem. 88, 1790-1796 (1984).
[CrossRef]

J. Phys. Chem. B (1)

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. Raman Spectro. (1)

T. Ichimura, N. Hayazawa, M. Hashimoto, Y. Inouye, and S. Kawata, "Local enhancement of coherent anti-Stokes Raman scattering by isolated gold nanoparticles," J. Raman Spectro. 34, 651-654 (2003).
[CrossRef]

J. Raman Spectrosc. (1)

E. O. Potma and X. S. Xie, "Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy," J. Raman Spectrosc. 34, 642-650 (2003).
[CrossRef]

Jpn. J. Appl. Phys. (1)

M. Hashimoto, T. Asada, T. Araki, Y. Inouye, and S. Kawata, "Automatic pulse duration control of picosecond laser using two-photon absorption detector," Jpn. J. Appl. Phys. 44, 3958-3961 (2005).
[CrossRef]

Microsc. Microanal. (1)

M. Hashimoto, T. Araki, and S. Kawata, "Multi-focus coherent anti-Stokes Raman scattering microscopy," Microsc. Microanal. 9(Suppl 2), 1090-1091 (2003).

Opt. Express (1)

Opt. Lett. (5)

Phys. Rev. E (1)

A. Fontes, K. Ajito, A. A. R. Neves, W. L. Moreira, A. A. de Thomaz, L. C. Barbosa, A. M. de Paula, and C. L. Cesar, "Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system," Phys. Rev. E 72, 012903 (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, 4142-4145 (1999).
[CrossRef]

Proc. Natl. Aca. Sci. (1)

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. Aca. Sci. 102, 16807-16812 (2005).
[CrossRef]

Science (1)

X. S. Xie, J. Yu, and W. Y. Yang, "Living cells as test tubes," Science 312, 228-230 (2006).
[CrossRef] [PubMed]

Supplementary Material (4)

» Media 1: MOV (333 KB)     
» Media 2: MOV (3031 KB)     
» Media 3: MOV (2673 KB)     
» Media 4: MOV (587 KB)     

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Figures (12)

Fig. 1.
Fig. 1.

Optical setup of the developed multi-focus CARS microscopy system: L, lenses; TL, tube lens; OL, objective lenses; TPD, two-photon detector; DSP, digital signal processor; GTI, Gires-Tournois interferometer.

Fig. 2.
Fig. 2.

CARS spectra of the polystyrene beads (solid line) and background (dashed line) obtained from a pixel of an image (33 ms/image). Strong resonance for the polystyrene beads was observed at 1000 cm-1, which corresponds to the phenyl ring breathing mode.

Fig. 3.
Fig. 3.

Video of real-time CARS images of the polystyrene beads (diameter of 3 µm) in water with a frame rate of 30 fps (Media 1). The observed molecular vibration was 1000 cm-1. Total incident laser intensities were 75.9 mW at 780 nm and 29.7 mW at 846 nm with 7 focal spots. The image size was 40 µm×40 µm.

Fig. 4.
Fig. 4.

Video of CARS spectral images of polystyrene beads (3 µm diameter) during wavelength scanning (Media 2). The image size was 40 µm×40 µm.

Fig. 5.
Fig. 5.

Real-time CARS spectral imaging of polystyrene beads (3 µm diameter) during wavelength scanning. (a) Temporal behavior of the signal from the two-photon absorption detector (TPD) that monitored the pulse duration of the ω 2 light, and the observed Raman shift. (b) CARS images of the polystyrene beads at each Raman shift. Total incident laser intensities were 75.9mWat 780 nm and 29.7mWat 846 nm with 7 focal spots. The images were obtained with a frame rate of 30 fps. The scale bar represents 10 µm.

Fig. 6.
Fig. 6.

Raman and CARS spectra of the DPPC lipid powder at 1442 cm-1. Strong resonance was observed at 1442 cm-1, which corresponds to the CH2 deformation mode.

Fig. 7.
Fig. 7.

CARS images of the DPPC MLVs at a Raman shift of 1442 cm-1 and 1486 cm-1. The total incident laser intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nm with 7 focal spots. The image acquisition time was 3 s (exposure time of 100 ms and 30 acquisitions). The scale bar represents 10 µm.

Fig. 8.
Fig. 8.

Video of real-time CARS images of the DPPC MLV obtained while varying the z-position (Media 3). The frame rate was 10 fps. The observed molecular vibration was 1442 cm-1. The total incident intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nm with 7 focal spots. The image size was 60 µm×60 µm.

Fig. 9.
Fig. 9.

Real-time CARS image of the DPPC MLV at z-positions of (a) 0 µm, (b) 5 µm, and (c) 10 µm. The observed molecular vibration was 1442 cm-1. The total incident intensities were 46.2mWat 780 nm and 77.4mWat 879 nm with 7 focal spots. The image acquisition time was 100 ms. The scale bar represents 5 µm.

Fig. 10.
Fig. 10.

Three-dimensional reconstruction of the CARS images of a DPPC MLV. The right bottom, left, and top panels represent a xy, yz, and zx optical slice taken at the equator of the DPPC MLV, respectively. The observed molecular vibration was 1442 cm-1. The incident total laser intensities were 46.2 mW at 780 nm and 77.4 mW at 879 nm with 7 focal spots. The total image acquisition time of the DPPC MLV was 7 s per 50 slices (100 ms per cross-sectional image). The scale bar represents 5 µm.

Fig. 11.
Fig. 11.

CARS images of the HeLa cells at a Raman shift of 2840 cm-1. The total incident laser intensities were 80.1 mW at 712 nm and 40.5 mW at 892 nm with 7 focal spots. The image acquisition time was 1 s (exposure time of 100 ms and 10 acquisitions). The scale bar represents 10 µm.

Fig. 12.
Fig. 12.

Video of real-time CARS images of the HeLa cells (Media 4). The frame rate was 10 fps. The observed molecular vibration was 2840 cm-1. The total incident laser intensities were 80.1 mW at 712 nm and 40.5 mW at 892 nm with 7 focal spots. The image size was 40 µm×40 µm.

Equations (1)

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