Abstract

In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.

© 2011 OSA

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
    [CrossRef] [PubMed]
  3. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  4. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt. 15(12), 3135–3139 (1976).
    [CrossRef] [PubMed]
  5. L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
    [CrossRef] [PubMed]
  6. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
    [CrossRef] [PubMed]
  7. C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
    [CrossRef]
  8. C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
    [CrossRef] [PubMed]
  9. V. Kasche and L. Lindqvist, “Reactions between the triplet state of fluorescein and oxygen,” J. Chem. Phys. 68(4), 817–823 (1964).
    [CrossRef]
  10. G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
    [CrossRef] [PubMed]
  11. T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
    [CrossRef] [PubMed]
  12. T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
    [CrossRef]
  13. A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
    [CrossRef]
  14. P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
    [CrossRef]
  15. K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
    [PubMed]
  16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
    [CrossRef]
  17. P. A. Quinto-Su and V. Venugopalan, “Mechanisms of laser cellular microsurgery,” Methods Cell Biol. 82, 113–151 (2007).
    [PubMed]
  18. B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
    [CrossRef] [PubMed]
  19. L. Sanche, “Low energy electron-driven damage in biomolecules,” Eur. Phys. J. D 35(2), 367–390 (2005).
    [CrossRef]
  20. U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
    [CrossRef] [PubMed]
  21. B. P. Yu, “Cellular defenses against damage from reactive oxygen species,” Physiol. Rev. 74(1), 139–162 (1994).
    [PubMed]
  22. K. Kuetemeyer, R. Rezgui, H. Lubatschowski, and A. Heisterkamp, “Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery,” Biomed. Opt. Express 1(2), 587–597 (2010).
    [CrossRef] [PubMed]
  23. A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
    [CrossRef] [PubMed]
  24. I. Gryczynski and J. R. Lakowicz, “Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation,” J. Fluoresc. 4(4), 331–336 (1994).
    [CrossRef]
  25. A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
    [CrossRef]
  26. M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
    [CrossRef]
  27. E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
    [CrossRef] [PubMed]
  28. M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
    [CrossRef] [PubMed]
  29. E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
    [CrossRef]
  30. R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
    [CrossRef] [PubMed]
  31. F. Bourgeois and A. Ben-Yakar, “Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans,” Opt. Express 15(14), 8521–8531 (2007).
    [CrossRef] [PubMed]
  32. K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
    [CrossRef]
  33. H. Görner, “Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA,” Photochem. Photobiol. 73(4), 339–348 (2001).
    [CrossRef] [PubMed]
  34. E. Olmo, “Nucleotype and cell size in vertebrates: a review,” Basic Appl. Histochem. 27(4), 227–256 (1983).
    [PubMed]
  35. F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
    [CrossRef] [PubMed]
  36. I. D. Johnson, “Practical considerations in the selection and application of fluorescent probes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer, 2006).

2010 (2)

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

K. Kuetemeyer, R. Rezgui, H. Lubatschowski, and A. Heisterkamp, “Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery,” Biomed. Opt. Express 1(2), 587–597 (2010).
[CrossRef] [PubMed]

2009 (1)

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

2007 (3)

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

F. Bourgeois and A. Ben-Yakar, “Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans,” Opt. Express 15(14), 8521–8531 (2007).
[CrossRef] [PubMed]

P. A. Quinto-Su and V. Venugopalan, “Mechanisms of laser cellular microsurgery,” Methods Cell Biol. 82, 113–151 (2007).
[PubMed]

2005 (3)

L. Sanche, “Low energy electron-driven damage in biomolecules,” Eur. Phys. J. D 35(2), 367–390 (2005).
[CrossRef]

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[CrossRef] [PubMed]

2004 (1)

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

2003 (1)

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

2002 (1)

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

2001 (4)

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
[CrossRef]

H. Görner, “Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA,” Photochem. Photobiol. 73(4), 339–348 (2001).
[CrossRef] [PubMed]

2000 (2)

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

1999 (1)

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

1998 (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

1997 (1)

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
[CrossRef] [PubMed]

1996 (3)

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
[CrossRef]

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

1995 (1)

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

1994 (4)

B. P. Yu, “Cellular defenses against damage from reactive oxygen species,” Physiol. Rev. 74(1), 139–162 (1994).
[PubMed]

I. Gryczynski and J. R. Lakowicz, “Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation,” J. Fluoresc. 4(4), 331–336 (1994).
[CrossRef]

K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
[CrossRef]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

1990 (2)

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1983 (1)

E. Olmo, “Nucleotype and cell size in vertebrates: a review,” Basic Appl. Histochem. 27(4), 227–256 (1983).
[PubMed]

1979 (1)

E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
[CrossRef]

1976 (1)

1964 (1)

V. Kasche and L. Lindqvist, “Reactions between the triplet state of fluorescein and oxygen,” J. Chem. Phys. 68(4), 817–823 (1964).
[CrossRef]

Amouyal, E.

