Abstract

We propose a two-photon microscope scheme capable of real-time, three-dimensional investigation of the electric activity pattern of neural networks or signal summation rules of individual neurons in a 0.6mm×0.6mm×0.2mm volume of the sample. The points of measurement are chosen according to a conventional scanning two-photon image, and they are addressed by separately adjustable optical fibers. This allows scanning at kilohertz repetition rates of as many as 100 data points. Submicrometer spatial resolution is maintained during the measurement similarly to conventional two-photon microscopy.

© 2007 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
    [CrossRef] [PubMed]
  2. B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
    [CrossRef] [PubMed]
  3. E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
    [CrossRef]
  4. L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).
  5. D. Strickland and G. Mourou, "Compression of amplified chirped optical pulses," Opt. Commun. 56, 219-221 (1985).
    [CrossRef]
  6. B. Proctor and F. W. Wise, "Quartz prism sequence for reduction of cubic phase in a mode-locked TiAl2O3 laser," Opt. Lett. 17, 1295-1297 (1992).
    [CrossRef] [PubMed]
  7. S. W. Clark, F. Ö. Ilday, and F. W. Wise, "Fiber delivery of femtosecond pulses from a Ti:sapphire laser," Opt. Lett. 26, 1320-1322 (2001).
    [CrossRef]
  8. For details on laser performance, visit http://www.fslasers.com.
  9. P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
    [CrossRef]
  10. S. Zeng, X. Lv, C. Zhan, W. R. Chen, W. Xiong, S. L. Jacques, and Q. Luo, "Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism," Opt. Lett. 31, 1091-1093 (2006).
    [CrossRef] [PubMed]
  11. R. Szipõcs, A. P. Kovács, and Z. Bor, "Dispersion measurement on crystals for ultrashort pulse generation with use of interference in the frequency domain," in Conference on Lasers and Electro-Optics, Vol. 11 of 1997 OSA Technical Digest Series (Optical Society of America, 1997), p. 133.
  12. A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
    [CrossRef]
  13. G. P. Agrawal, Nonlinear Fiber Optics (Academic, 2001).
  14. R. E. Sherriff, "Analytic expressions for group-delay dispersion and cubic dispersion in arbitrary prism sequences," J. Opt. Soc. Am. B 15, 1224-1230 (1998).
    [CrossRef]
  15. W. Göbel, A. Nimmerjahn, and F. Helmchen, "Distortion-free delivery of nanojoule femtosecond pulses from a Ti:sapphire laser through a hollow-core photonic crystal fiber," Opt. Lett. 29, 1285-1287 (2004).
    [CrossRef] [PubMed]
  16. K. Kuba and S. Nakayama, "Two-photon laser scanning microscopy: test of objective lenses and Ca+ probes," Neurosci. Res. (NY) 32, 281-294 (1998).
    [CrossRef]

2006

2005

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

2004

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

W. Göbel, A. Nimmerjahn, and F. Helmchen, "Distortion-free delivery of nanojoule femtosecond pulses from a Ti:sapphire laser through a hollow-core photonic crystal fiber," Opt. Lett. 29, 1285-1287 (2004).
[CrossRef] [PubMed]

2001

1999

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

1998

K. Kuba and S. Nakayama, "Two-photon laser scanning microscopy: test of objective lenses and Ca+ probes," Neurosci. Res. (NY) 32, 281-294 (1998).
[CrossRef]

R. E. Sherriff, "Analytic expressions for group-delay dispersion and cubic dispersion in arbitrary prism sequences," J. Opt. Soc. Am. B 15, 1224-1230 (1998).
[CrossRef]

1992

1990

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

1985

D. Strickland and G. Mourou, "Compression of amplified chirped optical pulses," Opt. Commun. 56, 219-221 (1985).
[CrossRef]

Agrawal, G. P.

G. P. Agrawal, Nonlinear Fiber Optics (Academic, 2001).

Barócsi, A.

