Abstract

We present a detailed analysis of the generation of second-harmonic radiation from biological membranes labeled with a styryl dye. In particular, we consider the high-numerical-aperture limit appropriate to high-resolution microscopy in which an excitation beam is tightly focused from the side onto a membrane surface. In this limit the active surface area that contributes to second-harmonic generation (SHG) depends only on the tightness of the beam focus and the SHG radiation is confined by phase matching into two well-defined off-axis lobes. We derive expressions for the SHG radiation power, angular distribution, and polarization dependence in the cases of ideal or nonideal molecular alignment in the membrane and uniaxiality of the molecular hyperpolarizability. We define an SHG cross section similar to that used in two-photon-excited fluorescence (TPEF) to permit direct comparison of the two imaging modalities. Finally, we corroborate our results with experiments based on the excitation of a styryl dye in giant unilamellar vesicles with a mode-locked Ti:sapphire laser.

© 2000 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. J. R. Lakowicz and I. Gryczynski, “Multiphoton excitation of biochemical fluorophores,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, p. 87.
  3. C. Xu and W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, p. 471.
  4. S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
    [CrossRef]
  5. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
    [CrossRef]
  6. J. I. Dadap, J. Shan, A. S. Weling, J. A. Misewich, A. Nahata, and T. F. Heinz, “Measurement of the vector character of electric fields by optical second-harmonic generation,” Opt. Lett. 24, 1059–1061 (1999).
    [CrossRef]
  7. R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Three-dimensional second-harmonic generation imaging with femtosecond laser pulses,” Opt. Lett. 23, 1209–1211 (1998).
    [CrossRef]
  8. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, “Second-harmonic tomography of tissues,” Opt. Lett. 22, 1323–1325 (1997).
    [CrossRef]
  9. G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
    [CrossRef]
  10. P. J. Campagnola, M. 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] [PubMed]
  11. D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5, 169–175 (1999).
    [CrossRef] [PubMed]
  12. M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
    [CrossRef]
  13. T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
    [CrossRef]
  14. N. Bloembergen, “Second harmonic reflected light,” Opt. Acta 13, 311–322 (1966).
    [CrossRef]
  15. Y. R. Shen, The Principles of Nonlinear Optics (Wiley, New York, 1984).
  16. R. Boyd, Nonlinear Optics (Academic, London, 1992).
  17. A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
    [CrossRef]
  18. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, “Membrane imaging by simultaneous second-harmonic generation and two photon microscopy,” Opt. Lett. 25, 320–322 (2000).
    [CrossRef]
  19. A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
    [CrossRef]
  20. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross section of molecular fluorophores with data from 690 nm to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
    [CrossRef]
  21. N. Bloembergen, Nonlinear Optics, 4th ed. (World Scientific, Singapore, 1965).
  22. M. Born and E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1993).
  23. J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998).
    [CrossRef]
  24. S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
    [CrossRef] [PubMed]
  25. T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
    [CrossRef]
  26. D. S. Chemla and J. Zyss, Nonlinear Optical Properties of Organic Molecules and Crystals (Academic, New York, 1984).
  27. J. L. Oudar, “Optical nonlinearities of conjugated molecules. Stilbene derivatives and highly polar aromatic compounds,” J. Chem. Phys. 67, 446–457 (1977).
    [CrossRef]
  28. L. M. Loew and L. L. Simpson, “Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer,” Biophys. J. 34, 353–365 (1981).
    [CrossRef] [PubMed]
  29. O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
    [CrossRef]
  30. C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
    [CrossRef]
  31. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in aplanetic system,” Proc. R. Soc. London, Ser. A 253, 358–379 (1959).
    [CrossRef]

2000 (1)

1999 (6)

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

J. I. Dadap, J. Shan, A. S. Weling, J. A. Misewich, A. Nahata, and T. F. Heinz, “Measurement of the vector character of electric fields by optical second-harmonic generation,” Opt. Lett. 24, 1059–1061 (1999).
[CrossRef]

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

P. J. Campagnola, M. 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] [PubMed]

D. Yelin and Y. Silberberg, “Laser scanning third-harmonic-generation microscopy in biology,” Opt. Express 5, 169–175 (1999).
[CrossRef] [PubMed]