E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
[CrossRef]

Baumgart, J.

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Bell, A. F.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Ben-Yakar, A.

Bernas, A.

E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
[CrossRef]

Bisby, R. H.

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

Botchway, S. W.

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

Boudaïffa, B.

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

Bourgeois, F.

Chen, T.-S.

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Clegg, R. M.

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Cloutier, P.

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

Crisostomo, A. G.

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

Cubitt, A. B.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Denk, W.

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dittrich, P. S.

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

Dumortier, L.

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Eggeling, C.

C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[CrossRef] [PubMed]

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

Epifanovsky, E.

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

Fischer, P.

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

Görner, H.

H. Görner, “Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA,” Photochem. Photobiol. 73(4), 339–348 (2001).
[CrossRef] [PubMed]

Grand, D.

E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
[CrossRef]

Grigorenko, B.

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

Gross, L. A.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Gryczynski, I.

I. Gryczynski and J. R. Lakowicz, “Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation,” J. Fluoresc. 4(4), 331–336 (1994).
[CrossRef]

Halbhuber, K. J.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

He, X.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Heikal, A. A.

A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
[CrossRef]

Heisterkamp, A.

K. Kuetemeyer, R. Rezgui, H. Lubatschowski, and A. Heisterkamp, “Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery,” Biomed. Opt. Express 1(2), 587–597 (2010).
[CrossRef] [PubMed]

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Hell, S. W.

Hennink, E. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

Hess, S. T.

A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
[CrossRef]

Hirschfeld, T.

Huels, M. A.

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

Hunting, D.

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

Hüttman, G.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

Kallio, K.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Kalninsh, K. K.

K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
[CrossRef]

Kasche, V.

V. Kasche and L. Lindqvist, “Reactions between the triplet state of fluorescein and oxygen,” J. Chem. Phys. 68(4), 817–823 (1964).
[CrossRef]

König, K.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

Krieg, R.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

Krylov, A. I.

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

Kuetemeyer, K.

Lakowicz, J. R.

I. Gryczynski and J. R. Lakowicz, “Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation,” J. Fluoresc. 4(4), 331–336 (1994).
[CrossRef]

Larsen, D. S.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Laubereau, A.

A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
[CrossRef]

Lindqvist, L.

V. Kasche and L. Lindqvist, “Reactions between the triplet state of fluorescein and oxygen,” J. Chem. Phys. 68(4), 817–823 (1964).
[CrossRef]

Loontiens, F. G.

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Lubatschowski, H.

K. Kuetemeyer, R. Rezgui, H. Lubatschowski, and A. Heisterkamp, “Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery,” Biomed. Opt. Express 1(2), 587–597 (2010).
[CrossRef] [PubMed]

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Luo, Q.-M.

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Maxwell, I. Z.

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Mazur, E.

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Nemukhin, A.

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

Ngezahayo, A.

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

Nikogosyan, D. N.

A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
[CrossRef]

Noack, J.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

Olmo, E.

E. Olmo, “Nucleotype and cell size in vertebrates: a review,” Basic Appl. Histochem. 27(4), 227–256 (1983).
[PubMed]

Ormö, M.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Paltauf, G.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

Parker, A. W.

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

Patterson, G. H.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Pestov, D. V.

K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
[CrossRef]

Peuckert, C.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

Piston, D. W.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Polyakov, I.

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

Quinto-Su, P. A.

P. A. Quinto-Su and V. Venugopalan, “Mechanisms of laser cellular microsurgery,” Methods Cell Biol. 82, 113–151 (2007).
[PubMed]

Regenfuss, P.

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Remington, S. J.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Reuther, A.

A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
[CrossRef]

Rezgui, R.

Riemann, I.

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

Rigler, R.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

Roshchina, Y. K.

K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
[CrossRef]

Sanche, L.

L. Sanche, “Low energy electron-driven damage in biomolecules,” Eur. Phys. J. D 35(2), 367–390 (2005).
[CrossRef]

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

Schwille, P.

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

Seidel, C. A. M.

C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[CrossRef] [PubMed]

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

Song, L.

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
[CrossRef] [PubMed]

Tanke, H. J.

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

Tirlapur, U. K.