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

Bor, Z.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

R. Szipõcs, A. P. Kovács, and Z. Bor, "Dispersion measurement on crystals for ultrashort pulse generation with use of interference in the frequency domain," in Conference on Lasers and Electro-Optics, Vol. 11 of 1997 OSA Technical Digest Series (Optical Society of America, 1997), p. 133.

Chen, W. R.

Clark, S. W.

Denk, W.

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

Göbel, W.

Görbe, M.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

Halas, J.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

Helmchen, F.

Ilday, F. Ö.

Jacques, S. L.

Jakab, L.

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

Kiss, J. P.

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

Klebniczki, J.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

Kovács, A. P.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

R. Szipõcs, A. P. Kovács, and Z. Bor, "Dispersion measurement on crystals for ultrashort pulse generation with use of interference in the frequency domain," in Conference on Lasers and Electro-Optics, Vol. 11 of 1997 OSA Technical Digest Series (Optical Society of America, 1997), p. 133.

Krol, J.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

Kuba, K.

K. Kuba and S. Nakayama, "Two-photon laser scanning microscopy: test of objective lenses and Ca+ probes," Neurosci. Res. (NY) 32, 281-294 (1998).
[CrossRef]

Kurdi, G.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

Lendvai, B.

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

Luo, Q.

Lv, X.

Maák, P.

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

Madarasz, P.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

Mayer, A.

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

Mourou, G.

D. Strickland and G. Mourou, "Compression of amplified chirped optical pulses," Opt. Commun. 56, 219-221 (1985).
[CrossRef]

Nakayama, S.

K. Kuba and S. Nakayama, "Two-photon laser scanning microscopy: test of objective lenses and Ca+ probes," Neurosci. Res. (NY) 32, 281-294 (1998).
[CrossRef]

Nimmerjahn, A.

Osvay, K.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

Proctor, B.

Richter, P.

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

Rózsa, B.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

Sherriff, R. E.

Strickland, D.

D. Strickland and G. Mourou, "Compression of amplified chirped optical pulses," Opt. Commun. 56, 219-221 (1985).
[CrossRef]

Strickler, J. H.

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

Szipocs, R.

R. Szipõcs, A. P. Kovács, and Z. Bor, "Dispersion measurement on crystals for ultrashort pulse generation with use of interference in the frequency domain," in Conference on Lasers and Electro-Optics, Vol. 11 of 1997 OSA Technical Digest Series (Optical Society of America, 1997), p. 133.

Valenta, L.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

Vizi, E. S.

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

Volosin, T.

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

Webb, W. W.

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

Wise, F. W.

Xiong, W.

Zelles, T.

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

Zeng, S.

Zhan, C.

Appl. Phys. B.

A. P. Kovács, K. Osvay, G. Kurdi, M. Görbe, J. Klebniczki, and Z. Bor, "Dispersion control of a pulse stretcher-compressor system with two-dimensional spectral interferometry," Appl. Phys. B. 80, 165-170 (2005).
[CrossRef]

Eur. J. Pharmacol.

E. S. Vizi, B. Rózsa, A. Mayer, J. P. Kiss, T. Zelles, and B. Lendvai, "Further evidence for the functional role of nonsynaptic nicotinic acetylcholine receptors," Eur. J. Pharmacol. 500, 499-508 (2004).
[CrossRef]

J. Neurosci.

B. Rózsa, T. Zelles, E. S. Vizi, and B. Lendvai, "Distance-dependent scaling of calcium transients evoked by backpropagating spikes and synaptic activity in dendrites of hippocampal interneurons," J. Neurosci. 24, 661-670 (2004).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

Neurosci. Res.

K. Kuba and S. Nakayama, "Two-photon laser scanning microscopy: test of objective lenses and Ca+ probes," Neurosci. Res. (NY) 32, 281-294 (1998).
[CrossRef]

Opt. Commun.

P. Maák, L. Jakab, A. Barócsi, and P. Richter, "Improved design method for acousto-optic light deflectors," Opt. Commun. 172, 297-324 (1999).
[CrossRef]

D. Strickland and G. Mourou, "Compression of amplified chirped optical pulses," Opt. Commun. 56, 219-221 (1985).
[CrossRef]

Opt. Lett.