O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
[CrossRef]

1998 (4)

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Three-dimensional second-harmonic generation imaging with femtosecond laser pulses,” Opt. Lett. 23, 1209–1211 (1998).
[CrossRef]

J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998).
[CrossRef]

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

1997 (2)

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, “Second-harmonic tomography of tissues,” Opt. Lett. 22, 1323–1325 (1997).
[CrossRef]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

1996 (2)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross section of molecular fluorophores with data from 690 nm to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

1992 (1)

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

1991 (1)

S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
[CrossRef] [PubMed]

1990 (1)

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

1986 (1)

C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
[CrossRef]

1983 (1)

T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
[CrossRef]

1981 (1)

L. M. Loew and L. L. Simpson, “Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer,” Biophys. J. 34, 353–365 (1981).
[CrossRef] [PubMed]

1977 (1)

J. L. Oudar, “Optical nonlinearities of conjugated molecules. Stilbene derivatives and highly polar aromatic compounds,” J. Chem. Phys. 67, 446–457 (1977).
[CrossRef]

1966 (1)

N. Bloembergen, “Second harmonic reflected light,” Opt. Acta 13, 311–322 (1966).
[CrossRef]

1959 (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in aplanetic system,” Proc. R. Soc. London, Ser. A 253, 358–379 (1959).
[CrossRef]

Alfano, R. R.

Beljonne, D.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Beratan, D. N.

S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
[CrossRef] [PubMed]

Blanchard-Desce, M.

Bloembergen, N.

N. Bloembergen, “Second harmonic reflected light,” Opt. Acta 13, 311–322 (1966).
[CrossRef]

Bouetvich, O.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Brakenhoff, G. J.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

Brédas, J. L.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Brochard, F.

O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
[CrossRef]

Burland, D.

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

Campagnola, P. J.

P. J. Campagnola, M. 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] [PubMed]

Chen, Z.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Cheng, L.-T.

S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
[CrossRef] [PubMed]

Dadap, J. I.

Denk, W.

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

Dirk, C. W.

C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
[CrossRef]

Friedman, N.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Gauderon, R.

Guo, Y.

Harris, D.

Heinz, T. F.

J. I. Dadap, J. Shan, A. S. Weling, J. A. Misewich, A. Nahata, and T. F. Heinz, “Measurement of the vector character of electric fields by optical second-harmonic generation,” Opt. Lett. 24, 1059–1061 (1999).
[CrossRef]

T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
[CrossRef]

Ho, P. P.

Khatchatouriants, A.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Kogej, T.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Lewis, A.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

P. J. Campagnola, M. 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] [PubMed]

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

Linial, M.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

Liu, F.

Loew, L.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Loew, L. M.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

P. J. Campagnola, M. 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] [PubMed]

L. M. Loew and L. L. Simpson, “Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer,” Biophys. J. 34, 353–365 (1981).
[CrossRef] [PubMed]

Lukins, P. B.

Maiti, S.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

Marder, S. R.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
[CrossRef] [PubMed]

Mertz, J.

Meyers, F.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Misewich, J. A.

Moreaux, L.

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, “Membrane imaging by simultaneous second-harmonic generation and two photon microscopy,” Opt. Lett. 25, 320–322 (2000).
[CrossRef]

O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
[CrossRef]

Muller, M.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

Nahata, A.

Oudar, J. L.

J. L. Oudar, “Optical nonlinearities of conjugated molecules. Stilbene derivatives and highly polar aromatic compounds,” J. Chem. Phys. 67, 446–457 (1977).
[CrossRef]

Peleg, G.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Perry, J. W.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Rice, J. E.

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in aplanetic system,” Proc. R. Soc. London, Ser. A 253, 358–379 (1959).
[CrossRef]

Rothman, Z.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Sacks, P.

Sandre, O.

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, “Membrane imaging by simultaneous second-harmonic generation and two photon microscopy,” Opt. Lett. 25, 320–322 (2000).
[CrossRef]

O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
[CrossRef]

Savage, H.

Schantz, S.

Shan, J.

Shear, J. B.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

Shelton, D. P.

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

Shen, Y. R.

T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
[CrossRef]

Sheppard, C. J. R.

