Abstract

It is shown that two-photon fluorescence images can be obtained throughout almost the entire gray matter of the mouse neocortex by using optically amplified femtosecond pulses. The achieved imaging depth approaches the theoretical limit set by excitation of out-of-focus fluorescence.

© 2003 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. F. Helmchen and W. Denk, Curr. Opin. Neurobiol. 12, 593 (2002).
    [CrossRef] [PubMed]
  3. W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
    [CrossRef] [PubMed]
  4. K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
    [CrossRef] [PubMed]
  5. D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
    [CrossRef]
  6. K. Konig, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, Opt. Lett. 24, 113 (1999).
    [CrossRef]
  7. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).
  8. A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
    [CrossRef] [PubMed]
  9. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
    [CrossRef]
  10. D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
    [CrossRef]
  11. M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
    [CrossRef]
  12. E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
    [CrossRef]
  13. S. H. Cho, F. X. Kartner, U. Morgner, E. P. Ippen, J. G. Fujimoto, J. E. Cunningham, and W. H. Knox, Opt. Lett. 26, 560 (2001).
    [CrossRef]
  14. M. Ramaswamy, M. Ulman, J. Paye, and J. G. Fujimoto, Opt. Lett. 18, 1822 (1993).
    [CrossRef] [PubMed]
  15. T. B. Norris, Opt. Lett. 17, 1009 (1992).
    [CrossRef] [PubMed]
  16. W. Denk, “Optical beam power controller using a tiltable birefringent plate,” U.S. patent6,249,379B1 (June19, 2001).
  17. J. P. Ying, F. Liu, and R. R. Alfano, Appl. Opt. 38, 2151 (1999).
    [CrossRef]
  18. P. Theer and W. Denk are preparing a manuscript on the fundamental imaging-depth limit in two-photon microscopy.
  19. J. W. McLean, J. D. Freeman, and R. E. Walker, Appl. Opt. 37, 4701 (1998).
    [CrossRef]
  20. H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
    [CrossRef]
  21. M. J. Booth, M. A. A. Neil, and T. Wilson, J. Opt. Soc. Am. A 19, 2112 (2002).
    [CrossRef]

2002 (2)

2001 (5)

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
[CrossRef] [PubMed]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
[CrossRef]

S. H. Cho, F. X. Kartner, U. Morgner, E. P. Ippen, J. G. Fujimoto, J. E. Cunningham, and W. H. Knox, Opt. Lett. 26, 560 (2001).
[CrossRef]

1999 (5)

K. Konig, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, Opt. Lett. 24, 113 (1999).
[CrossRef]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

J. P. Ying, F. Liu, and R. R. Alfano, Appl. Opt. 38, 2151 (1999).
[CrossRef]

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

1998 (3)

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

J. W. McLean, J. D. Freeman, and R. E. Walker, Appl. Opt. 37, 4701 (1998).
[CrossRef]

1994 (1)

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

1993 (1)

1992 (1)

1990 (1)

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

Alfano, R. R.

Baur, D.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

Beaurepaire, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
[CrossRef]

Becker, T. W.

Booth, M. J.

Brakenhoff, G. J.

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Cho, S. H.

Cunningham, J. E.

Delaney, K. R.

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, Curr. Opin. Neurobiol. 12, 593 (2002).
[CrossRef] [PubMed]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

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

P. Theer and W. Denk are preparing a manuscript on the fundamental imaging-depth limit in two-photon microscopy.

W. Denk, “Optical beam power controller using a tiltable birefringent plate,” U.S. patent6,249,379B1 (June19, 2001).

Fischer, P.

Fittinghoff, D. N.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

Freeman, J. D.

Fujimoto, J. G.

Gelperin, A.

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Halbhuber, K. J.

Hell, S. W.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

Helmchen, F.

F. Helmchen and W. Denk, Curr. Opin. Neurobiol. 12, 593 (2002).
[CrossRef] [PubMed]

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Hopt, A.