Science

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

Other

L. Valenta, T. Volosin, J. Krol, J. Halas, P. Madarasz, B. Rózsa, and E. S. Vizi, "Application of precision engineering constructions in precision laboratory equipments," Gépészet ISBN 963 214 7480 pp. 723-727 (2004).

R. Szipõcs, A. P. Kovács, and Z. Bor, "Dispersion measurement on crystals for ultrashort pulse generation with use of interference in the frequency domain," in Conference on Lasers and Electro-Optics, Vol. 11 of 1997 OSA Technical Digest Series (Optical Society of America, 1997), p. 133.

G. P. Agrawal, Nonlinear Fiber Optics (Academic, 2001).

For details on laser performance, visit http://www.fslasers.com.

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Figures (7)

Fig. 1
Fig. 1

(a) Dendritic spines to be measured in a 3D volume (projection of 53 two-photon images in the z direction) and (b) scheme of the AO switch, optical fibers, and imaging system, which addresses the points of interest for two-photon excitation.

Fig. 2
Fig. 2

Experimental setup for random-access 3D two-photon measurements.

Fig. 3
Fig. 3

(a) Measured laser spectrum: λ 0 = 795 n m , Δ λ 20 n m . (b) Measured interferometric autocorrelation function at the laser output: Δ τ 55 f s (slightly chirped pulse).

Fig. 4
Fig. 4

(a) Dispersion curve of the ZnSe prism calculated from the measured delay values. (b) Measured dispersion curve of the Proctor and Wise four-prism sequence (L1, L2, and L3 are distances between the prisms and d1, d2, d3, and d4 are the distances of the beam propagating in the prism and the prism tip).

Fig. 5
Fig. 5

(a) Recompression of ∼1 ps stretched pulse to 40 f s by the highly dispersive imaging system. (b) Pulse duration remains 40 f s at different pulse energies: 40, 20, 10 mW after the imaging system.

Fig. 6
Fig. 6

(a) Input and output spectra of femtosecond laser pulses transmitted through the chirped pulse fiber delivery system and (b) measured autocorrelation trace at the output (dispersion compensating unit included) at 38 mW.

Fig. 7
Fig. 7

(a) Two-photon microscope image of 10 μ m diameter fluorescent beads measured in different depths of the sample. (The different focal planes were addressed by moving the fiber end positions along the z direction and the xy scanning was realized by translating the sample in the given focal plane.) (b) Intensity distribution along the lateral diameter (x) of a bead for evaluation of the resolution.

Equations (66)

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

0.6 m m × 0.6 m m × 0.2 m m
( < 1 m s )
( P 1 , P 2 , , P n )
0.6 m m × 0.6 m m × 0.2 m m
C a 2 +
( Δ λ )
20 f s
1 n J
Δ τ 25 f s
O B J 1
O B J 2
( G D D 14,000 f s 2 )
795 n m
20 n m
G D D 2700 f s 2
( A O x , A O y )
T e O 2
50 90 M H z
10 × 10
A O x
( 50 %
A O y
( > 80 % )
5 × 2
40 %
1 3 μ s
10 × 10
1500 f s 2
( P x , P y )
805 n m
38 °
60 ° 65 °
( L i , i = 1 , , n )
( L 1 )
O B J 1
4 ×
N A 0.13
O B J 2
40 ×
N A 0.8
P M T 1
P M T 2
( M 4 , M 5 )
( L 2 )
( O B J 2 )
1 p s
40 f s
40 f s
( E 1 n J )
100 f s
25 f s
40 n m
130 m m
12 m m
G D D 10, 000 f s 2 )
10 μ m
1 μ m
0.6 m m × 0.6 m m × 0.2 m m
0.6 μ m
0.6 m m × 0.6 m m × 0.2 m m
λ 0 = 795 n m
Δ λ 20 n m
Δ τ 55 f s
40 f s
40 f s
10 μ m

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