Sheres, M.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Silberberg, Y.

Simpson, L. L.

L. M. Loew and L. L. Simpson, “Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer,” Biophys. J. 34, 353–365 (1981).
[CrossRef] [PubMed]

Squier, J.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

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]

Tom, H. W. K.

T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
[CrossRef]

Treinin, M.

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Twieg, R. J.

C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
[CrossRef]

Wagniére, G.

C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
[CrossRef]

Webb, W. W.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross section of molecular fluorophores with data from 690 nm to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

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

Wei, M.

P. J. Campagnola, M. 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] [PubMed]

Weling, A. S.

Willets, A.

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

Williams, R. M.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

Wilson, K. R.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in aplanetic system,” Proc. R. Soc. London, Ser. A 253, 358–379 (1959).
[CrossRef]

Xu, C.

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross section of molecular fluorophores with data from 690 nm to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

Yelin, D.

Zhadin, N.

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

Zipfel, W. R.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

Biophys. J. (2)

P. J. Campagnola, M. 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] [PubMed]

L. M. Loew and L. L. Simpson, “Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer,” Biophys. J. 34, 353–365 (1981).
[CrossRef] [PubMed]

Chem. Phys. (1)

A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouetvich, Z. Rothman, L. Loew, and M. Sheres, “Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans,” Chem. Phys. 245, 133–144 (1999).
[CrossRef]

Chem. Phys. Lett. (1)

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, “Mechanisms for enhancement of two-photon absorption in donor–acceptor conjugated chromophores,” Chem. Phys. Lett. 298, 1–6 (1998).
[CrossRef]

Eur. Phys. J. D (1)

J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998).
[CrossRef]

J. Am. Chem. Soc. (1)

C. W. Dirk, R. J. Twieg, and G. Wagniére, “The contribution of π electrons to second harmonic generation in organic molecules,” J. Am. Chem. Soc. 108, 5387–5395 (1986).
[CrossRef]

J. Chem. Phys. (2)

J. L. Oudar, “Optical nonlinearities of conjugated molecules. Stilbene derivatives and highly polar aromatic compounds,” J. Chem. Phys. 67, 446–457 (1977).
[CrossRef]

A. Willets, J. E. Rice, D. Burland, and D. P. Shelton, “Problems in the comparison of theoretical and experimental hyperpolarizabilities,” J. Chem. Phys. 97, 7590–7599 (1992).
[CrossRef]

J. Microsc. (1)

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–269 (1998).
[CrossRef]

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

Opt. Acta (1)

N. Bloembergen, “Second harmonic reflected light,” Opt. Acta 13, 311–322 (1966).
[CrossRef]

Opt. Express (1)

Opt. Lett. (4)

Phys. Rev. A (1)

T. F. Heinz, H. W. K. Tom, and Y. R. Shen, “Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation,” Phys. Rev. A 28, 1883–1885 (1983).
[CrossRef]

Proc. Natl. Acad. Sci. USA (3)

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, “Non-linear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
[CrossRef]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. USA 93, 10, 763–10, 768 (1996).
[CrossRef]

O. Sandre, L. Moreaux, and F. Brochard, “Dynamics of transient pores in stretched vesicles,” Proc. Natl. Acad. Sci. USA 96, 10, 588–10, 596 (1999).
[CrossRef]

Proc. R. Soc. London, Ser. A (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in aplanetic system,” Proc. R. Soc. London, Ser. A 253, 358–379 (1959).
[CrossRef]

Science (3)

S. R. Marder, D. N. Beratan, and L.-T. Cheng, “Approaches for optimizing the first hyperpolarizability of conjugated organic molecules,” Science 252, 103–106 (1991).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring serotonin distribution in live cells with three-photon excitation,” Science 24, 530–532 (1997).
[CrossRef]

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

Other (7)

J. R. Lakowicz and I. Gryczynski, “Multiphoton excitation of biochemical fluorophores,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, p. 87.

