Abstract

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50–200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.

© 1998 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  2. P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
    [CrossRef]
  3. K. König, L. Hong, M. W. Berns, and B. J. Tromberg, Nature (London) 377, 20 (1995).
    [CrossRef]
  4. K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
    [CrossRef]
  5. R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).
  6. C. Xu and W. W. Webb, J. Opt. Soc. Am. B 13, 481 (1996).
    [CrossRef]
  7. P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
    [CrossRef]
  8. Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
    [CrossRef]
  9. M. Booth and S. W. Hell, J. Microsc. 190, 298 (1998).
    [CrossRef] [PubMed]
  10. D. J. Arndt-Jovin and T. M. Jovin, J. Histochem. Cytochem. 25, 585 (1977).
    [CrossRef] [PubMed]
  11. A. Schönle and S. W. Hell, Opt. Lett. 23, 325 (1998).
    [CrossRef]
  12. M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).
  13. A. K. Kirsch, C. K. Meyer, and T. M. Jovin, in Integration of Optical Techniques in Scanning Microscopy, E. Kohen and J. G. Hirschberg, eds. (Plenum, New York, 1996), p. 317.
  14. W. Denk and K. Svoboda, Neuron 18, 351 (1997).
    [CrossRef] [PubMed]
  15. J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
    [CrossRef]
  16. Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
    [CrossRef]

1998 (3)

1997 (3)

W. Denk and K. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

1996 (1)

1995 (3)

Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

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

1994 (1)

P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
[CrossRef]

1991 (1)

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

1990 (1)

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

1977 (1)

D. J. Arndt-Jovin and T. M. Jovin, J. Histochem. Cytochem. 25, 585 (1977).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

D. J. Arndt-Jovin and T. M. Jovin, J. Histochem. Cytochem. 25, 585 (1977).
[CrossRef] [PubMed]

Baumann, P. H.

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

Berns, M. W.

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

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

Bewersdorf, J.

Booth, M.

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

Denk, W.

W. Denk and K. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

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, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).

Fox, M. H.

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

Gratton, E.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Halbhuber, K. J.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Hänninen, P. E.

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
[CrossRef]

Hell, S. W.

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

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

J. Bewersdorf, R. Pick, and S. W. Hell, Opt. Lett. 23, 655 (1998).
[CrossRef]

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
[CrossRef]

Hong, L.

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

Jovin, T. M.

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

D. J. Arndt-Jovin and T. M. Jovin, J. Histochem. Cytochem. 25, 585 (1977).
[CrossRef] [PubMed]

A. K. Kirsch, C. K. Meyer, and T. M. Jovin, in Integration of Optical Techniques in Scanning Microscopy, E. Kohen and J. G. Hirschberg, eds. (Plenum, New York, 1996), p. 317.

Kimel, S.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Kirsch, A. K.

A. K. Kirsch, C. K. Meyer, and T. M. Jovin, in Integration of Optical Techniques in Scanning Microscopy, E. Kohen and J. G. Hirschberg, eds. (Plenum, New York, 1996), p. 317.

König, K.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

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

Krasieva, T.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Liang, H.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Liu, Y.

Mantulin, W. W.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Meyer, C. K.

A. K. Kirsch, C. K. Meyer, and T. M. Jovin, in Integration of Optical Techniques in Scanning Microscopy, E. Kohen and J. G. Hirschberg, eds. (Plenum, New York, 1996), p. 317.

Pick, R.

Robert-Nicoud, M.

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

Sauerbrey, R.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).

Schönle, A.

Schrader, M.

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

Simon, U.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).

So, P. T. C.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Soini, E.

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
[CrossRef]

Sonek, G. J.

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

Strickler, J. H.

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

Svaasand, L. O.

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Svoboda, K.

W. Denk and K. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

Tromberg, B. J.

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Y. Liu, G. J. Sonek, M. W. Berns, K. König, and B. J. Tromberg, Opt. Lett. 20, 2246 (1995).
[CrossRef]

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

Webb, W. W.

C. Xu and W. W. Webb, J. Opt. Soc. Am. B 13, 481 (1996).
[CrossRef]

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

Wei, X.

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

Wolleschensky, R.

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).

Xu, C.

Zhang, Z.

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

Bioimaging (1)

P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, Bioimaging 3, 70 (1995).
[CrossRef]

J. Cell Sci. (1)

M. H. Fox, D. J. Arndt-Jovin, T. M. Jovin, P. H. Baumann, and M. Robert-Nicoud, J. Cell Sci. 99, 247 (1991).

J. Histochem. Cytochem. (1)

D. J. Arndt-Jovin and T. M. Jovin, J. Histochem. Cytochem. 25, 585 (1977).
[CrossRef] [PubMed]

J. Microsc. (2)

P. E. Hänninen, E. Soini, and S. W. Hell, J. Microsc. 176, 222 (1994).
[CrossRef]

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

J. Opt. Soc. Am. B (1)

Jpn. J. Appl. Phys. (1)

Z. Zhang, G. J. Sonek, X. Wei, M. W. Berns, and B. J. Tromberg, Jpn. J. Appl. Phys. 36, 1598 (1997).
[CrossRef]

Nature (London) (1)

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

Neuron (1)

W. Denk and K. Svoboda, Neuron 18, 351 (1997).
[CrossRef] [PubMed]

Opt. Lett. (3)

Proc. SPIE (1)

K. König, H. Liang, S. Kimel, L. O. Svaasand, B. J. Tromberg, T. Krasieva, M. W. Berns, K. J. Halbhuber, P. T. C. So, W. W. Mantulin, and E. Gratton, Proc. SPIE 2983, 37 (1997).
[CrossRef]

Science (1)

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

Other (2)

R. Wolleschensky, T. Feurer, R. Sauerbrey, and U. Simon, “Characterization and optimization of a laser-scanning microscope in the femtosecond regime,” Appl. Phys. B (to be published).

A. K. Kirsch, C. K. Meyer, and T. M. Jovin, in Integration of Optical Techniques in Scanning Microscopy, E. Kohen and J. G. Hirschberg, eds. (Plenum, New York, 1996), p. 317.

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Figures (4)

Fig. 1
Fig. 1

Cw two-photon far-field microscopy of a DAPI-stained polytene chromosome recorded with cw excitation at 647 nm by an ArKr laser (total exposure, 4 s).

Fig. 2
Fig. 2

Living BBI-342-labeled (15-µM BBI-342 in phos-phate-buffered saline) 3T3 Balb/c fibroblast nuclei imaged in a closed perfusion chamber and averaged over two consecutive 1-s frames (total exposure, 0.5 s).

Fig. 3
Fig. 3

SNOM fluorescence image of a BBI-342-stained and dried polytene chromosome.

Fig. 4
Fig. 4

Double-logarithmic plots of fluorescence as a function of laser power for a BBI-342-stained polytene chromosome (observed with SNOM) and calf thymus DNA + DAPI (far-field laser scanning microscope).

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ϕfl0.561/τfσ2Pave2/ωA2,

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