Abstract

One of the main challenges in understanding the central nervous system is to measure the network dynamics of neuronal assemblies, while preserving the computational role of individual neurons. However, this is not possible with current techniques. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

© 2008 Optical Society of America

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    [CrossRef] [PubMed]

2008 (2)

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

2007 (3)

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

J. Mapelli and E. D'Angelo, "The spatial organization of long-term synaptic plasticity at the input stage of cerebellum," J. Neurosci. 27, 1285-1296 (2007).
[CrossRef] [PubMed]

2006 (5)

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

V. Iyer, T. M. Hoogland, and P. Saggau, "Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy," J. Neurophysiol. 95, 535-545 (2006).
[CrossRef]

L. Sacconi, D. A. Dombeck, and W. W. Webb, "Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials," Proc. Natl. Acad. Sci. U S A 103, 3124-3129 (2006).
[CrossRef] [PubMed]

S. Shoham, D. H. O'Connor, and R. Segev, "How silent is the brain: is there a "dark matter" problem in neuroscience?," J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. 192, 777-784 (2006).
[CrossRef]

2005 (4)

D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb, "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy," J. Neurophysiol. 94, 3628-3636 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

G. D. Reddy and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10, 064038 (2005).
[CrossRef]

2004 (3)

H. Nishiyama and D. J. Linden, "Differential maturation of climbing fiber innervation in cerebellar vermis," J. Neurosci. 24, 3926-3932 (2004).
[CrossRef] [PubMed]

A. Grinvald and R. Hildesheim, "VSDI: a new era in functional imaging of cortical dynamics," Nat. Rev. Neurosci. 5, 874-885 (2004).
[CrossRef] [PubMed]

D. A. Dombeck, M. Blanchard-Desce, and W. W. Webb, "Optical recording of action potentials with second-harmonic generation microscopy," J. Neurosci. 24, 999-1003 (2004).
[CrossRef] [PubMed]

2003 (5)

T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz, "Mechanics of membrane potential sensing with second-harmonic generation micrsocopy," J. Biomed. Opt. 8, 428-431 (2003).
[CrossRef] [PubMed]

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-60 (2003).
[CrossRef] [PubMed]

S. D. Antic, "Action potentials in basal and oblique dendrites of rat neocortical pyramidal neurons," J. Physiol. 550, 35-50 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences" Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

T. Knopfel, K. Tomita, R. Shimazaki, and R. Sakai, "Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins," Methods 30, 42-48 (2003).
[CrossRef] [PubMed]

2002 (1)

U. Egert, D. Heck, and A. Aertsen, "Two-dimensional monitoring of spiking networks in acute brain slices," Exp. Brain Res. 142, 268-274 (2002).
[CrossRef] [PubMed]

2000 (3)

1997 (3)

J. E. Gonzalez and R. Y. Tsien, "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer," Chem. Biol. 4, 269-277 (1997).
[CrossRef] [PubMed]

M. S. Siegel and E. Y. Isacoff, "A genetically encoded optical probe of membrane voltage," Neuron 19, 735-741 (1997).
[CrossRef] [PubMed]

A. Bullen, S. S. Patel, and P. Saggau, "High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators," Biophys. J. 73, 477-491 (1997).
[CrossRef] [PubMed]

1993 (1)

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

1991 (1)

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

1980 (2)

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices," J. Physiol. 305, 197-213 (1980).
[PubMed]

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices," J. Physiol. 305, 171-195 (1980).
[PubMed]

1969 (2)

J. C. Eccles, "The cerebellum as a computer: patterns in space and time," J. Physiol. 229, 1-32 (1969).

D. Marr, "A theory of cerebellar cortex," J. Physiol. 202, 437-470 (1969).

Aertsen, A.

U. Egert, D. Heck, and A. Aertsen, "Two-dimensional monitoring of spiking networks in acute brain slices," Exp. Brain Res. 142, 268-274 (2002).
[CrossRef] [PubMed]

Antic, S.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Antic, S. D.

S. D. Antic, "Action potentials in basal and oblique dendrites of rat neocortical pyramidal neurons," J. Physiol. 550, 35-50 (2003).
[CrossRef] [PubMed]

Bamberg, E.

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

Barchi, J. R.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Blanchard-Desce, M.

