Abstract

The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.

© 2003 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |

  1. G. Dolino, �??Direct observation of ferroelectric domains in TGS with second-harmonic light,�?? Appl. Phys. Lett. 22, 123-124 (1973).
    [CrossRef]
  2. J. N. Gannaway and C. J. R. Sheppard, �??Second-harmonic imaging in the scanning optical microscope,�?? Opt. Quantum Electron. 10, 435 (1978).
    [CrossRef]
  3. Y. R. Shen, �??Surface properties probed by 2nd harmonic and sum frequency generation,�?? Nature 337, 519 (1989).
    [CrossRef]
  4. C.-K. Sun, S.-W. Chu, S.-P. Tai, S. Keller, U. K. Mishra, and S. P. DenBaars, �??Scanning second-harmonicgeneration and third-harmonic-generation microscopy of GaN,�?? Appl. Phys. Lett. 77, 2331-2333 (2000).
    [CrossRef]
  5. C. -K. Sun, S. W. Chu, S. P. Tai, S. Keller, A. Abare, U. K. Mishira, and S. P. DenBaars, �??Mapping Piezoelectric-Field Distribution in Gallium Nitride with Scanning Second-Harmonic Generation Microscopy,�?? Scanning 23, 182 (2001).
    [CrossRef] [PubMed]
  6. P. J. Campagnola, M. -D. 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]
  7. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, �??Membrane imaging by simultaneous secondharmonic generation and two-photon microscopy,�?? Opt. Lett. 25, 320-322 (2000).
    [CrossRef]
  8. G. Peleg, A. Lewis, M. Linial, and L. M. Loew, �??Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites,�?? Proc. Natl. Acad. Sci. 96, 6700-6704 (1999).
    [CrossRef] [PubMed]
  9. I. Freund, M. Deutsch, and A. Sprecher, �??Connective Tissue Polarity: Optical Second-harmonic Microscopy, Crossed-beam Summation, and Small-angle Scattering in Rat-tail Tendon,�?? Biophys. J. 50, 693-712 (1986).
    [CrossRef] [PubMed]
  10. Y. Guo, P. P. Ho, A. Tirksliunas, F. Liu, and R. R. Alfano, �??Optical harmonic generation from animal tissues by the use of picosecond and femtosecond laser pulses,�?? Opt. Lett. 22, 1323-1325 (1997).
    [CrossRef]
  11. S.-W. Chu, I-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, �??Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy,�?? J. Microscopy 208, 190-200 (2002).
    [CrossRef]
  12. S.-W. Chu, I-S. Chen, T.-M. Liu, B.-L. Lin, P.-C. Cheng, and C.-K. Sun, �??Multi-modal Nonlinear Spectral Microscopy Based on a Femtosecond Cr:forsterite Laser,�?? Opt. Lett. 26, 1909-1911 (2001).
    [CrossRef]
  13. Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, �??Subsurface tumor progression investigated by noninvasive optical second harmonic tomography,�?? Proc. Natl. Acad. Sci. 96, 10854-10856 (1999).
    [CrossRef] [PubMed]
  14. W. Denk, J. H. Strickler, and W. W. Webb, �??Two photon laser scanning fluorescence microscopy,�?? Science 248, 73 (1990).
    [CrossRef] [PubMed]
  15. K. König, T. W. Becker, P. Fischer, I. Riemann, and K.-J. Halbhuber, �??Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,�?? Opt. Lett. 24, 113-115 (1999).
    [CrossRef]
  16. A. Vogel, J. Noack, G. Hüttmann, and G. Paltauf, �??Femtosecond-laser-produced low-density plasmas in transparent biological media: A tool for the creation of chemical, thermal and thermomechanical effects below the optical breakdown threshold,�?? Proc. SPIE 4633A, 1-15 (2002).
  17. R. Shack, B. Bell, D. Hillman, R. Kingston, A. Landesman, R. Shoemaker, D. Vukobratovich, and P.H. Bartels, �??Ultrafast laser scanner microscope-first performance test (biological application),�?? IEEE International Workshop on Physics and Engineering in Medical Imaging. viii + 292, 49-57 (1982).
  18. M. Kobayashi, K. Fujita, T. Kaneko, T. Takamatsu, O. Nakamura, and S. Kawata, �??Second-harmonicgeneration microscope with a microlens array scanner,�?? Opt. Lett. 27, 1324-1326 (2002).
    [CrossRef]
  19. Private communication with OLYMPUS OPTICAL CO., LTD.
  20. Gavin D. Reid and Klaas Wynne, �??Ultrafast laser technology and spectroscopy,�?? in Encyclopedia of Analytical Chemistry, R.A. Meyers ed. (John Wiley & Sons Ltd, Chichester, UK. 2000)