C. Xu and W. W. Webb, “Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1997), Vol. 5, p. 471.

Y. R. Shen, The Principles of Nonlinear Optics (Wiley, New York, 1984).

R. Boyd, Nonlinear Optics (Academic, London, 1992).

D. S. Chemla and J. Zyss, Nonlinear Optical Properties of Organic Molecules and Crystals (Academic, New York, 1984).

N. Bloembergen, Nonlinear Optics, 4th ed. (World Scientific, Singapore, 1965).

M. Born and E. Wolf, Principles of Optics, 6th ed. (Pergamon, Oxford, 1993).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (9)

Fig. 1
Fig. 1

Coordinate system defining the SHG emission direction. The membrane surface (shaded; z=0) is approximated to be planar at the length scales considered. The focused excitation beam propagates in the +x direction. The resultant SHG is mostly confined to an interaction area schematically depicted as a dotted ellipsoid and is radiated in the directions defined by θ and φ (thick arrow), with polarization components parallel to (E2ωp) and perpendicular to (E2ωs) the emission plane (shown dashed).

Fig. 2
Fig. 2

Left, an excitation beam propagating in the x direction and polarized along the z axis is focused (side-on) onto the membrane of a labeled lipid vesicle. Only a small surface area (thick segment; side view) of this much larger vesicle contributes to SHG. Phase matching between the SHG and excitation fields causes the SHG radiation to be double peaked in the forward direction. Right, far-field power distribution of the SHG radiation.

Fig. 3
Fig. 3

Experimental layout: a Ti:sapphire laser beam is focused into a sample with a microscope objective (MO). The transmitted SHG is collected with a condenser (C), bandpass (BP) filtered, and detected with a photomultiplier tube (PMT). The transmitted laser light is blocked with a colored glass filter (F). The TPEF from the sample is epicollected, discriminated with a dichroic mirror (DM), bandpass (BP) filtered, and detected with a PMT. Three-dimensional images are formed by scanning the laser focal spot in the lateral directions with galvanometer-mounted mirrors, and in the axial direction by translating the MO.

Fig. 4
Fig. 4

Simultaneous (a) TPEF and (b) SHG images of three vesicles labeled with Di-6-ASPBS that have adhered to form a foam. The radiating Di-6-ASPBS molecules are more-or-less symmetrically distributed in the adherence regions between the vesicles. In the left region, a symmetric distribution results in a nearly perfect cancellation of SHG. In the right region, the cancellation is imperfect because of a disparity in labeling density. As opposed to SHG radiation, TPEF is independent of molecular distribution because it is incoherent.

Fig. 5
Fig. 5

CCD image of the back aperture of the SHG collection objective. The excitation beam is scanned only over a small portion of an equatorial slice of a GUV membrane. The double-peaked angular distribution of the SHG radiation is apparent.

Fig. 6
Fig. 6

Stack of CCD images of the back aperture of the SHG collection objective. The excitation beam, incident from above in the insets, is scanned through full cross sections of a GUV at various latitudes. The equatorial cross section is scanned for image (a), leading to a single annular ring at θpeak, whereas latitudes below the equator are scanned for images (b) and (c). The CCD images were integrated over times long enough to permit multiple scans. From image (a) we deduce that θpeak24°.

Fig. 7
Fig. 7

Variation of SHG power along the equator of a GUV labeled with Di-6-ASPBS. Excitation is linearly polarized. A polarizer is inserted in the detection path and is oriented parallel (PSHG) or perpendicular (PSHG) to the excitation polarization. Plots are shown as a function of angle ϕ between the normal to the membrane plane (z axis) and the excitation polarization direction. The dashed trace corresponds to the cos6 ϕ dependence expected for perfectly aligned uniaxial molecules.

Fig. 8
Fig. 8

Variations of the ratio θpeak/θNA [where θNA =sin-1(NA/n)] and of the parameter (1-ξ2)1/2/ξ2 as a function of the NA of a water-immersion microscope objective, assuming a uniformly backfilled aperture and linear polarization. The plots are obtained from full numerical evaluations valid for arbitrary NA. For low NA, θpeakθNA/2.

Fig. 9
Fig. 9

Spatial profile of the phase-anomaly parameter ξ (shades) and of the normalized intensity-squared distribution (contours) about the focal center (0, 0) of an 880-nm-wavelength laser beam focused by water-immersion objective with a NA of 0.9. The axial propagation direction is x; the lateral direction is y. Profiles were calculated assuming a uniformly backfilled aperture and linear polarization. ξ is relatively constant within the major portion of the intensity-squared profile.

