Abstract

We report in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The measurements have 16µm spatial and 3-ms temporal resolution and reach depths of 150 µm below the cortical surface. The depth-dependent differential-fluorescence optical sections of activity are consistent with known cortical architecture and represent an important step toward in vivo measurement of functioning complex neural networks. Our observations employ a custom gradient-index lens probe and voltage-sensitive dye fluorescence; the use of epi-illumination rather than dark-field illumination provides the dramatic signal-to-noise improvement necessary for fast three-dimensional imaging.

© 2004 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
    [CrossRef]
  2. A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
    [CrossRef] [PubMed]
  3. L. B. Cohen and B. M. Salzberg, Rev. Physiol. Biochem. Pharmacol. 83, 35 (1978).
  4. R. Yuste, F. Lanni, and A. Konnerth, Imaging Neurons (Cold Spring Harbor Laboratory, New York, 2000).
  5. D. Contreras and R. Llinas, J. Neurosci 21, 9403 (2001).
    [PubMed]
  6. F. Bevilacqua, D. Piguet, J. D. Gross, B. J. Tromberg, and C. Depeursinge, Appl. Opt. 38, 4939 (1999).
    [CrossRef]
  7. R. Andrew and B. Mac Vicar, Neurosci. 62, 371 (1994).
    [CrossRef]
  8. J. Holthoff and O. Witte, J. Neurosci. 16, 2740 (1996).
    [PubMed]
  9. C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).
  10. R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
    [CrossRef] [PubMed]
  11. K. H. Kim, C. Buehler, and P. T. C. So, Appl. Opt. 38, 6004 (1999).
    [CrossRef]
  12. K. Svoboda, F. Helmchen, W. Denk, and D. Tank, Nat. Neurosci. 2, 65 (1999).
    [CrossRef] [PubMed]
  13. M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
    [CrossRef] [PubMed]
  14. A. G. Yodh and D. A. Boas, Biomedical Photonics, T. Vo-Dinh, ed. (CRC Press, Boca Raton, Fla., 2003).
  15. D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
    [CrossRef] [PubMed]
  16. J. Jung and M. Schnitzer, Opt. Lett. 28, 902 (2003).
    [CrossRef] [PubMed]
  17. H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
    [CrossRef] [PubMed]
  18. K. R. Castleman, Digital Image Processing (Prentice-Hall, Englewood Cliffs, N.J., 1979).
  19. L. Cauller and B. Connors, J. Neurosci. 14, 751 (1994).
    [PubMed]

2003 (2)

C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).

J. Jung and M. Schnitzer, Opt. Lett. 28, 902 (2003).
[CrossRef] [PubMed]

2001 (2)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

D. Contreras and R. Llinas, J. Neurosci 21, 9403 (2001).
[PubMed]

1999 (4)

F. Bevilacqua, D. Piguet, J. D. Gross, B. J. Tromberg, and C. Depeursinge, Appl. Opt. 38, 4939 (1999).
[CrossRef]

K. H. Kim, C. Buehler, and P. T. C. So, Appl. Opt. 38, 6004 (1999).
[CrossRef]

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

D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
[CrossRef] [PubMed]

1997 (1)

R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
[CrossRef] [PubMed]

1996 (1)

J. Holthoff and O. Witte, J. Neurosci. 16, 2740 (1996).
[PubMed]

1994 (2)

L. Cauller and B. Connors, J. Neurosci. 14, 751 (1994).
[PubMed]

R. Andrew and B. Mac Vicar, Neurosci. 62, 371 (1994).
[CrossRef]

1992 (1)

H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
[CrossRef] [PubMed]

1991 (1)

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

1986 (1)

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

1978 (1)

L. B. Cohen and B. M. Salzberg, Rev. Physiol. Biochem. Pharmacol. 83, 35 (1978).

Andrew, R.

R. Andrew and B. Mac Vicar, Neurosci. 62, 371 (1994).
[CrossRef]

Beaurepaire, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

Bevilacqua, F.

Blonder, G. E.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Boas, D. A.

A. G. Yodh and D. A. Boas, Biomedical Photonics, T. Vo-Dinh, ed. (CRC Press, Boca Raton, Fla., 2003).

Buehler, C.

Castleman, K. R.

K. R. Castleman, Digital Image Processing (Prentice-Hall, Englewood Cliffs, N.J., 1979).

Cauller, L.

L. Cauller and B. Connors, J. Neurosci. 14, 751 (1994).
[PubMed]

Chaigneau, E.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

Charpak, S.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

Cohen, L. B.

L. B. Cohen and B. M. Salzberg, Rev. Physiol. Biochem. Pharmacol. 83, 35 (1978).

Connors, B.

L. Cauller and B. Connors, J. Neurosci. 14, 751 (1994).
[PubMed]

Contreras, D.

D. Contreras and R. Llinas, J. Neurosci 21, 9403 (2001).
[PubMed]

Denk, W.

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

Depeursinge, C.

Ebner, T. J.

H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
[CrossRef] [PubMed]

Elias, S. A.

H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
[CrossRef] [PubMed]

Frostig, R. D.

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

George, J. S.

D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
[CrossRef] [PubMed]

Gilbert, C. D.

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

Grinvald, A.

C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

Gross, J. D.

Helmchen, F.

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

Holthoff, J.

J. Holthoff and O. Witte, J. Neurosci. 16, 2740 (1996).
[PubMed]

Jung, J.

Kim, K. H.

Kleinfeld, D.

R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
[CrossRef] [PubMed]

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Konnerth, A.