D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb, "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy," J. Neurophysiol. 94, 3628-3636 (2005).
[CrossRef] [PubMed]

D. A. Dombeck, M. Blanchard-Desce, and W. W. Webb, "Optical recording of action potentials with second-harmonic generation microscopy," J. Neurosci. 24, 999-1003 (2004).
[CrossRef] [PubMed]

T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz, "Mechanics of membrane potential sensing with second-harmonic generation micrsocopy," J. Biomed. Opt. 8, 428-431 (2003).
[CrossRef] [PubMed]

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, "Membrane imaging by simultaneous second harmonic generation and two photon microscopy," Opt. Lett. 25, 320-322 (2000).
[CrossRef]

L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, "Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy," Opt. Lett. 25, 320-322 (2000).
[CrossRef]

Blonder, G. E.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

Bouevitch, O.

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

Bourdieu, L.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Boyden, E. S.

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

Bullen, A.

A. Bullen, S. S. Patel, and P. Saggau, "High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators," Biophys. J. 73, 477-491 (1997).
[CrossRef] [PubMed]

Campagnola, P. J.

P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-60 (2003).
[CrossRef] [PubMed]

Chatenay, D.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Cohen, L. B.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Contreras, D.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

D'Angelo, E.

J. Mapelli and E. D'Angelo, "The spatial organization of long-term synaptic plasticity at the input stage of cerebellum," J. Neurosci. 27, 1285-1296 (2007).
[CrossRef] [PubMed]

Deisseroth, K.

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Dieudonne, S.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Dombeck, D. A.

L. Sacconi, D. A. Dombeck, and W. W. Webb, "Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials," Proc. Natl. Acad. Sci. U S A 103, 3124-3129 (2006).
[CrossRef] [PubMed]

D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb, "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy," J. Neurophysiol. 94, 3628-3636 (2005).
[CrossRef] [PubMed]

D. A. Dombeck, M. Blanchard-Desce, and W. W. Webb, "Optical recording of action potentials with second-harmonic generation microscopy," J. Neurosci. 24, 999-1003 (2004).
[CrossRef] [PubMed]

Eccles, J. C.

J. C. Eccles, "The cerebellum as a computer: patterns in space and time," J. Physiol. 229, 1-32 (1969).

Egert, U.

U. Egert, D. Heck, and A. Aertsen, "Two-dimensional monitoring of spiking networks in acute brain slices," Exp. Brain Res. 142, 268-274 (2002).
[CrossRef] [PubMed]

Eisenthal, K. B.

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

Falk, C. X.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Fisher, J. A.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Gonzalez, J. E.

J. E. Gonzalez and R. Y. Tsien, "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer," Chem. Biol. 4, 269-277 (1997).
[CrossRef] [PubMed]

Grinvald, A.

A. Grinvald and R. Hildesheim, "VSDI: a new era in functional imaging of cortical dynamics," Nat. Rev. Neurosci. 5, 874-885 (2004).
[CrossRef] [PubMed]

Heck, D.

U. Egert, D. Heck, and A. Aertsen, "Two-dimensional monitoring of spiking networks in acute brain slices," Exp. Brain Res. 142, 268-274 (2002).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods 2, 932-940 (2005).
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A. Grinvald and R. Hildesheim, "VSDI: a new era in functional imaging of cortical dynamics," Nat. Rev. Neurosci. 5, 874-885 (2004).
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Hoogland, T. M.

V. Iyer, T. M. Hoogland, and P. Saggau, "Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy," J. Neurophysiol. 95, 535-545 (2006).
[CrossRef]

Isacoff, E. Y.

M. S. Siegel and E. Y. Isacoff, "A genetically encoded optical probe of membrane voltage," Neuron 19, 735-741 (1997).
[CrossRef] [PubMed]

Iyer, V.

V. Iyer, T. M. Hoogland, and P. Saggau, "Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy," J. Neurophysiol. 95, 535-545 (2006).
[CrossRef]

Ji, N.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Jiang, J.

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

Kim, G. H.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Kleinfeld, D.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

Knopfel, T.

T. Knopfel, K. Tomita, R. Shimazaki, and R. Sakai, "Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins," Methods 30, 42-48 (2003).
[CrossRef] [PubMed]

Kosterin, P.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Kremer, Y.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Krichevsky, O.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Lam, Y. W.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

LaPorta, A.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
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Leger, J. F.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Lewis, A.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

Linden, D. J.

H. Nishiyama and D. J. Linden, "Differential maturation of climbing fiber innervation in cerebellar vermis," J. Neurosci. 24, 3926-3932 (2004).
[CrossRef] [PubMed]

Llinas, R.

