Abstract

Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.

© 2004 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |

  1. B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Essential Cell Biology (Taylor and Francis, New York, 1997).
  2. R. L. Amy and R. Storb, �??Selective mitochondrial damage by a ruby laser microbeam: an electron microscope study,�?? Science 150, 756-757 (1965).
    [CrossRef] [PubMed]
  3. M. W. Berns, J. Aist, J. Edwards, K. Strahs, J. Girton, P. McNeil, J. B. Rattner, M. Kitzes, M. Hammerwilson, L. H. Liaw, A. Siemens, M. Koonce, S. Peterson, S. Brenner, J. Burt, R. Walter, P. J. Bryant, D. Vandyk, J. Couclombe, T. Cahill, and G. S. Bern, �??Laser microsurgery in cell and developmental biology,�?? Science 213, 505-513 (1981).
    [CrossRef] [PubMed]
  4. M. W. Berns, W. H. Write, and R. W. Steubing, �??Laser microbeam as a tool in cell biology,�?? Int. Rev. Cytol. 129, 1-44 (1991).
    [CrossRef] [PubMed]
  5. K. O. Greulich, Micromanipulation by light in biology and medicine, (Birkhähser-Verlag, Basel, Switzerland, 1999).
  6. V. Venygopalan, A. Guerra III, K. Hahen, and A. Vogel, �??Role of laser-induced plasma formation in pulse cellular microsurgery and micromaniplation,�?? Phys. Rev. Lett. 88, 078103 (2002).
    [CrossRef]
  7. W. Denk, J. H. Strickler, and W. W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  8. J. M. Squirell, D. L. Wokosin, J. G. White, and B. D. Bavister, �??Long-term two-photon fluorescence imaging of mammalian embryos without compromising vitality,�?? Nat. Biotechnolo. 17, 763-767 (1999).
    [CrossRef]
  9. Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, �??Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,�?? J. Microsc. 185, 9-20 (1997).
    [CrossRef] [PubMed]
  10. K. König, P. T. C. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, �??Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes,�?? Opt. Lett. 22, 135-136 (1997).
    [CrossRef] [PubMed]
  11. K. König, T. W. Becker, P. Fischer, I. Riemann, and K. �??J. Halbhuber, �??Pulse-length dependence of cellularresponse to intense near-infrared laser pulses in multiphoton microscopes,�?? Opt. Lett. 24, 113-115 (1999).
    [CrossRef]
  12. H. J. Kester, D. Baur, R. Uhl, and S. W. Hell, �??Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,�?? Biophys. J. 77, 2226-2236 (1999).
    [CrossRef]
  13. K. König, �??Laser tweezers and multiphoton microscopes in life sciences,�?? Histochem. Cell Biol. 114, 79-92 (2000).
    [PubMed]
  14. H. Oehring, I. Riedmann, P. Fisher, K. -J. Halbhuber, and K. König, �??Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses,�?? Scanning 22, 263-270 (2000).
    [CrossRef] [PubMed]
  15. U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. Halbhuber, �??Femtosecond near-infrared laser pulse elicit generation of reactive oxygen species in mammalian cells leading to apotosis-like death,�?? Exp. Cell Res. 263, 88-97 (2001).
    [CrossRef] [PubMed]
  16. K. König, W. Riemann, and W. Fritzsche, �??Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,�?? Opt. Lett. 26, 819-821 (2001).
    [CrossRef]
  17. U. K. Tirlapur and K. König, �??Targeted transfection by femtosecond laser,�?? Nature 418, 290-291 (2002).
    [CrossRef] [PubMed]
  18. U. K. Tirlapur and K. König �??Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability,�?? The Plant J. 31, 365-374 (2002).
    [CrossRef]
  19. N. Shen, C. B. Schaffer, D. Datta, and E. Mazur, �??Photodisruption in biological tissues and single cells using femtosecond laser pulses,�?? Conference on Lasers and Electro-Optics (Baltimore, MD, 2001) 403-404.
  20. N. Shen, M. Colvin, F. Genin, T. Huser, G. A. Cortopassi, T. Stearns, P. LeDuc, D. E. Ingber, and E. Mazur, �??Using femtosecond laser subcellular surgery to study cell biology,�?? Biophys. J. 86, 520A-520A, Part 2 Suppl. S (2004).
  21. T. Higashi, E. Nagamori, T. Sone, S. Matsunaga, and K. Fukui, �??A novel transfection method for mammalian cells using calcium alginate microbeads,�?? J. Biosci. Bioeng. 97, 191-195 (2004).
  22. N. Benvenisty and L. Reshef, �??Direct introduction of genes into rats and expression of the genes,�?? Proc. Natl. Acad. Sci. USA 83, 9551-9555 (1986).
    [CrossRef] [PubMed]
  23. B. C. Stuart, M. D. Feit, S. Herman, A.M. Rubenchik, B. W. Shore, and M. D. Perry, �??Optical ablation by high-power short-pulse lasers,�?? J. Opt. Soc. Am. B 13, 459-467 (1996).
    [CrossRef]
  24. M. Lenzner, J. Krüger, S. Sartania, Z. Cheng, Ch. Spielmann, G. Mourou, W. Kautek, and F. Krausz, �??Femtosecond optical breakdown in dielectrics,�?? Phys. Rev. Lett. 80, 4076-4079 (1998).
    [CrossRef]
  25. A. P. Joglekar, H. �??H. Liu, E. Meyhöfer, G. Mourou, and A. J. Hunt, Poc. Natl. Acad. Sci. USA 101, 5856-5861 (2004).
    [CrossRef]
  26. P. A. Barnes and K. E. Rieckoff, �??Laser induced underwater sparks,�?? Appl. Phys. Lett. 13, 282-284 (1968).
    [CrossRef]
  27. B. Zysset, J. G. Fujimoto and T. F. Deutsch, �??Time-resolved measurement of picosecond optical-breakdown,�?? Appl. Phys. B 48, 139- 147 (1989).
    [CrossRef]
  28. E. N. Glezer, C. B. Schaffer, N. Nishimura and E. Mazur, �??Minimally disruptive laser-induced breakdown in water,�?? Opt. Lett. 22, 1817-1879 (1997).
    [CrossRef]
  29. C. B. Schaffer, N. Nishimura, E. N. Glezer, A. M.-T. Kim, and E. Mazur, �??Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds,�?? Opt. Express. 10, 196-203 (2002), <a href=http://www.opticsexpress.org/abstract.cfm?URI=OPEX-10-3-196">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-10-3-196</a>
    [CrossRef] [PubMed]
  30. E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, �??Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,�?? Biophys. J. 77, 2837-2849 (1999).
    [CrossRef] [PubMed]
  31. J. White and E. H. Stelzer, �??Photobleaching GFP reveals protein dynamics inside live cells,�?? Trends Cell Biol. 9, 61-65 (1999).
    [CrossRef] [PubMed]

