Abstract

We demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics.

© 2000 Optical Society of America

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Errata

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, "Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy: errata," Opt. Lett. 25, 678-678 (2000)
https://www.osapublishing.org/ol/abstract.cfm?uri=ol-25-9-678

References

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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  6. S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
    [CrossRef] [PubMed]
  7. T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).
  8. O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
    [CrossRef]
  9. L. M. Loew and L. L. Simpson, Biophys. J. 34, 353 (1981).
    [CrossRef]
  10. D. S. Chemla and J. Zyss, eds., Nonlinear Optical Properties of Organic Molecules and Crystals (Academic, New York, 1984), Vol. 1.
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  12. Y. R. Shen, The Principles of Nonlinear Optics (Wiley, New York, 1984).

1999 (3)

P. J. Campagnola, M. Wei, and L. M. Loew, Biophys. J. 76, A95 (1999).

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
[CrossRef]

1998 (2)

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, Opt. Lett. 23, 1209 (1998).
[CrossRef]

1997 (1)

1991 (1)

S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
[CrossRef] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1981 (1)

L. M. Loew and L. L. Simpson, Biophys. J. 34, 353 (1981).
[CrossRef]

1977 (1)

J. L. Oudar and D. S. Chemla, J. Chem. Phys. 66, 2664 (1977).

Alfano, R. R.

Beljonne, D.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

Beratan, D. N.

S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
[CrossRef] [PubMed]

Brédas, J. L.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

Brochard, F.

O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
[CrossRef]

Campagnola, P. J.

P. J. Campagnola, M. Wei, and L. M. Loew, Biophys. J. 76, A95 (1999).

Chemla, D. S.

J. L. Oudar and D. S. Chemla, J. Chem. Phys. 66, 2664 (1977).

Cheng, L.-T.

S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Gauderon, R.

Guo, Y.

Harris, D.

Ho, P. P.

Kogej, T.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

Lewis, A.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

Linial, M.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

Liu, F.

Loew, L. M.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

P. J. Campagnola, M. Wei, and L. M. Loew, Biophys. J. 76, A95 (1999).

L. M. Loew and L. L. Simpson, Biophys. J. 34, 353 (1981).
[CrossRef]

Lukins, P. B.

Marder, S. R.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
[CrossRef] [PubMed]

Meyers, F.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

Moreaux, L.

O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
[CrossRef]

Oudar, J. L.

J. L. Oudar and D. S. Chemla, J. Chem. Phys. 66, 2664 (1977).

Peleg, G.

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

Perry, J. W.

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

Sacks, P.

Sandre, O.

O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
[CrossRef]

Savage, H.

Schantz, S.

Shen, Y. R.

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

Sheppard, C. J. R.

Simpson, L. L.

L. M. Loew and L. L. Simpson, Biophys. J. 34, 353 (1981).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wei, M.

P. J. Campagnola, M. Wei, and L. M. Loew, Biophys. J. 76, A95 (1999).

Zhadin, N.

Biophys. J. (2)

P. J. Campagnola, M. Wei, and L. M. Loew, Biophys. J. 76, A95 (1999).

L. M. Loew and L. L. Simpson, Biophys. J. 34, 353 (1981).
[CrossRef]

Chem. Phys. Lett. (1)

T. Kogej, D. Beljonne, F. Meyers, J. W. Perry, S. R. Marder, and J. L. Brédas, Chem. Phys. Lett. 298, 1 (1998).

J. Chem. Phys. (1)

J. L. Oudar and D. S. Chemla, J. Chem. Phys. 66, 2664 (1977).

Opt. Lett. (2)

Proc. Natl. Acad. Sci. USA (2)

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, Proc. Natl. Acad. Sci. USA 96, 6700 (1999).
[CrossRef]

O. Sandre, L. Moreaux, and F. Brochard, Proc. Natl. Acad. Sci. USA 96, 10,588 (1999).
[CrossRef]

Science (2)

S. R. Marder, D. N. Beratan, and L.-T. Cheng, Science 252, 103 (1991).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other (2)

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

D. S. Chemla and J. Zyss, eds., Nonlinear Optical Properties of Organic Molecules and Crystals (Academic, New York, 1984), Vol. 1.

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Figures (3)

Fig. 1
Fig. 1

Experimental layout: Ti:sapphire laser light is focused into a sample with a microscope objective (MO; 0.9-N.A. water immersion; Olympus). The transmitted SHG is collected with a condenser (C; 1.4-N.A., Olympus), bandpass (BP) filtered, and detected with a photomultiplier tube (PMT). The transmitted laser light is blocked with a CuSO4 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 of the laser focal spot in the XY directions with galvanometer-mounted mirrors and in the Z direction by translation of the microscope objective.

Fig. 2
Fig. 2

Left, an excitation beam propagating in the z direction and polarized along the y 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. The surface area is defined by N/Ns, where N is the effective number of radiating molecules and Ns is their surface density (molecules/unit membrane area). Right, the SHG radiation is double peaked in the forward direction with a far-field power distribution given by Sθ,φ. The peaks are separated in the yz plane by 2θpeak60°.

Fig. 3
Fig. 3

(a) SHG and (b) TPEF images of two adhering vesicles labeled with Di-6-ASPBS (equatorial slice), excited at 880 nm. The total acquisition time for the images was 1.5 s, for an excitation power at the sample of <1 mW. The adhesion area where the membranes are fused exhibits a centrosymmetric molecular distribution wherein TPEF is allowed but SHG is not. The corresponding forward-detected emission spectra of SHG (left peak) and TPEF (right peak) are shown in (c). The large Stokes shift in fluorescence gives rise to a large spectral separation between the two emission peaks. An expanded view of the SHG peak is shown in the inset (4-nm resolution).

Equations (4)

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P2ωθ,φ=ω42π2n03c5N2β2Sθ,φIω2,
Θ=38πθ,φdΩ3πξ24kω2wxwz1-ξ2.
σSHG=4ω53π2n03c5β2 m4/photon s-1,
PSHGPTPEFΘNσSHGσTPEF.

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