Equations (46)

Equations on this page are rendered with MathJax. Learn more.

μ 2ω=½βEω2zˆ,
E2ω(ψ)=-μ2ωω2πε0c2r sin(ψ)exp(-2iω[t])ψˆ,
P2ω(ψ)=316πσSHGsin2(ψ)Iω2,
σSHG=4n2ωω53πnω2ε03c5|β|2
PSHG=½σSHG Iω2
PTPEF=½σTPEF Iω2,
μ2ω,i(x, y)=½Eω2(x, y)j,kβijkεˆjεˆk,
Eω(x, y)=-iEω exp-x2wx2-y2wy2+iξkω x,
E2ω(0)(θ, φ)=E2ω(0)p(θ, φ)E2ω(0)s(θ, φ)=ηr M·μ 2ω(0),
Mθˆφˆ=-sin θcos θ sin φcos θ cos φ0cos φ-sin φ.
E2ω(θ, φ)
=ηNsr  M·μ2ω(x, y)×exp[-ik2ω(x cos θ+y sin θ sin φ)]dx dy,
E2ω(θ, φ)=NA(θ, φ)E2ω(0)(θ, φ),
N=π2wxwyNs,
A(θ, φ)=exp-k2ω28[wx2(cos θ-ξ)2+wy2(sin θ sin φ)2]
P2ωp,s(θ, φ)=½n2ωε0cr2|E2ωp,s(θ, φ)|2,
PSHG=PSHGp+PSHGs=4π3n2ωε0cη2N2×[Θxμ2ω,x(0)2+Θyμ2ω,y(0)2+Θzμ2ω,z(0)2],
Θx=38π A2(θ, φ)sin3 θdθdφ,
Θy=38π A2(θ, φ)[1-sin2 θ sin2 φ]sin θdθdφ,
Θz=38π A2(θ, φ)[1-sin2 θ cos2 φ]sin θdθdφ.
βxxz=βxzx=βyyz=βyzyβ+,
βxyz=βxzy=-βyxz=-βyzxβ-,
βzxx=βzyyβt,
βzzzβz,
β+=cos3 αB++½sin2 α cos α(BZ-BT),
β-=½3 cos2 α-1B-,
βt=cos3 αBT+½sin2 α cos α(BZ+BT-2B+),
βz=cos3 αBZ+sin2 α cos α(BT+2B+),
B+=½(βXXZ+βYYZ),
B-=½(βXYZ-βYXZ),
BT=½(βZXX+βZYY),
BZ=βZZZ.
μ2ω(0)=½ Eω2(β- sin 2ϕ, β+ sin 2ϕ, βz cos2 ϕ+βt sin2 ϕ).
μ2ω(0)=½βzEω2zˆ,
P2ωp(θ, φ)=316πσSHG N2A2(θ, φ)cos2 θ cos2 φIω2,
P2ωs(θ, φ)=316πσSHG N2A2(θ, φ)sin2 φIω2,
PSHG=½ΘzN2σSHG Iω2.
PSHGPTPEF=22ΘzN σSHGσTPEF,
Θz3ξ2k2ω2wxwy1-ξ2.
μ 2ω(0)=½ Eω2(0, 0, βz cos2 ϕ).
E2ωE2ω=cos(φ-ϕ)-sin(φ-ϕ)sin(φ-ϕ)cos(φ-ϕ)E2ωpE2ωs.
PSHG=4π3n2ωε0cη2N2μ2ω,z(0)2[(Θz-2Θz)cos2 ϕ+Θz],
PSHG=4π3n2ωε0cη2N2μ2ω,z(0)2[(Θz-2Θz)sin2 ϕ+Θz],
Θz=38π  A2(θ, φ)×[(1-cos θ)2 sin2 φ cos2 φ]sin θdθdφ.
βZZZ(-2ω, ω, ω)
=22μeg2Δμ1(ωeg-2ω+iΓ)(ωeg-ω+iΓ)+1(ωeg-ω+iΓ)(ωeg+ω-iΓ)+1(ωeg+2ω-iΓ)(ωeg+ω-iΓ),

Metrics