Abstract

The influence of the pulse length, τ, of ultrashort laser pulses at 780 and 920  nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with 60μs pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a P2/τ dependence (P, mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.

© 1999 Optical Society of America

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    [CrossRef]

1998

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef] [PubMed]

S. W. Hell, M. J. Booth, S. Wilms, C. M. Schnetter, A. K. Kirsch, D. J. Arndt Jovin, and T. M. Jovin, Opt. Lett. 23, 1238 (1998).
[CrossRef]

J. Bewersdorf and S. W. Hell, J. Microsc. 1, 28 (1998).

A. Schönle and S. Hell, Opt. Lett. 23, 325 (1998).
[CrossRef]

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

1997

K. König, P. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

1996

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Opt. Lett. 21, 1090 (1996).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

1995

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1985

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Arndt Jovin, D. J.

Bahlmann, K.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Berns, M. W.

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Opt. Lett. 21, 1090 (1996).
[CrossRef]

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

Bewersdorf, J.

J. Bewersdorf and S. W. Hell, J. Microsc. 1, 28 (1998).

Booth, M. J.

Centonze, V. E.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Chapman, C. F.

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Cheng, D.

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Crittenden, S.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Cunningham, M. L.

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Feurer, T.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

Giovanazzi, S. M.

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Gratton, E.

K. König, P. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Gryczynski, I.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Hell, S.

Hell, S. W.

J. Bewersdorf and S. W. Hell, J. Microsc. 1, 28 (1998).

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef] [PubMed]

S. W. Hell, M. J. Booth, S. Wilms, C. M. Schnetter, A. K. Kirsch, D. J. Arndt Jovin, and T. M. Jovin, Opt. Lett. 23, 1238 (1998).
[CrossRef]

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Johnson, J. S.

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Jovin, T. M.

Kirsch, A. K.

König, K.

K. König, P. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Opt. Lett. 21, 1090 (1996).
[CrossRef]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

Lakowicz, J. R.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Liang, H.

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Opt. Lett. 21, 1090 (1996).
[CrossRef]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

Liu, Y.

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Maiti, S.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Malak, H.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Mantulin, W. W.

K. König, P. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Peak, M. J.

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Sauerbrey, R.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

Schnetter, C. M.

Schönle, A.

Schrader, M.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Shear, J. B.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

Simon, U.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

So, P.

K. König, P. So, W. W. Mantulin, and E. Gratton, Opt. Lett. 22, 135 (1997).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Soini, A.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

Sonek, G. J.

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Tromberg, B. J.

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Opt. Lett. 21, 1090 (1996).
[CrossRef]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

Webb, W. W.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

White, J.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Williams, R. M.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

Wilms, S.

Wokosin, D. L.

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Wolleschensky, R.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

Xu, C.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

Zipfel, W. R.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

Appl. Phys. B

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, Appl. Phys. B 67, 87 (1998).
[CrossRef]

Bioimaging

D. L. Wokosin, V. E. Centonze, S. Crittenden, and J. White, Bioimaging 4, 208 (1996).
[CrossRef]

Biophys. J.

Y. Liu, D. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, Biophys. J. 68, 2137 (1995).
[CrossRef] [PubMed]

J. Biomed. Opt.

S. W. Hell, K. Bahlmann, M. Schrader, A. Soini, H. Malak, I. Gryczynski, and J. R. Lakowicz, J. Biomed. Opt. 1, 71 (1996).
[CrossRef] [PubMed]

J. Microsc.

M. J. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
[CrossRef] [PubMed]

K. König, P. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, J. Microsc. 183, 197 (1996).

J. Bewersdorf and S. W. Hell, J. Microsc. 1, 28 (1998).

Nature (London)

K. König, H. Liang, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
[CrossRef]

Opt. Lett.

Photochem. Photobiol.

M. L. Cunningham, J. S. Johnson, S. M. Giovanazzi, and M. J. Peak, Photochem. Photobiol. 42(2), 125 (1985).
[CrossRef]

Proc. Natl. Acad. Sci. USA

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, Proc. Natl. Acad. Sci. USA 93, 10,763 (1996).
[CrossRef]

Science

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, Science 275, 530 (1997).
[CrossRef] [PubMed]

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Figures (2)

Fig. 1
Fig. 1

Scheme of the multiphoton laser scanning microscope, with an out-of-cavity pulse-stretching unit consisting of two SF14 prisms and two blazed gratings: NDF1, NDF2, neutral-density filters; BE, beam expander; M’s, mirrors; P, powermeter.

Fig. 2
Fig. 2

(a) Mean power of the 780-nm pulses used to induce cell damage versus pulse length. ●, mean power at which the onset of visible morphological damage occurs (the bars indicate standard deviation); △, mean power at which cloning efficiency is reduced to 50%. (b) Relative power versus the square root of pulse broadening M. Curve fitting reveals a nearly linear relation y=0.82x+0.13,R=0.99. The symbols are defined in (a).

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