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices," J. Physiol. 305, 197-213 (1980).
[PubMed]

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices," J. Physiol. 305, 171-195 (1980).
[PubMed]

Loew, L. M.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
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P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-60 (2003).
[CrossRef] [PubMed]

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

Magee, J. C.

N. Ji, J. C. Magee, and E. Betzig, "High-speed, low-photodamage nonlinear imaging using passive pulse splitters," Nat. Methods 5, 197-202 (2008).
[CrossRef] [PubMed]

Mapelli, J.

J. Mapelli and E. D'Angelo, "The spatial organization of long-term synaptic plasticity at the input stage of cerebellum," J. Neurosci. 27, 1285-1296 (2007).
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Marr, D.

D. Marr, "A theory of cerebellar cortex," J. Physiol. 202, 437-470 (1969).

Mertz, J.

Millard, A. C.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

Mongin, O.

T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz, "Mechanics of membrane potential sensing with second-harmonic generation micrsocopy," J. Biomed. Opt. 8, 428-431 (2003).
[CrossRef] [PubMed]

Moreaux, L.

Nagel, G.

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

Nemet, B.

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

Nikolenko, V.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

Nishiyama, H.

H. Nishiyama and D. J. Linden, "Differential maturation of climbing fiber innervation in cerebellar vermis," J. Neurosci. 24, 3926-3932 (2004).
[CrossRef] [PubMed]

Nuriya, M.

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

Obaid, A. L.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

O'Connor, D. H.

S. Shoham, D. H. O'Connor, and R. Segev, "How silent is the brain: is there a "dark matter" problem in neuroscience?," J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. 192, 777-784 (2006).
[CrossRef]

Patel, S. S.

A. Bullen, S. S. Patel, and P. Saggau, "High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators," Biophys. J. 73, 477-491 (1997).
[CrossRef] [PubMed]

Pinevsky, I.

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

Pons, T.

T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz, "Mechanics of membrane potential sensing with second-harmonic generation micrsocopy," J. Biomed. Opt. 8, 428-431 (2003).
[CrossRef] [PubMed]

Poskanzer, K. E.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

Raccuia-Behling, F.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

Reddy, G. D.

G. D. Reddy and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10, 064038 (2005).
[CrossRef]

Sacconi, L.

L. Sacconi, D. A. Dombeck, and W. W. Webb, "Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials," Proc. Natl. Acad. Sci. U S A 103, 3124-3129 (2006).
[CrossRef] [PubMed]

D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb, "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy," J. Neurophysiol. 94, 3628-3636 (2005).
[CrossRef] [PubMed]

Saggau, P.

V. Iyer, T. M. Hoogland, and P. Saggau, "Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy," J. Neurophysiol. 95, 535-545 (2006).
[CrossRef]

G. D. Reddy and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10, 064038 (2005).
[CrossRef]

A. Bullen, S. S. Patel, and P. Saggau, "High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators," Biophys. J. 73, 477-491 (1997).
[CrossRef] [PubMed]

Sakai, R.

T. Knopfel, K. Tomita, R. Shimazaki, and R. Sakai, "Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins," Methods 30, 42-48 (2003).
[CrossRef] [PubMed]

Salome, R.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Salzberg, B. M.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Sandre, O.

Segev, R.

S. Shoham, D. H. O'Connor, and R. Segev, "How silent is the brain: is there a "dark matter" problem in neuroscience?," J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. 192, 777-784 (2006).
[CrossRef]

Shimazaki, R.

T. Knopfel, K. Tomita, R. Shimazaki, and R. Sakai, "Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins," Methods 30, 42-48 (2003).
[CrossRef] [PubMed]

Shoham, S.

S. Shoham, D. H. O'Connor, and R. Segev, "How silent is the brain: is there a "dark matter" problem in neuroscience?," J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. 192, 777-784 (2006).
[CrossRef]

Siegel, M. S.

M. S. Siegel and E. Y. Isacoff, "A genetically encoded optical probe of membrane voltage," Neuron 19, 735-741 (1997).
[CrossRef] [PubMed]

Slusher, R. E.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

Stepnoski, R. A.

R. A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G. E. Blonder, R. E. Slusher, and D. Kleinfeld, "Noninvasive detection of changes in membrane potential in cultured neurons by light scattering," Proc. Natl. Acad. Sci. U S A 88, 9382-9386 (1991).
[CrossRef] [PubMed]

Sugimori, M.

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices," J. Physiol. 305, 171-195 (1980).
[PubMed]

R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices," J. Physiol. 305, 197-213 (1980).
[PubMed]

Teisseyre, T. Z.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

Tomita, K.

