Abstract

In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.

© 2015 Optical Society of America

Full Article  |  PDF Article
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]

2015 (2)

I. Costantini, J. P. Ghobril, A. P. Di Giovanna, A. L. Allegra Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5, 9808 (2015).
[Crossref] [PubMed]

A. L. Allegra Mascaro, L. Silvestri, L. Sacconi, and F. S. Pavone, “Towards a comprehensive understanding of brain machinery by correlative microscopy,” J. Biomed. Opt. 20(6), 061105 (2015).
[Crossref] [PubMed]

2014 (3)

M. E. Sheffield and D. A. Dombeck, “Calcium transient prevalence across the dendritic arbour predicts place field properties,” Nature 517(7533), 200–204 (2014).
[Crossref] [PubMed]

A. J. Schain, R. A. Hill, and J. Grutzendler, “Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy,” Nat. Med. 20(4), 443–449 (2014).
[Crossref] [PubMed]

L. Silvestri, A. L. Allegra Mascaro, I. Costantini, L. Sacconi, and F. S. Pavone, “Correlative two-photon and light sheet microscopy,” Methods 66(2), 268–272 (2014).
[Crossref] [PubMed]

2013 (3)

A. J. Canty, L. Huang, J. S. Jackson, G. E. Little, G. Knott, B. Maco, and V. De Paola, “In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits,” Nat. Commun. 4, 2038 (2013).
[Crossref] [PubMed]

A. L. Allegra Mascaro, P. Cesare, L. Sacconi, G. Grasselli, G. Mandolesi, B. Maco, G. W. Knott, L. Huang, V. De Paola, P. Strata, and F. S. Pavone, “In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex,” Proc. Natl. Acad. Sci. U.S.A. 110(26), 10824–10829 (2013).
[Crossref] [PubMed]

A. Holtmaat, J. Randall, and M. Cane, “Optical imaging of structural and functional synaptic plasticity in vivo,” Eur. J. Pharmacol. 719(1-3), 128–136 (2013).
[Crossref] [PubMed]

2012 (6)

T. Knöpfel, “Genetically encoded optical indicators for the analysis of neuronal circuits,” Nat. Rev. Neurosci. 13(10), 687–700 (2012).
[PubMed]

R. J. Zatorre, R. D. Fields, and H. Johansen-Berg, “Plasticity in gray and white: neuroimaging changes in brain structure during learning,” Nat. Neurosci. 15(4), 528–536 (2012).
[Crossref] [PubMed]

J. Liu, K. Dietz, J. M. DeLoyht, X. Pedre, D. Kelkar, J. Kaur, V. Vialou, M. K. Lobo, D. M. Dietz, E. J. Nestler, J. Dupree, and P. Casaccia, “Impaired adult myelination in the prefrontal cortex of socially isolated mice,” Nat. Neurosci. 15(12), 1621–1623 (2012).
[Crossref] [PubMed]

A. Bria and G. Iannello, “TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images,” BMC Bioinformatics 13(1), 316 (2012).
[Crossref] [PubMed]

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
[Crossref] [PubMed]

2011 (2)

J. Binding, J. Ben Arous, J. F. Léger, S. Gigan, C. Boccara, and L. Bourdieu, “Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy,” Opt. Express 19(6), 4833–4847 (2011).
[Crossref] [PubMed]

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

2010 (1)

K. A. Nave, “Myelination and support of axonal integrity by glia,” Nature 468(7321), 244–252 (2010).
[Crossref] [PubMed]

2009 (1)

A. Holtmaat and K. Svoboda, “Experience-dependent structural synaptic plasticity in the mammalian brain,” Nat. Rev. Neurosci. 10(9), 647–658 (2009).
[Crossref] [PubMed]

2008 (1)

R. J. Franklin and C. Ffrench-Constant, “Remyelination in the CNS: from biology to therapy,” Nat. Rev. Neurosci. 9(11), 839–855 (2008).
[Crossref] [PubMed]

2007 (1)

C. E. Brown, P. Li, J. D. Boyd, K. R. Delaney, and T. H. Murphy, “Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke,” J. Neurosci. 27(15), 4101–4109 (2007).
[Crossref] [PubMed]

2003 (1)

L. Bø, C. A. Vedeler, H. I. Nyland, B. D. Trapp, and S. J. Mørk, “Subpial demyelination in the cerebral cortex of multiple sclerosis patients,” J. Neuropathol. Exp. Neurol. 62(7), 723–732 (2003).
[PubMed]

2000 (1)

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, “Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP,” Neuron 28(1), 41–51 (2000).
[Crossref] [PubMed]

Allegra Mascaro, A. L.

