Abstract

A laser scanning microscope using third-harmonic generation as a probe is shown to produce high-resolution images of transparent biological specimens. Third harmonic light is generated by a tightly focused short-pulse laser beam and collected point-by-point to form a digital image. Demonstrations with two biological samples are presented. Live neurons in a cell culture are imaged with clear and detailed images, including organelles at the threshold of optical resolution. Internal organelles of yeast cells are also imaged, demonstrating the ability of the technique for cellular and intracellular imaging.

© 1999 Optical Society of America

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References

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  1. T. Wilson, Confocal microscopy, (Academic, London, 1990).
  2. W. Denk, J. H. Stricker, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  3. 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 275, 530–532 (1997).
    [CrossRef] [PubMed]
  4. M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).
  5. D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
    [CrossRef]
  6. R. Hellwarth and P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Comm. 12, 318–322 (1974).
    [CrossRef]
  7. J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. and Quant. Elect. 10, 435–439 (1978).
    [CrossRef]
  8. 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]
  9. G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
    [CrossRef]
  10. Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
    [CrossRef]
  11. R. Boyd, Nonlinear Optics, (Academic, New York, 1992).
  12. M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
    [CrossRef] [PubMed]
  13. J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, “Third harmonic generation microscopy,” Opt. Express 3, 315–324 (1998). http://www.opticsexpress.org/oearchive/source/5872.htm
    [CrossRef] [PubMed]
  14. D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
    [CrossRef]
  15. Y. R. Shen, The principles of nonlinear optics, (Wiley, New York, 1984), Chap. 27.

1999 (1)

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[CrossRef]

1998 (3)

1997 (3)

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 275, 530–532 (1997).
[CrossRef] [PubMed]

M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

1996 (2)

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[CrossRef]

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

1990 (1)

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

1978 (1)

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. and Quant. Elect. 10, 435–439 (1978).
[CrossRef]

1974 (1)

R. Hellwarth and P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Comm. 12, 318–322 (1974).
[CrossRef]

Bahlmann, K.

M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).

Barad, Y.

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[CrossRef]

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Bouevitch, O.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

Boyd, R.

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

Brakenhoff, G. J.

M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
[CrossRef] [PubMed]

J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, “Third harmonic generation microscopy,” Opt. Express 3, 315–324 (1998). http://www.opticsexpress.org/oearchive/source/5872.htm
[CrossRef] [PubMed]

Centonze, V. E.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[CrossRef]

Christensen, P.

R. Hellwarth and P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Comm. 12, 318–322 (1974).
[CrossRef]

Crittenden, S.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[CrossRef]

Denk, W.

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

Eizenberg, H.

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Gannaway, J. N.

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. and Quant. Elect. 10, 435–439 (1978).
[CrossRef]

Gauderon, R.

Hell, S. W.

M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).

Hellwarth, R.

R. Hellwarth and P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Comm. 12, 318–322 (1974).
[CrossRef]

Horowitz, M.

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Lewis, A.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

Linial, M.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

Loew, L.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

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 275, 530–532 (1997).
[CrossRef] [PubMed]

Muller, M.

Müller, M.

M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
[CrossRef] [PubMed]

Parnas, D.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

Patel, J. S.

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[CrossRef]

Peleg, G.

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

Schrader, M.

M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).

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 275, 530–532 (1997).
[CrossRef] [PubMed]

Shen, Y. R.

Y. R. Shen, The principles of nonlinear optics, (Wiley, New York, 1984), Chap. 27.

Sheppard, C. J. R.

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. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. and Quant. Elect. 10, 435–439 (1978).
[CrossRef]

Silberberg, Y.

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[CrossRef]

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

Sqier, J.

M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
[CrossRef] [PubMed]

Squier, J. A.

Stricker, J. H.

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

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 275, 530–532 (1997).
[CrossRef] [PubMed]

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

White, J.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[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 275, 530–532 (1997).
[CrossRef] [PubMed]

Wilson, K. R.

M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
[CrossRef] [PubMed]

J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, “Third harmonic generation microscopy,” Opt. Express 3, 315–324 (1998). http://www.opticsexpress.org/oearchive/source/5872.htm
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, Confocal microscopy, (Academic, London, 1990).

Wokosin, D. L.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[CrossRef]

Yelin, D.

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[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 275, 530–532 (1997).
[CrossRef] [PubMed]

Appl. Phys. Lett. (2)

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third-harmonic generation,” Appl. Phys. Lett. 70, 922–924 (1997).
[CrossRef]

D. Yelin, Y. Silberberg, Y. Barad, and J. S. Patel, “Depth resolved imaging of nematic liquid crystals by third-harmonic microscopy,” Appl. Phys. Lett. 74, 3107–3109 (1999).
[CrossRef]

Bioimaging (2)

G. Peleg, A. Lewis, O. Bouevitch, L. Loew, D. Parnas, and M. Linial, “Gigantic optical non-linearities from nanopartical-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems,” Bioimaging 4, 215–224 (1996).
[CrossRef]

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, “Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser,” Bioimaging 4, 208–214 (1996).
[CrossRef]

J. Microsc (1)

M. Müller, J. Sqier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc 191, 266–274 (1998).
[CrossRef] [PubMed]

Opt. and Quant. Elect. (1)

J. N. Gannaway and C. J. R. Sheppard, “Second-harmonic imaging in the scanning optical microscope,” Opt. and Quant. Elect. 10, 435–439 (1978).
[CrossRef]

Opt. Comm. (1)

R. Hellwarth and P. Christensen, “Nonlinear optical microscopic examination of structure in polycrystalline ZnSe,” Opt. Comm. 12, 318–322 (1974).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Optik (1)

M. Schrader, K. Bahlmann, and S. W. Hell, “Three-photon-excitation microscopy: theory, experiment and applications,” Optik 104, 116–124 (1997).

Science (2)

W. Denk, J. H. Stricker, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[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 275, 530–532 (1997).
[CrossRef] [PubMed]

Other (3)

T. Wilson, Confocal microscopy, (Academic, London, 1990).

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

Y. R. Shen, The principles of nonlinear optics, (Wiley, New York, 1984), Chap. 27.

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Figures (7)

Fig. 1.
Fig. 1.

Optical setup for laser scanning THG microscopy.

Fig. 2.
Fig. 2.

THG images of neurons in a cell culture. The size of the cell’s soma is about 15 µm.

Fig. 3.
Fig. 3.

Sectioning of live neurons in a cell culture. Each image is a horizontal section of the neuron’s soma. The sections are separated by 0.5 µm, where the top-left section is closer to the glass substrate and the bottom-right section is the top of the cell. The dimensions of each image are 20×20 µm.

Fig. 4.
Fig. 4.

Vertical sectioning of the neurons in Fig. 3. The bright nucleolus, the dark nucleus and organelles outside the nucleus can be seen.

Fig. 5.
Fig. 5.

Sectioning of live neurons. This cell has two nucleoli. The dimensions of each image are 20×20 µm.

Fig. 6.
Fig. 6.

THG images of three dendritic spines on a single dendrite.

Fig. 7.
Fig. 7.

THG images of yeast cell.

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