T. Knopfel, K. Tomita, R. Shimazaki, and R. Sakai, "Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins," Methods 30, 42-48 (2003).
[CrossRef] [PubMed]

Tsien, R. Y.

J. E. Gonzalez and R. Y. Tsien, "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer," Chem. Biol. 4, 269-277 (1997).
[CrossRef] [PubMed]

Wachowiak, M.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Webb, W. W.

L. Sacconi, D. A. Dombeck, and W. W. Webb, "Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials," Proc. Natl. Acad. Sci. U S A 103, 3124-3129 (2006).
[CrossRef] [PubMed]

D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb, "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy," J. Neurophysiol. 94, 3628-3636 (2005).
[CrossRef] [PubMed]

D. A. Dombeck, M. Blanchard-Desce, and W. W. Webb, "Optical recording of action potentials with second-harmonic generation microscopy," J. Neurosci. 24, 999-1003 (2004).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences" Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Wei, M. D.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

Welle, C. G.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences" Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Wuskell, J. P.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

Wyart, C.

R. Salome, Y. Kremer, S. Dieudonne, J. F. Leger, O. Krichevsky, C. Wyart, D. Chatenay, and L. Bourdieu, "Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors," J. Neurosci. Methods 154, 161-174 (2006).
[CrossRef] [PubMed]

Yan, P.

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

Yodh, A. G.

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

Yuste, R.

V. Nikolenko, K. E. Poskanzer, and R. Yuste, "Two-photon photostimulation and imaging of neural circuits," Nat. Methods 4, 943-950 (2007).
[CrossRef] [PubMed]

M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, "Imaging membrane potential in dendritic spines," Proc. Natl. Acad. Sci. U S A 103, 786-790 (2006).
[CrossRef] [PubMed]

Zecevic, D.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Zhang, F.

E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, "Millisecond-timescale, genetically targeted optical control of neural activity," Nat. Neurosci. 8, 1263-1268 (2005).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences" Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Zochowski, M.

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. (1)

S. Shoham, D. H. O'Connor, and R. Segev, "How silent is the brain: is there a "dark matter" problem in neuroscience?," J. Comp. Physiol. A Neuropathol. Sens. Neural Behav. Physiol. 192, 777-784 (2006).
[CrossRef]

Biol. Bull. (1)

M. Zochowski, M. Wachowiak, C. X. Falk, L. B. Cohen,Y. W. Lam, S. Antic, and D. Zecevic, "Imaging membrane potential with voltage-sensitive dyes," Biol. Bull. 198, 1-21 (2000).
[CrossRef] [PubMed]

Biophys. J. (2)

O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, "Probing membrane potential with non-linear optics," Biophys. J. 65, 672-679 (1993).
[CrossRef] [PubMed]

A. Bullen, S. S. Patel, and P. Saggau, "High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators," Biophys. J. 73, 477-491 (1997).
[CrossRef] [PubMed]

Chem. Biol. (1)

J. E. Gonzalez and R. Y. Tsien, "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer," Chem. Biol. 4, 269-277 (1997).
[CrossRef] [PubMed]

Exp. Brain Res. (1)

U. Egert, D. Heck, and A. Aertsen, "Two-dimensional monitoring of spiking networks in acute brain slices," Exp. Brain Res. 142, 268-274 (2002).
[CrossRef] [PubMed]

J. Biomed. Opt. (3)

G. D. Reddy and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10, 064038 (2005).
[CrossRef]

T. Pons, L. Moreaux, O. Mongin, M. Blanchard-Desce, and J. Mertz, "Mechanics of membrane potential sensing with second-harmonic generation micrsocopy," J. Biomed. Opt. 8, 428-431 (2003).
[CrossRef] [PubMed]

T. Z. Teisseyre, A. C. Millard, P. Yan, J. P. Wuskell, M. D. Wei, A. Lewis, and L. M. Loew, "Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light," J. Biomed. Opt. 12, 044001 (2007).
[CrossRef] [PubMed]

J. Neurophysiol. (3)

V. Iyer, T. M. Hoogland, and P. Saggau, "Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy," J. Neurophysiol. 95, 535-545 (2006).
[CrossRef]

J. A. Fisher, J. R. Barchi, C. G. Welle, G. H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg, "Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ," J. Neurophysiol. 99, 1545-53 (2008).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Random access second-harmonic generation (RASH) microscope. A fiber laser provided the excitation light, which comprised 200 fs width pulses at 80 MHz repetition rate. The laser beam was adjusted for optimal linear polarization via a half-wave (λ/2) plate. Beam passes were made through 45° AOM for angular spreading pre-compensation. A second half-wave (λ/2) plate was placed after the AOM to optimize the diffraction efficiencies of the 2 orthogonally mounted AODs (AOD-x and AOD-y). A scanning lens (SL) and a microscope tube lens (TL) expanded the beam before it was focused onto the specimen by the objective lens. The SHG signal was collected by an oil immersion condenser, band-pass filtered (BFP) and focalized by a collection lens (CL) into a GaAsP PMT.