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

Tonge, P. J.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Tsien, R. Y.

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

van Grondelle, R.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

van Stokkum, I. H.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Varma, C. A.

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

Vengris, M.

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Venugopalan, V.

P. A. Quinto-Su and V. Venugopalan, “Mechanisms of laser cellular microsurgery,” Methods Cell Biol. 82, 113–151 (2007).
[PubMed]

Verhoeven, J. W.

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

Vogel, A.

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

Volkmer, A.

C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[CrossRef] [PubMed]

Webb, W. W.

A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wichmann, J.

Widengren, J.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

Young, I. T.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

Yu, B. P.

B. P. Yu, “Cellular defenses against damage from reactive oxygen species,” Physiol. Rev. 74(1), 139–162 (1994).
[PubMed]

Zechel, A.

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Zeng, S.-Q.

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Zhang, Z.-H.

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Zhou, W.

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Anal. Chem. (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef]

Appl. Opt. (1)

Appl. Phys. B (2)

P. S. Dittrich and P. Schwille, “Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation,” Appl. Phys. B 73(8), 829–837 (2001).
[CrossRef]

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005).
[CrossRef]

Basic Appl. Histochem. (1)

E. Olmo, “Nucleotype and cell size in vertebrates: a review,” Basic Appl. Histochem. 27(4), 227–256 (1983).
[PubMed]

Biochem. Biophys. Res. Commun. (1)

T.-S. Chen, S.-Q. Zeng, Q.-M. Luo, Z.-H. Zhang, and W. Zhou, “High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy,” Biochem. Biophys. Res. Commun. 291(5), 1272–1275 (2002).
[CrossRef] [PubMed]

Biochemistry (1)

F. G. Loontiens, P. Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, “Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities,” Biochemistry 29(38), 9029–9039 (1990).
[CrossRef] [PubMed]

Biomed. Opt. Express (1)

Biophys. J. (3)

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[CrossRef] [PubMed]

L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[CrossRef] [PubMed]

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Cell. Mol. Biol. (Noisy-le-grand) (1)

K. König, I. Riemann, P. Fischer, and K. J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. (Noisy-le-grand) 45(2), 195–201 (1999).
[PubMed]

Chem. Phys. (1)

A. A. Heikal, S. T. Hess, and W. W. Webb, “Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity,” Chem. Phys. 274(1), 37–55 (2001).
[CrossRef]

ChemPhysChem (1)

C. Eggeling, A. Volkmer, and C. A. M. Seidel, “Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy,” ChemPhysChem 6(5), 791–804 (2005).
[CrossRef] [PubMed]

Chin. Phys. Lett. (1)

T.-S. Chen, S.-Q. Zeng, W. Zhou, and Q.-M. Luo, “A quantitative theory model of a photobleaching mechanism,” Chin. Phys. Lett. 20(11), 1940–1943 (2003).
[CrossRef]

Eur. Phys. J. D (1)

L. Sanche, “Low energy electron-driven damage in biomolecules,” Eur. Phys. J. D 35(2), 367–390 (2005).
[CrossRef]

Exp. Cell Res. (1)

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. J. Halbhuber, “Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,” Exp. Cell Res. 263(1), 88–97 (2001).
[CrossRef] [PubMed]

J. Chem. Phys. (3)

A. Reuther, D. N. Nikogosyan, and A. Laubereau, “Primary photochemical processes in thymine in concentrated aqueous solution studied by femtosecond UV spectroscopy,” J. Chem. Phys. 100(13), 5570–5577 (1996).
[CrossRef]

V. Kasche and L. Lindqvist, “Reactions between the triplet state of fluorescein and oxygen,” J. Chem. Phys. 68(4), 817–823 (1964).
[CrossRef]

E. Epifanovsky, I. Polyakov, B. Grigorenko, A. Nemukhin, and A. I. Krylov, “The effect of oxidation on the electronic structure of the green fluorescent protein chromophore,” J. Chem. Phys. 132(11), 115104 (2010).
[CrossRef] [PubMed]

J. Fluoresc. (1)

I. Gryczynski and J. R. Lakowicz, “Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation,” J. Fluoresc. 4(4), 331–336 (1994).
[CrossRef]

J. Photochem. Photobiol. Chem. (1)

K. K. Kalninsh, D. V. Pestov, and Y. K. Roshchina, “Absorption and fluorescence spectra of the probe Hoechst 33258,” J. Photochem. Photobiol. Chem. 83(1), 39–47 (1994).
[CrossRef]