R. Yuste, F. Lanni, and A. Konnerth, Imaging Neurons (Cold Spring Harbor Laboratory, New York, 2000).

Lanni, F.

R. Yuste, F. Lanni, and A. Konnerth, Imaging Neurons (Cold Spring Harbor Laboratory, New York, 2000).

LaPorta, A.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Lieke, E.

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

Llinas, R.

D. Contreras and R. Llinas, J. Neurosci 21, 9403 (2001).
[PubMed]

Mac Vicar, B.

R. Andrew and B. Mac Vicar, Neurosci. 62, 371 (1994).
[CrossRef]

Mertz, J.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

Oheim, M.

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

Peterson, C. C. H.

C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).

Piguet, D.

Raccuia-Behling, F.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Rector, D. M.

D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
[CrossRef] [PubMed]

Rogers, R. F.

D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
[CrossRef] [PubMed]

Sakmann, B.

C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).

Salzberg, B. M.

L. B. Cohen and B. M. Salzberg, Rev. Physiol. Biochem. Pharmacol. 83, 35 (1978).

Schnitzer, M.

Slusher, R. E.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

So, P. T. C.

Stepnoski, R. A.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Svoboda, K.

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

Tank, D.

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

Tank, D. W.

R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
[CrossRef] [PubMed]

Tromberg, B. J.

Weisel, T. N.

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

Witte, O.

J. Holthoff and O. Witte, J. Neurosci. 16, 2740 (1996).
[PubMed]

Yae, H.

H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
[CrossRef] [PubMed]

Yodh, A. G.

A. G. Yodh and D. A. Boas, Biomedical Photonics, T. Vo-Dinh, ed. (CRC Press, Boca Raton, Fla., 2003).

Yuste, R.

R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
[CrossRef] [PubMed]

R. Yuste, F. Lanni, and A. Konnerth, Imaging Neurons (Cold Spring Harbor Laboratory, New York, 2000).

Appl. Opt. (2)

Cereb. Cortex (1)

R. Yuste, D. W. Tank, and D. Kleinfeld, Cereb. Cortex 7, 546 (1997).
[CrossRef] [PubMed]

J. Neurosci (1)

D. Contreras and R. Llinas, J. Neurosci 21, 9403 (2001).
[PubMed]

J. Neurosci. (3)

J. Holthoff and O. Witte, J. Neurosci. 16, 2740 (1996).
[PubMed]

C. C. H. Peterson, A. Grinvald, and B. Sakmann, J. Neurosci. 23, 1298 (2003).

L. Cauller and B. Connors, J. Neurosci. 14, 751 (1994).
[PubMed]

J. Neurosci. Methods (3)

M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak, J. Neurosci. Methods 111, 29 (2001).
[CrossRef] [PubMed]

D. M. Rector, R. F. Rogers, and J. S. George, J. Neurosci. Methods 91, 135 (1999).
[CrossRef] [PubMed]

H. Yae, S. A. Elias, and T. J. Ebner, J. Neurosci. Methods 42, 195 (1992).
[CrossRef] [PubMed]

Nat. Neurosci. (1)

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

Nature (1)

A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Weisel, Nature 324, 361 (1986).
[CrossRef] [PubMed]

Neurosci. (1)

R. Andrew and B. Mac Vicar, Neurosci. 62, 371 (1994).
[CrossRef]

Opt. Lett. (1)

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

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, Proc. Natl. Acad. Sci. USA 88, 9382 (1991).
[CrossRef]

Rev. Physiol. Biochem. Pharmacol. (1)

L. B. Cohen and B. M. Salzberg, Rev. Physiol. Biochem. Pharmacol. 83, 35 (1978).

Other (3)

R. Yuste, F. Lanni, and A. Konnerth, Imaging Neurons (Cold Spring Harbor Laboratory, New York, 2000).

K. R. Castleman, Digital Image Processing (Prentice-Hall, Englewood Cliffs, N.J., 1979).

A. G. Yodh and D. A. Boas, Biomedical Photonics, T. Vo-Dinh, ed. (CRC Press, Boca Raton, Fla., 2003).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (3)

Fig. 1
Fig. 1

Optics setup for, a, epifluorescence and, b, dark-field illumination schemes. Inset in b, detail of perimeter fiber illumination. c, Cross-section of RH795-stained brain slice (top, bright field; bottom, fluorescence) revealing uniform staining. d, Time course of events for single trial. BS, beam splitter; CL, collimating objective; RO, relay objective; EF, emission filter; VM, vibrating mirror; IF, illuminating fibers.

Fig. 2
Fig. 2

Neural activity in response to, a, cortical and, b, thalamic stimulation. Stimulation at 150 ms for a, b and 0 ms for e, f. Time stamps for b as in a, f as in e. Frames from left to right: ΔF/F one frame after stimulation, halfway up the rising edge of the response, and maximum ΔF/F. Traces of averaged, thresholded activity (c, cortical, d, thalamic) show trends as a function of depth. e, illumination Epi-illumination and, f, dark-field-illumination responses to thalamic activity. g, Signal-to-noise comparison.

Fig. 3
Fig. 3

Peak response ΔF/F images at three focal planes 0, 75, and 150 µm below the cortex surface during the spread of electrical stimulation (a, cortical; b, thalamic). c, Known thalamocortical and, d, intracortical projections. Shading shows approximate range of GRIN lens measurements with respect to cortical layers and indicates the strength relative to the amplitude of the responses to, c, cortical and, d, thalamic stimulation.

Metrics