Abstract

We achieve depth-resolved polarization microscopy by measuring third-harmonic generation induced by a tightly focused circularly polarized beam. In crystals exhibiting strong birefringence this signal is dominated by positively phase-matched third-harmonic generation. This process occurs in only optically anisotropic media, in which the birefringence compensates for the phase mismatch between the fundamental and the third harmonic induced by dispersion. Both the intensity and the polarization of the emitted signal provide information on the local optical anisotropy. We demonstrate the technique by imaging biogenic crystals in sea urchin larval spicules.

© 2003 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  7. D. Yelin and Y. Silberberg, Opt. Express 5, 169 (1999), http://www.opticsexpress.org.
    [CrossRef] [PubMed]
  8. D. Yelin and Y. Silberberg, Miscrosc. Anal. 80, 15 (2000).
  9. A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  16. R. Boyd, Nonlinear Optics (Academic, New York, 1992).
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    [CrossRef]
  19. E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
    [CrossRef]
  20. A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
    [CrossRef]
  21. Note that, because of the large positive phase mismatch in calcite, the THG signal as derived in relation 2 becomes exceedingly small when objectives with a small N.A. are used. We have verified this experimentally by using 0.25-N.A. and 0.57-N.A. objectives.
  22. D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
    [CrossRef]
  23. B. R. Masters, ed., Selected Papers on Multiphoton Excitation Microscopy (SPIE, Bellingham, Washington, 2003).

2002 (3)

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

2001 (3)

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

J. R. Kuhn, Z. Wu, and M. Poenie, Biophys. J. 80, 972 (2001).
[CrossRef] [PubMed]

V. V. Yakovlev and S. V. Govorkov, Appl. Phys. Lett. 79, 4136 (2001).
[CrossRef]

2000 (1)

D. Yelin and Y. Silberberg, Miscrosc. Anal. 80, 15 (2000).

1999 (2)

1998 (2)

1997 (3)

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

1996 (3)

R. Oldenburg, Nature 381, 811 (1996).
[CrossRef]

A. M. Glazer, J. G. Lewis, and W. Kaminsky, Proc. R. Soc. London Ser. A 452, 2751 (1996), and references therein.
[CrossRef]

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

1990 (1)

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

1988 (1)

A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
[CrossRef]

Addadi, L.

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

Aizenberg, J.

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Beniash, E.

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

Bouevitch, O.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Boyd, R.

R. Boyd, Nonlinear Optics (Academic, New York, 1992).

Brakenhoff, G. J.

Cheng, J.

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

de Boeij, W. P.

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Denk, W.

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

Dudovich, N.

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Glazer, A. M.

A. M. Glazer, J. G. Lewis, and W. Kaminsky, Proc. R. Soc. London Ser. A 452, 2751 (1996), and references therein.
[CrossRef]

Govorkov, S. V.

V. V. Yakovlev and S. V. Govorkov, Appl. Phys. Lett. 79, 4136 (2001).
[CrossRef]

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Jia, Y. K.

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Kaminsky, W.

A. M. Glazer, J. G. Lewis, and W. Kaminsky, Proc. R. Soc. London Ser. A 452, 2751 (1996), and references therein.
[CrossRef]

Korokotian, E.

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

Kuhn, J. R.

J. R. Kuhn, Z. Wu, and M. Poenie, Biophys. J. 80, 972 (2001).
[CrossRef] [PubMed]

Levenson, M. D.

M. D. Levenson, Introduction to Nonlinear Laser Spectroscopy (Academic, New York, 1982).

Lewis, A.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Lewis, J. G.

A. M. Glazer, J. G. Lewis, and W. Kaminsky, Proc. R. Soc. London Ser. A 452, 2751 (1996), and references therein.
[CrossRef]

Linial, M.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Loew, L.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Maiti, S.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Muller, M.

Oldenburg, R.

R. Oldenburg, Nature 381, 811 (1996).
[CrossRef]

Oron, D.

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

Ossig, F.

A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
[CrossRef]

Parnas, D.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Peleg, G.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Penzkofer, A.

A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
[CrossRef]

Poenie, M.

J. R. Kuhn, Z. Wu, and M. Poenie, Biophys. J. 80, 972 (2001).
[CrossRef] [PubMed]

Potma, E. O.

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Qiu, P.

A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
[CrossRef]

Segal, M.

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

Shear, J. B.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Silberberg, Y.

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

D. Yelin and Y. Silberberg, Miscrosc. Anal. 80, 15 (2000).

D. Yelin and Y. Silberberg, Opt. Express 5, 169 (1999), http://www.opticsexpress.org.
[CrossRef] [PubMed]

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Squier, J.

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. 191, 266 (1998).
[CrossRef]

Squier, J. A.

