Abstract

Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2–3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×106 while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage.

© 2016 Optical Society of America

Full Article  |  PDF Article
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References

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    [Crossref] [PubMed]

2014 (4)

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

A. P. Wojtovich and T. H. Foster, “Optogenetic control of ros production,” Redox Biology 2, 368–376 (2014).
[Crossref] [PubMed]

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

2013 (1)

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

2012 (1)

2011 (2)

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Bio. Opt. Express 2, 805–816 (2011).
[Crossref]

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

2010 (1)

M. L. Circu and T. Y. Aw, “Reactive oxygen species, cellular redox systems, and apoptosis,” Free Radical Biol. Med. 48, 749–762 (2010).
[Crossref]

2009 (1)

H.-W. Wang, Y.-H. Wei, and H.-W. Guo, “Reduced nicotinamide adenine dinucleotide (nadh) fluorescence for the detection of cell death,” Anti-Cancer Agents in Med. Chem. 91012 (2009).
[Crossref]

2008 (1)

P. Ball, “Water as an active constituent in cell biology,” Chem. Rev. 108, 74–108 (2008).
[Crossref]

2007 (1)

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

2006 (1)

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

2005 (2)

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38, 97–166 (2005).
[Crossref]

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

2001 (3)

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, 88–97 (2001).
[Crossref] [PubMed]

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, 829–837 (2001).
[Crossref]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[Crossref] [PubMed]

2000 (1)

J. R. Lakowicz, “On spectral relaxation in proteins,” Photochem. Photobiol. 72, 421–437 (2000).
[Crossref] [PubMed]

1999 (3)

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

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]

1997 (1)

1996 (1)

1995 (2)

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, “Cell damage by near-IR microbeams,” Nature 377, 20–21 (1995).
[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, 2588–2600 (1995).
[Crossref] [PubMed]

1984 (1)

Aldwayyan, a. S.

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

AlObiadi, a. a.

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

AlSalhi, M. S.

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

Andersen, P. E.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Andresen, E. F.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Atif, M.

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

Aw, T. Y.

M. L. Circu and T. Y. Aw, “Reactive oxygen species, cellular redox systems, and apoptosis,” Free Radical Biol. Med. 48, 749–762 (2010).
[Crossref]

Ball, P.

P. Ball, “Water as an active constituent in cell biology,” Chem. Rev. 108, 74–108 (2008).
[Crossref]

Becker, T. W.

Bergman, K.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

Berns, M. W.

Block, S. M.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

Boyd, R. W.

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

Chadd, E. H.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

Chang, C.-Y.

Chang, N.-S.

Chen, S.-J.

Cheng, L.-C.

Chirico, G.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38, 97–166 (2005).
[Crossref]

Cho, K.-C.

Circu, M. L.

M. L. Circu and T. Y. Aw, “Reactive oxygen species, cellular redox systems, and apoptosis,” Free Radical Biol. Med. 48, 749–762 (2010).
[Crossref]

Collini, M.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38, 97–166 (2005).
[Crossref]

Delannoy, L.

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Diaspro, A.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38, 97–166 (2005).
[Crossref]

Didier, M. E. P.

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[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, 829–837 (2001).
[Crossref]

Dong, C. Y.

Donitzky, C.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

Dutto, F.

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Fischer, P.

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]

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

Flyvbjerg, H.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Foster, T. H.

A. P. Wojtovich and T. H. Foster, “Optogenetic control of ros production,” Redox Biology 2, 368–376 (2014).
[Crossref] [PubMed]

Galli, R.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Geiger, K. D.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Goehukasyan, V. V.

V. V. Goehukasyan and A. A. Haikal, Natural Biomarkers for Cellular Matabolism (CRC Press, 2014).

Gratton, E.

Grigiene, J.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Guo, H.-W.

H.-W. Wang, Y.-H. Wei, and H.-W. Guo, “Reduced nicotinamide adenine dinucleotide (nadh) fluorescence for the detection of cell death,” Anti-Cancer Agents in Med. Chem. 91012 (2009).
[Crossref]

Hagedorn, P. H.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Haikal, A. A.

V. V. Goehukasyan and A. A. Haikal, Natural Biomarkers for Cellular Matabolism (CRC Press, 2014).

Halbhuber, K.

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

Halbhuber, K. J.

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, 88–97 (2001).
[Crossref] [PubMed]

Heisterkamp, A.

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Bio. Opt. Express 2, 805–816 (2011).
[Crossref]

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, 2588–2600 (1995).
[Crossref] [PubMed]

Hoover, E. E.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Hopt, A.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[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, 1015–1047 (2005).
[Crossref]

Jourdain, P.

Kalies, S.