Appl. Phys. B

B. Zysset, J. G. Fujimoto and T. F. Deutsch, �??Time-resolved measurement of picosecond optical-breakdown,�?? Appl. Phys. B 48, 139- 147 (1989).
[CrossRef]

Appl. Phys. Lett.

P. A. Barnes and K. E. Rieckoff, �??Laser induced underwater sparks,�?? Appl. Phys. Lett. 13, 282-284 (1968).
[CrossRef]

Biophys. J.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, �??Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery,�?? Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

H. J. Kester, D. Baur, R. Uhl, and S. W. Hell, �??Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,�?? Biophys. J. 77, 2226-2236 (1999).
[CrossRef]

N. Shen, M. Colvin, F. Genin, T. Huser, G. A. Cortopassi, T. Stearns, P. LeDuc, D. E. Ingber, and E. Mazur, �??Using femtosecond laser subcellular surgery to study cell biology,�?? Biophys. J. 86, 520A-520A, Part 2 Suppl. S (2004).

CLEO 2001

N. Shen, C. B. Schaffer, D. Datta, and E. Mazur, �??Photodisruption in biological tissues and single cells using femtosecond laser pulses,�?? Conference on Lasers and Electro-Optics (Baltimore, MD, 2001) 403-404.

Exp. Cell Res.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. Halbhuber, �??Femtosecond near-infrared laser pulse elicit generation of reactive oxygen species in mammalian cells leading to apotosis-like death,�?? Exp. Cell Res. 263, 88-97 (2001).
[CrossRef] [PubMed]

Histochem. Cell Biol.

K. König, �??Laser tweezers and multiphoton microscopes in life sciences,�?? Histochem. Cell Biol. 114, 79-92 (2000).
[PubMed]

Int. Rev. Cytol.

M. W. Berns, W. H. Write, and R. W. Steubing, �??Laser microbeam as a tool in cell biology,�?? Int. Rev. Cytol. 129, 1-44 (1991).
[CrossRef] [PubMed]

J. Biosci. Bioeng.

T. Higashi, E. Nagamori, T. Sone, S. Matsunaga, and K. Fukui, �??A novel transfection method for mammalian cells using calcium alginate microbeads,�?? J. Biosci. Bioeng. 97, 191-195 (2004).

J. Microsc.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, �??Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,�?? J. Microsc. 185, 9-20 (1997).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

Nat. Biotechnolo.

J. M. Squirell, D. L. Wokosin, J. G. White, and B. D. Bavister, �??Long-term two-photon fluorescence imaging of mammalian embryos without compromising vitality,�?? Nat. Biotechnolo. 17, 763-767 (1999).
[CrossRef]

Nature

U. K. Tirlapur and K. König, �??Targeted transfection by femtosecond laser,�?? Nature 418, 290-291 (2002).
[CrossRef] [PubMed]

Opt. Express.