Fig. 2.
Fig. 2.

Staining and imaging. a) A photograph of a sagittal cerebellar slice unstained, b) Cerebellar slice after incubation with FM4-64 (100 µg/ml) showing the dye loading throughout the tissue. c, d) Upper traces show sodium spikes typically recorded in the PC soma after current injection. Lower traces show calcium spikes coming from the dendritic compartment of the PC. These current clamp recording show normal physiological conditions in the unstained (c) and stained (d) condition. In these measurements, PCs were hyperpolarized at a holding potential of -70 mV. e) The four panels show traces of voltage transient (black line) in a single PC following stimulation of climbing fibers (green line) at different holding potentials (-60, -70, -80, -90 mV). For each holding potential, 20 sequential trials are shown superimposed. Red trace shows the average of the 20 trials. f) SHG image of cerebellar slice at three different depths of 10, 50 and 100 µm, oriented with the granule cell layer on the left and the molecular layer on the right. Examples of SHG signals from a PC (red arrow), granule cell (yellow arrow) and interneuron (blue arrow). The images were acquired with the same laser power across all three depths.

Fig. 3.
Fig. 3.

Optical recording of APs induced by afferent fibers stimulation. a) Traces of APs in a single PC following stimulation of climbing fibers (green line). 20 trials are shown superimposed. b) Simultaneous SHG recording of APs in 20 trials, corresponding to the electrophysiological trace in a). Integration time 100 µs, sampling frequency 2 kHz. c) The average of the 20 trials are presented for both electrical (red trace) and SHG (blue trace) signals, showing how well the SHG changes track the membrane potential. d) Electrical traces of 20 trials demonstrating jitter in the response of PCs to the afferent stimulation. e) Simultaneous recording of SHG in 20 trials showing the commensurate jitter in the optical recordings. Integration time 100 µs, sampling frequency 2 kHz. f) Averaged traces of both electrical and SHG signals, showing the effect of jitter on broadening and attenuation of APs when multiple trials are averaged. In these measurements, PCs were hyperpolarized at a holding potential of -70 mV. One second was allowed between the 20 individual multi-line scans.

Fig. 4.
Fig. 4.

Optical multi-unit recording of stimulated electrical activity. a) SHG image of a cerebellar slice taken at a depth of 90 µm. The multi-unit SHG recording was carried out from the lines drawn (dotted red) on the 5 PCs, with the integration time per membrane pass indicated. b) Multiplexed recording of APs from the 5 PCs following stimulation of climbing fibers (green lines). Each trace represents the average of 20 trials for each PCs. The time resolution is 0.47 ms. c) Superposition of SHG traces corrected for multiplex delay is shown. PC5 is not shown since it was quiescent.

Fig. 5.
Fig. 5.

Optical multi-unit recording of spontaneous electrical activity. a) SHG image from a cerebellar slice taken at a depth of 50 µm. The multi-unit SHG recording was carried out from the lines drawn (dotted red) on PC1 and PC2, with the integration time per membrane pass indicated. PC1 was also measured simultaneously by electrophysiology (shadow of electrode can be seen below PC1). b) SHG signal from PC1 showing a spontaneous AP recorded in a single trial. Each point represents 0.535 ms. Confidence intervals are drawn indicating the probability of the noise crossing thresholds. The probability of the noise crossing a threshold of 0.92 is 0.003%, indicating the event shown is an AP (see panel e). c) SHG signal from PC2, suggesting that no spontaneous activity was detected in this 150 ms sampling time. d) Simultaneous electrical recording of PC1, corresponding to the SHG trace shown in panel b. The electrical recording of spontaneous activity in PC1 before e) and after f) the SHG signal collection.

Tables (1)

Tables Icon

Table 1. Electrophysiological properties of PCs. Input resistance, AP duration, firing threshold and AP amplitude between the stained (n = 8) and unstained (n = 8) slices. The AP amplitude was measured respect to the firing threshold. The numbers represent mean ± standard deviation.

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