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Bio. Opt. Express 2, 805–816 (2011).
[Crossref]

Kasimova, M. R.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Kirsch, M.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Knig, K.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

Koch, E.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

König, K.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[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, 88–97 (2001).
[Crossref] [PubMed]

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]

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

K. König, P. T. So, W. W. Mantulin, and E. Gratton, “Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes,” Opt. Lett. 22, 135–136 (1997).
[Crossref] [PubMed]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, “Cell damage in near-infrared multimode optical traps as a result of multiphoton absorption,” Opt. Lett. 21, 1090 (1996).
[Crossref] [PubMed]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, “Cell damage by near-IR microbeams,” Nature 377, 20–21 (1995).
[Crossref] [PubMed]

Krab, K.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [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, 88–97 (2001).
[Crossref] [PubMed]

Kuetemeyer, K.

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Bio. Opt. Express 2, 805–816 (2011).
[Crossref]

Lakowicz, J. R.

J. R. Lakowicz, “On spectral relaxation in proteins,” Photochem. Photobiol. 72, 421–437 (2000).
[Crossref] [PubMed]

Le Harzic, R.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

Liang, H.

Lin, C.-Y.

Liou, G. F.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

Macias-Romero, C.

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Magistretti, P.

Mantulin, W. W.

Marquet, P.

Masters, B. R.

B. R. Masters and S. Peter, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008).

Moller, I. M.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Neher, E.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[Crossref] [PubMed]

Neuman, K. C.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

Noack, J.

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

Paltauf, G.

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

Pawley, J.

J. Pawley, Handbook of Biological Confocal Microscopy (Springer Science, 2006).
[Crossref]

Peter, S.

B. R. Masters and S. Peter, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008).

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, 88–97 (2001).
[Crossref] [PubMed]

Radenovic, A.

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

Riemann, I.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

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]

Roke, S.

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Sanders, D. J.

Schackert, G.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[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, 829–837 (2001).
[Crossref]

So, P. T.

Song, L.

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

Squier, J. A.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Steiner, G.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Tanke, H. J.

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

Tarun, O. B.

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, 88–97 (2001).
[Crossref] [PubMed]

Tromberg, B. J.

Uckermann, O.

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[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, 1015–1047 (2005).
[Crossref]

Wang, H.-W.

H.-W. Wang, Y.-H. Wei, and H.-W. Guo, “Reduced nicotinamide adenine dinucleotide (nadh) fluorescence for the detection of cell death,” Anti-Cancer Agents in Med. Chem. 91012 (2009).
[Crossref]

Wei, Y.-H.

H.-W. Wang, Y.-H. Wei, and H.-W. Guo, “Reduced nicotinamide adenine dinucleotide (nadh) fluorescence for the detection of cell death,” Anti-Cancer Agents in Med. Chem. 91012 (2009).
[Crossref]

Wllner, C.

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

Wojtovich, A. P.

A. P. Wojtovich and T. H. Foster, “Optogenetic control of ros production,” Redox Biology 2, 368–376 (2014).
[Crossref] [PubMed]

Xu, C.

Yen, W.-C.

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, 2588–2600 (1995).
[Crossref] [PubMed]

Zubkovs, V.

C. Macias-Romero, M. E. P. Didier, P. Jourdain, P. Marquet, P. Magistretti, O. B. Tarun, V. Zubkovs, A. Radenovic, and S. Roke, “High throughput second harmonic imaging for label-free biological applications,” Opt. Express 22, 31102–31112 (2014).
[Crossref]

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Anti-Cancer Agents in Med. Chem. (1)

H.-W. Wang, Y.-H. Wei, and H.-W. Guo, “Reduced nicotinamide adenine dinucleotide (nadh) fluorescence for the detection of cell death,” Anti-Cancer Agents in Med. Chem. 91012 (2009).
[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, 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, 1015–1047 (2005).
[Crossref]

Bio. Opt. Express (1)

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, “Mechanisms of high-order photobleaching and its relationship to intracellular ablation,” Bio. Opt. Express 2, 805–816 (2011).
[Crossref]

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, 2588–2600 (1995).
[Crossref] [PubMed]

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, “Characterization of photodamage to escherichia coli in optical traps,” Biophys. J. 77, 2856–2863 (1999).
[Crossref] [PubMed]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
[Crossref] [PubMed]

Cell. Mol. Biol. (1)

K. König, I. Riemann, P. Fischer, and K. Halbhuber, “Intracellular nanourgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

Chem. Rev. (1)

P. Ball, “Water as an active constituent in cell biology,” Chem. Rev. 108, 74–108 (2008).
[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, 88–97 (2001).
[Crossref] [PubMed]

Free Radical Biol. Med. (1)

M. L. Circu and T. Y. Aw, “Reactive oxygen species, cellular redox systems, and apoptosis,” Free Radical Biol. Med. 48, 749–762 (2010).
[Crossref]