C. B. Schaffer, N. Nishimura, E. N. Glezer, A. M.-T. Kim, and E. Mazur, �??Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds,�?? Opt. Express. 10, 196-203 (2002), <a href=http://www.opticsexpress.org/abstract.cfm?URI=OPEX-10-3-196">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-10-3-196</a>
[CrossRef] [PubMed]

Opt. Lett.

Phys. Rev. Lett.

M. Lenzner, J. Krüger, S. Sartania, Z. Cheng, Ch. Spielmann, G. Mourou, W. Kautek, and F. Krausz, �??Femtosecond optical breakdown in dielectrics,�?? Phys. Rev. Lett. 80, 4076-4079 (1998).
[CrossRef]

V. Venygopalan, A. Guerra III, K. Hahen, and A. Vogel, �??Role of laser-induced plasma formation in pulse cellular microsurgery and micromaniplation,�?? Phys. Rev. Lett. 88, 078103 (2002).
[CrossRef]

Poc. Natl. Acad. Sci. USA

A. P. Joglekar, H. �??H. Liu, E. Meyhöfer, G. Mourou, and A. J. Hunt, Poc. Natl. Acad. Sci. USA 101, 5856-5861 (2004).
[CrossRef]

Proc. Natl. Acad. Sci.

N. Benvenisty and L. Reshef, �??Direct introduction of genes into rats and expression of the genes,�?? Proc. Natl. Acad. Sci. USA 83, 9551-9555 (1986).
[CrossRef] [PubMed]

Scanning

H. Oehring, I. Riedmann, P. Fisher, K. -J. Halbhuber, and K. König, �??Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses,�?? Scanning 22, 263-270 (2000).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 248, 73-76 (1990).
[CrossRef] [PubMed]

R. L. Amy and R. Storb, �??Selective mitochondrial damage by a ruby laser microbeam: an electron microscope study,�?? Science 150, 756-757 (1965).
[CrossRef] [PubMed]

M. W. Berns, J. Aist, J. Edwards, K. Strahs, J. Girton, P. McNeil, J. B. Rattner, M. Kitzes, M. Hammerwilson, L. H. Liaw, A. Siemens, M. Koonce, S. Peterson, S. Brenner, J. Burt, R. Walter, P. J. Bryant, D. Vandyk, J. Couclombe, T. Cahill, and G. S. Bern, �??Laser microsurgery in cell and developmental biology,�?? Science 213, 505-513 (1981).
[CrossRef] [PubMed]

The Plant J.

U. K. Tirlapur and K. König �??Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability,�?? The Plant J. 31, 365-374 (2002).
[CrossRef]

Trends Cell Biol.

J. White and E. H. Stelzer, �??Photobleaching GFP reveals protein dynamics inside live cells,�?? Trends Cell Biol. 9, 61-65 (1999).
[CrossRef] [PubMed]

Other

K. O. Greulich, Micromanipulation by light in biology and medicine, (Birkhähser-Verlag, Basel, Switzerland, 1999).

B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Essential Cell Biology (Taylor and Francis, New York, 1997).

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 (5)

Fig. 1.
Fig. 1.

Schematic of the experimental setup. OB, DM, BP, and PMT denote objective lens, dichroic mirror, bandpass filter, and photomulutiplier tube, respectively.

Fig. 2.
Fig. 2.

Confocal images of the cells (a) before and (b) after laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. The white circles and arrows indicate individual HeLa cells and target mitochondria, respectively. Additional red fluorescence in Fig. 2b is derived from propidium iodide (PI). The laser pulses were focused inside cells α and β at energies of 7 nJ/pulse and 3 nJ /pulse, respectively.

Fig. 3
Fig. 3

Confocal images of HeLa cell before and after fs-laser irradiation. (a) and (b) confocal images of the HeLa cell before and after femtosecond laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. A target mitochondrion is indicated by a white arrow. (c) and (d) magnified views of square areas indicated in (a) and (b), respectively. The center of the dotted circles show target mitochondria.

Fig. 4.
Fig. 4.

(a) and (b) stacked three-dimensional confocal images at different depths before and after femtosecond laser irradiation. Red fluorescence shows mitochondria of HeLa cells stained with MitoTracker Red. A target mitochondrion is indicated by a white arrow. Twenty-one images were obtained by translation of the objective lens by 6 μm in steps of 300 nm. (c) and (d) magnified views of square areas indicated in (a) and (b), respectively. The center of the dotted circles show target mitochondria.

Fig. 5.
Fig. 5.

(a) Confocal images of laser-irradiated cells before and after restaining. Yellow fluorescence shows mitochondria visualized by EYFP. Target mitochondrion is indicated by a white arrow. (c) magnified view of square area indicated in (a) before femtosecond laser irradiation. (b) confocal image obtained after femtosecond laser irradiation. (d) magnified view of square area in (b). (e) and (f) confocal images obtained after restaining by MitoTracker Red. (e) confocal image obtained by excitation with the Ar+ laser. (f) confocal image obtained by excitation with the He-Ne laser. Dotted circles show target mitochondria.

Metrics