J. Phys. Chem. A (1)

M. Vengris, I. H. van Stokkum, X. He, A. F. Bell, P. J. Tonge, R. van Grondelle, and D. S. Larsen, “Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution,” J. Phys. Chem. A 108(21), 4587–4598 (2004).
[CrossRef]

Methods Cell Biol. (2)

A. Heisterkamp, J. Baumgart, I. Z. Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski, “Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics,” Methods Cell Biol. 82, 293–307 (2007).
[CrossRef] [PubMed]

P. A. Quinto-Su and V. Venugopalan, “Mechanisms of laser cellular microsurgery,” Methods Cell Biol. 82, 113–151 (2007).
[PubMed]

Neuron (1)

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18(3), 351–357 (1997).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Photochem. Photobiol. (3)

H. Görner, “Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA,” Photochem. Photobiol. 73(4), 339–348 (2001).
[CrossRef] [PubMed]

E. Amouyal, A. Bernas, and D. Grand, “On the photoionization energy threshold of tryptophan in aqueous solutions,” Photochem. Photobiol. 29(6), 1071–1077 (1979).
[CrossRef]

R. H. Bisby, A. G. Crisostomo, S. W. Botchway, and A. W. Parker, “Nanoscale hydroxyl radical generation from multiphoton ionization of tryptophan,” Photochem. Photobiol. 85(1), 353–357 (2009).
[CrossRef] [PubMed]

Physiol. Rev. (1)

B. P. Yu, “Cellular defenses against damage from reactive oxygen species,” Physiol. Rev. 74(1), 139–162 (1994).
[PubMed]

Science (3)

B. Boudaïffa, P. Cloutier, D. Hunting, M. A. Huels, and L. Sanche, “Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons,” Science 287(5458), 1658–1660 (2000).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

M. Ormö, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington, “Crystal structure of the Aequorea victoria green fluorescent protein,” Science 273(5280), 1392–1395 (1996).
[CrossRef] [PubMed]

Other (1)

I. D. Johnson, “Practical considerations in the selection and application of fluorescent probes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer, 2006).

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Figures (7)

Fig. 1.
Fig. 1.

Schematic set-up for multiphoton imaging and logging of photobleaching kinetics.

Fig. 2.
Fig. 2.

Wavelength-dependence of the multiphoton-order for Hoechst 33342 and eGFP. Each data point represents the mean ± standard deviation of at least five experiments. The multiphoton-order was about two over the whole wavelength range for eGFP, while it increased from two up to three for Hoechst.

Fig. 3.
Fig. 3.

Wavelength-dependence of the photobleaching-order for eGFP at different laser-parameters. The dotted line illustrates its behaviour. Each data point represents the mean ± standard deviation of at least five experiments. While the photobleaching-order was independent of repetition rate and NA, there was a step increase of one at about 840 nm.

Fig. 4.
Fig. 4.

Wavelength-dependence of the photobleaching-order for Hoechst at different repetition rates. The dotted line illustrates its behaviour. Each data point represents the mean ± standard deviation of at least five experiments. The bleaching-order varied slightly around two from 720 up to 900 nm and increased to three at 950 nm.

Fig. 5.
Fig. 5.

Correlation of ROS formation and high-order photobleaching. (a) Multiphoton images from different points of time during photobleaching. ROS concentration increased during the drop in Hoechst fluorescence. Scale bar: 8 µm. (b) Time-dependence of ROS formation in comparison to Hoechst photobleaching for three half-life periods. Each data point represents the mean ± standard deviation of at least five cells. The half-life period of photobleaching and the half-saturation value of ROS formation were approximately at the same time (yellow box).

Fig. 6.
Fig. 6.

Influence of eGFP photobleaching on the degradation of Hoechst molecules in its environment. (a) Multiphoton images of Hoechst and eGFP before and after photobleaching of eGFP. Scale bar: 16 µm. (b) Loss of relative Hoechst fluorescence intensity referred to an absolute bleaching of eGFP for different half-life periods and both repetition rates. Each data point represents the mean ± standard error of at least eight experiments. The loss of Hoechst fluorescence intensity referred to an absolute bleaching of eGFP is within the range of 50–80%.

Fig. 7.
Fig. 7.

Schematic of high-order photobleaching and ablation of Hoechst molecules. Violet arrows correspond to wavelengths from 720 up to 920 nm, yellow arrows to 920 nm and above. In photobleaching two or three photons evoke the excitation of Hoechst and another two photons are sequentially absorbed in saturated one photon transitions, while in ablation multiphoton-ionization occurs by the quasi-simultaneous absorption of four or five photons.

Equations (1)

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r LDP r H 3 · C · R loss · 3 4 π 3

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