A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
[CrossRef] [PubMed]

Ippen, E. P.

Kartner, F. X.

Kleinfeld, D.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Knox, W. H.

Koester, H. J.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

Konig, K.

Krestel, H. E.

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

Liu, F.

Mayford, M.

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

McLean, J. W.

Mertz, J.

E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
[CrossRef]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Millard, A. C.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

Mitra, P. P.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Morgner, U.

Muller, M.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

Neher, E.

A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
[CrossRef] [PubMed]

Neil, M. A. A.

Norris, T. B.

Oheim, M.

E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
[CrossRef]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

Paye, J.

Ramaswamy, M.

Riemann, I.

Seeburg, P. H.

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

Simon, U.

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

Sprengel, R.

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

Squier, J.

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

Squier, J. A.

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

Stowbridge, B. W.

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Strickler, J. H.

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

Svoboda, K.

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

Tank, D. W.

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Theer, P.

P. Theer and W. Denk are preparing a manuscript on the fundamental imaging-depth limit in two-photon microscopy.

Uhl, R.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

Ulman, M.

Walker, R. E.

Webb, W. W.

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

Wilson, T.

Wolleschensky, R.

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

Ying, J. P.

Yuste, R.

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

Appl. Opt. (2)

Biophys. J. (2)

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, Biophys. J. 77, 2226 (1999).

A. Hopt and E. Neher, Biophys. J. 80, 2029 (2001).
[CrossRef] [PubMed]

Curr. Opin. Neurobiol. (1)

F. Helmchen and W. Denk, Curr. Opin. Neurobiol. 12, 593 (2002).
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

D. N. Fittinghoff, A. C. Millard, J. A. Squier, and M. Muller, IEEE J. Quantum Electron. 35, 479 (1999).
[CrossRef]

J. Microsc. (Oxford) (1)

M. Muller, J. Squier, R. Wolleschensky, U. Simon, and G. J. Brakenhoff, J. Microsc. (Oxford) 191, 141 (1998).
[CrossRef]

J. Neurosci. Methods (2)

W. Denk, K. R. Delaney, A. Gelperin, D. Kleinfeld, B. W. Stowbridge, D. W. Tank, and R. Yuste, J. Neurosci. Methods 54, 151 (1994).
[CrossRef] [PubMed]

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 112, 205 (2001).
[CrossRef]

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

Nat. Neurosci. (1)

K. Svoboda, F. Helmchen, W. Denk, and D. W. Tank, Nat. Neurosci. 2, 65 (1999).
[CrossRef] [PubMed]

Nucleic Acids Res. (1)

H. E. Krestel, M. Mayford, P. H. Seeburg, and R. Sprengel, Nucleic Acids Res. 29, E39 (2001).
[CrossRef]

Opt. Commun. (1)

E. Beaurepaire, M. Oheim, and J. Mertz, Opt. Commun. 188, 25 (2001).
[CrossRef]

Opt. Lett. (4)

Proc. Natl. Acad. Sci. USA (1)

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef]

Science (1)

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

Other (2)

W. Denk, “Optical beam power controller using a tiltable birefringent plate,” U.S. patent6,249,379B1 (June19, 2001).

P. Theer and W. Denk are preparing a manuscript on the fundamental imaging-depth limit in two-photon microscopy.

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Figures (1)

Fig. 1
Fig. 1

Volume images of (a) a tissue phantom, (b) stained cerebral blood vessels, and (c) fluorescent-protein-labeled neurons. Shown are xz projections and single xy planes at three depths (z positions) for each sample. Each data set consists of 415 z sections (256×256 pixels, averaged over three scans, 1.95 frames/s) taken at increments of 3 µm up to a depth of 1150 µm below the surface. The horizontal striping in the deepest xy image in (c) is due to heartbeat-induced brain motion.

Equations (2)

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

zmax=ls lnP¯γ1/fτ,
SB=2πNA2λnlsz2 exp-2z/ls,

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