J. Appl. Phys. (1)

R. Le Harzic, I. Riemann, K. König, C. Wllner, C. Donitzky, K. Knig, C. Wllner, and C. Donitzky, “Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345 nm,” J. Appl. Phys. 102, 114701 (2007).
[Crossref]

Laser Physics (1)

M. S. AlSalhi, M. Atif, a. a. AlObiadi, and a. S. Aldwayyan, “Photodynamic damage study of hela cell line using ala,” Laser Physics 21, 733–739 (2011).
[Crossref]

Nano Lett. (1)

C. Macias-Romero, M. E. P. Didier, V. Zubkovs, L. Delannoy, F. Dutto, A. Radenovic, and S. Roke, “Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales,” Nano Lett. 14, 2552–2557 (2014).
[Crossref] [PubMed]

Nat. Photonics (1)

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7, 93–101 (2013).
[Crossref] [PubMed]

Nature (1)

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, “Cell damage by near-IR microbeams,” Nature 377, 20–21 (1995).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Lett. (3)

Photochem. Photobiol. (1)

J. R. Lakowicz, “On spectral relaxation in proteins,” Photochem. Photobiol. 72, 421–437 (2000).
[Crossref] [PubMed]

PLoS One (1)

R. Galli, O. Uckermann, E. F. Andresen, K. D. Geiger, E. Koch, G. Schackert, G. Steiner, and M. Kirsch, “Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues,” PLoS One 9, e110295 (2014).
[Crossref] [PubMed]

Q. Rev. Biophys. (1)

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38, 97–166 (2005).
[Crossref]

Redox Biology (1)

A. P. Wojtovich and T. H. Foster, “Optogenetic control of ros production,” Redox Biology 2, 368–376 (2014).
[Crossref] [PubMed]

The Plant Cell (1)

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Moller, “The free nadh concentration is kept constant in plant mitochondria under different metabolic conditions,” The Plant Cell 18, 688–698 (2006).
[Crossref] [PubMed]

Other (4)

V. V. Goehukasyan and A. A. Haikal, Natural Biomarkers for Cellular Matabolism (CRC Press, 2014).

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

J. Pawley, Handbook of Biological Confocal Microscopy (Springer Science, 2006).
[Crossref]

B. R. Masters and S. Peter, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008).

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Figures (3)

Fig. 1
Fig. 1

Simulations of heat flow and photobleaching experiments for different illuminating repetition rates (a) Schematic of a focused pulsed Gaussian beam. (b) Calculated temperature rise in the same volume of water following a 80 MHz (scanning illumination, red), and a wide-field illumination (blue) with the same deposited and absorbed peak intensity (1 J/cm3). Less heat is accumulated in the water when illuminating with the wide-field and lower repetition rate system. (c) Two-photon fluorescence (2PF) intensity decay due to photobleaching of a solution of phenolsulfonphthalein (phenol red) as a function of time. The solution is illuminated at constant peak intensity (83 GW/cm2) and cumulative fluence (20×106 pulses) using different repetition rates (and hence different powers and recording times). The inset shows the photobleaching decay rates obtained from the 2PF intensity decay as a function of the illumination repetition rate (fitted with a quadratic function). For a constant peak intensity, the photobleaching decay rate depends quadratically on the repetition rate and hence on the power.

Fig. 2
Fig. 2

Proliferation assay of human embryonic kidney (HEK) 293 cells after illumination with 1035 nm, 170 fs, 200 kHz laser pulses with listed peak intensities. (a) Timeline of the experiment. (b) Histogram of the proliferation efficiency on the first, second, and third day after 30 s exposures with different peak intensities. (c) Histogram of the proliferation efficiency for the same peak intensity (60 GW/cm2) but different exposure times. The inset shows a phase contrast image of the control (unexposed) cells three days after exposure (scale bar: 50 µm). The error bars represent the standard deviation of the cell count.

Fig. 3
Fig. 3

(a) Examples of the endogenous 2PF intensity decays in the HEK cells for different illuminating peak intensities. (b) Decay rates obtained from fitting the intensity decay of the endogenous 2PF with an exponential function. (c) Percentage of 2PF intensity lost after 30 s of exposure with the different peak intensities. (d) Percentage of 2PF intensity lost over time when exposing the cells during 370 s to 60 GW/cm2. The red markers represent the exposure times used in the proliferation experiment shown in Fig. 2(c). The error bars and shaded area in (d) represent the standard deviation from 20–30 measurements.

Tables (1)

Tables Icon

Table 1 Results of different cell viability studies, including the current one, as a function of illuminating peak intensities.

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

Δ T ( x , y , z , t ) = n = 0 int ( t * f ) 0 τ p A 2 8 π 2 C p [ κ ( t t n / f ) ] 3 / 2 e 2 ( x 2 + y 2 ) a 2 2 z 2 b 2 ( x x ) 2 + ( y y ) 2 + ( z z ) 2 4 κ ( t t n / f ) d x d y d z d t .

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