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.

© 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. M�ller, 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.

Other

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

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]

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

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]

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

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]

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]

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]

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

M. M�ller, 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]

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. 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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