Stricker, J. H.

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

van Haastert, P. J. M.

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Webb, W. W.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

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

Weiner, S.

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

Wiersma, D. A.

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Williams, R. M.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Wilson, K. R.

Wu, Z.

J. R. Kuhn, Z. Wu, and M. Poenie, Biophys. J. 80, 972 (2001).
[CrossRef] [PubMed]

Xie, X. S.

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Yakovlev, V. V.

V. V. Yakovlev and S. V. Govorkov, Appl. Phys. Lett. 79, 4136 (2001).
[CrossRef]

Yelin, D.

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

D. Yelin and Y. Silberberg, Miscrosc. Anal. 80, 15 (2000).

D. Yelin and Y. Silberberg, Opt. Express 5, 169 (1999), http://www.opticsexpress.org.
[CrossRef] [PubMed]

Zheng, G.

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

Zipfel, W. R.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Zumbusch, A.

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Appl. Phys. B (2)

A. Penzkofer, F. Ossig, and P. Qiu, Appl. Phys. B 47, 71 (1988).
[CrossRef]

D. Yelin, D. Oron, E. Korokotian, M. Segal, and Y. Silberberg, Appl. Phys. B 74, S97 (2002).
[CrossRef]

Appl. Phys. Lett. (2)

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

V. V. Yakovlev and S. V. Govorkov, Appl. Phys. Lett. 79, 4136 (2001).
[CrossRef]

Bioimaging (1)

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, Bioimaging 4, 215 (1996).
[CrossRef]

Biophys. J. (2)

J. R. Kuhn, Z. Wu, and M. Poenie, Biophys. J. 80, 972 (2001).
[CrossRef] [PubMed]

J. Cheng, Y. K. Jia, G. Zheng, and X. S. Xie, Biophys. J. 83, 502 (2002).
[CrossRef] [PubMed]

J. Microsc. (1)

M. Muller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, J. Microsc. 191, 266 (1998).
[CrossRef]

Miscrosc. Anal. (1)

D. Yelin and Y. Silberberg, Miscrosc. Anal. 80, 15 (2000).

Nature (2)

N. Dudovich, D. Oron, and Y. Silberberg, Nature 418, 512 (2002).
[CrossRef] [PubMed]

R. Oldenburg, Nature 381, 811 (1996).
[CrossRef]

Opt. Express (2)

Phys. Rev. Lett. (1)

A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett. 82, 4142 (1999).
[CrossRef]

Proc. Natl. Acad. Sci. USA (1)

E. O. Potma, W. P. de Boeij, P. J. M. van Haastert, and D. A. Wiersma, Proc. Natl. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

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

A. M. Glazer, J. G. Lewis, and W. Kaminsky, Proc. R. Soc. London Ser. A 452, 2751 (1996), and references therein.
[CrossRef]

Proc. R. Soc. London Ser. B (1)

E. Beniash, J. Aizenberg, L. Addadi, and S. Weiner, Proc. R. Soc. London Ser. B 264, 461 (1997).
[CrossRef]

Science (2)

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

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Other (4)

R. Boyd, Nonlinear Optics (Academic, New York, 1992).

M. D. Levenson, Introduction to Nonlinear Laser Spectroscopy (Academic, New York, 1982).

B. R. Masters, ed., Selected Papers on Multiphoton Excitation Microscopy (SPIE, Bellingham, Washington, 2003).

Note that, because of the large positive phase mismatch in calcite, the THG signal as derived in relation 2 becomes exceedingly small when objectives with a small N.A. are used. We have verified this experimentally by using 0.25-N.A. and 0.57-N.A. objectives.

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Figures (2)

Fig. 1
Fig. 1

Experimental THG measurements from a thick x-cut calcite crystal when the input beam is focused either at the air–calcite interface (dashed curve) or within the calcite crystal (solid curve). a, THG intensity as a function of the relative angle between the input (linear) polarization and the ordinary axis of the crystal. b, THG intensity observed with circularly polarized illumination as a function of the relative angle between the analyzer polarization and the crystal ordinary axis.

Fig. 2
Fig. 2

Images of a single larval spicule spread on a glass slide. a, Image obtained with linearly polarized light, showing THG from both bulk birefringent media and interfaces. b, Image obtained with circularly polarized light, showing THG from only bulk birefringent media. c, Image obtained with circularly polarized light, with an analyzer oriented along the vertical direction. d, Same as c, with an analyzer oriented along the horizontal direction. Images c and d, which are given on the same intensity scale, indicate that throughout the larval spicule the crystal orientation along the horizontal axis is maintained.

Equations (2)

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A3=i2πgωncA13dzχ3zexpiΔkz1+2iz/b2,
A3bΔkexp-bΔk/2,

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