Appl. Phys. Lett. (2)

G. Dolino, �??Direct observation of ferroelectric domains in TGS with second-harmonic light,�?? Appl. Phys. Lett. 22, 123-124 (1973).
[CrossRef]

C.-K. Sun, S.-W. Chu, S.-P. Tai, S. Keller, U. K. Mishra, and S. P. DenBaars, �??Scanning second-harmonicgeneration and third-harmonic-generation microscopy of GaN,�?? Appl. Phys. Lett. 77, 2331-2333 (2000).
[CrossRef]

Biophys. J. (2)

P. J. Campagnola, M. -D. 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]

I. Freund, M. Deutsch, and A. Sprecher, �??Connective Tissue Polarity: Optical Second-harmonic Microscopy, Crossed-beam Summation, and Small-angle Scattering in Rat-tail Tendon,�?? Biophys. J. 50, 693-712 (1986).
[CrossRef] [PubMed]

J. Microscopy (1)

S.-W. Chu, I-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, �??Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy,�?? J. Microscopy 208, 190-200 (2002).
[CrossRef]

Nature (1)

Y. R. Shen, �??Surface properties probed by 2nd harmonic and sum frequency generation,�?? Nature 337, 519 (1989).
[CrossRef]

Opt. Lett. (5)

Opt. Quantum Electron. (1)

J. N. Gannaway and C. J. R. Sheppard, �??Second-harmonic imaging in the scanning optical microscope,�?? Opt. Quantum Electron. 10, 435 (1978).
[CrossRef]

Proc. Natl. Acad. Sci. (2)

G. Peleg, A. Lewis, M. Linial, and L. M. Loew, �??Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites,�?? Proc. Natl. Acad. Sci. 96, 6700-6704 (1999).
[CrossRef] [PubMed]

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, �??Subsurface tumor progression investigated by noninvasive optical second harmonic tomography,�?? Proc. Natl. Acad. Sci. 96, 10854-10856 (1999).
[CrossRef] [PubMed]

Proc. SPIE (1)

A. Vogel, J. Noack, G. Hüttmann, and G. Paltauf, �??Femtosecond-laser-produced low-density plasmas in transparent biological media: A tool for the creation of chemical, thermal and thermomechanical effects below the optical breakdown threshold,�?? Proc. SPIE 4633A, 1-15 (2002).

Scanning (1)

C. -K. Sun, S. W. Chu, S. P. Tai, S. Keller, A. Abare, U. K. Mishira, and S. P. DenBaars, �??Mapping Piezoelectric-Field Distribution in Gallium Nitride with Scanning Second-Harmonic Generation Microscopy,�?? Scanning 23, 182 (2001).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, �??Two photon laser scanning fluorescence microscopy,�?? Science 248, 73 (1990).
[CrossRef] [PubMed]

Other (3)

R. Shack, B. Bell, D. Hillman, R. Kingston, A. Landesman, R. Shoemaker, D. Vukobratovich, and P.H. Bartels, �??Ultrafast laser scanner microscope-first performance test (biological application),�?? IEEE International Workshop on Physics and Engineering in Medical Imaging. viii + 292, 49-57 (1982).

Private communication with OLYMPUS OPTICAL CO., LTD.

Gavin D. Reid and Klaas Wynne, �??Ultrafast laser technology and spectroscopy,�?? in Encyclopedia of Analytical Chemistry, R.A. Meyers ed. (John Wiley & Sons Ltd, Chichester, UK. 2000)

Supplementary Material (3)

» Media 1: MOV (688 KB)     
» Media 2: MOV (1154 KB)     
» Media 3: MOV (782 KB)     

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 (4)

Fig. 1.
Fig. 1.

Nonlinear emission spectra showing the SHG intensity difference from mouse muscle fibers excited by (A) the 82-MHz and (B) the 2-GHz Ti:sapphire lasers.

Fig. 2.
Fig. 2.

(705 KB) Real-time (15 fps) SHG microscopic images in live zebrafish muscular tissue with the 2-GHz, 150mW Ti:sapphire laser.

Fig. 3.
Fig. 3.

(1182 KB, 800 KB) Time series of SHG in vivo microscopic images in live fish muscle tissue with the 82-MHz, 115mW Ti:sapphire laser showing the dramatic optical damage induced by the excessively strong peak intensity. Scale bar: 10 µm.

Fig. 4.
Fig. 4.

The SHG microscopic image in fish muscle tissue with the 82-MHz, 8mW Ti:sapphire laser. With the same parameter settings as those in Fig. 2, only noise can be seen. Scale bar: 10 µm.

Metrics