I. Costantini, J. P. Ghobril, A. P. Di Giovanna, A. L. Allegra Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5, 9808 (2015).
[Crossref] [PubMed]

A. L. Allegra Mascaro, L. Silvestri, L. Sacconi, and F. S. Pavone, “Towards a comprehensive understanding of brain machinery by correlative microscopy,” J. Biomed. Opt. 20(6), 061105 (2015).
[Crossref] [PubMed]

L. Silvestri, A. L. Allegra Mascaro, I. Costantini, L. Sacconi, and F. S. Pavone, “Correlative two-photon and light sheet microscopy,” Methods 66(2), 268–272 (2014).
[Crossref] [PubMed]

A. L. Allegra Mascaro, P. Cesare, L. Sacconi, G. Grasselli, G. Mandolesi, B. Maco, G. W. Knott, L. Huang, V. De Paola, P. Strata, and F. S. Pavone, “In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex,” Proc. Natl. Acad. Sci. U.S.A. 110(26), 10824–10829 (2013).
[Crossref] [PubMed]

Amunts, K.

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
[Crossref] [PubMed]

Arganda-Carreras, I.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Armstrong, D. D.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Aronica, E.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Avanzini, G.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Axer, M.

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
[Crossref] [PubMed]

Bahlmann, K.

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
[Crossref] [PubMed]

Barkovich, A. J.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Battaglia, G.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Becker, A.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Ben Arous, J.

Bernstein, M.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, “Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP,” Neuron 28(1), 41–51 (2000).
[Crossref] [PubMed]

Binding, J.

Blümcke, I.

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Bø, L.

L. Bø, C. A. Vedeler, H. I. Nyland, B. D. Trapp, and S. J. Mørk, “Subpial demyelination in the cerebral cortex of multiple sclerosis patients,” J. Neuropathol. Exp. Neurol. 62(7), 723–732 (2003).
[PubMed]

Boccara, C.

Bourdieu, L.

Boyd, J. D.

C. E. Brown, P. Li, J. D. Boyd, K. R. Delaney, and T. H. Murphy, “Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke,” J. Neurosci. 27(15), 4101–4109 (2007).
[Crossref] [PubMed]

Breuer, L.

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
[Crossref] [PubMed]

Bria, A.

A. Bria and G. Iannello, “TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images,” BMC Bioinformatics 13(1), 316 (2012).
[Crossref] [PubMed]

Brown, C. E.

C. E. Brown, P. Li, J. D. Boyd, K. R. Delaney, and T. H. Murphy, “Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke,” J. Neurosci. 27(15), 4101–4109 (2007).
[Crossref] [PubMed]

Cane, M.

A. Holtmaat, J. Randall, and M. Cane, “Optical imaging of structural and functional synaptic plasticity in vivo,” Eur. J. Pharmacol. 719(1-3), 128–136 (2013).
[Crossref] [PubMed]

Canty, A. J.

A. J. Canty, L. Huang, J. S. Jackson, G. E. Little, G. Knott, B. Maco, and V. De Paola, “In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits,” Nat. Commun. 4, 2038 (2013).
[Crossref] [PubMed]

Casaccia, P.

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I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
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Wallace, M.

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, “Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP,” Neuron 28(1), 41–51 (2000).
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Zatorre, R. J.

R. J. Zatorre, R. D. Fields, and H. Johansen-Berg, “Plasticity in gray and white: neuroimaging changes in brain structure during learning,” Nat. Neurosci. 15(4), 528–536 (2012).
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Zilles, K.

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
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BMC Bioinformatics (1)

A. Bria and G. Iannello, “TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images,” BMC Bioinformatics 13(1), 316 (2012).
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Epilepsia (1)

I. Blümcke, M. Thom, E. Aronica, D. D. Armstrong, H. V. Vinters, A. Palmini, T. S. Jacques, G. Avanzini, A. J. Barkovich, G. Battaglia, A. Becker, C. Cepeda, F. Cendes, N. Colombo, P. Crino, J. H. Cross, O. Delalande, F. Dubeau, J. Duncan, R. Guerrini, P. Kahane, G. Mathern, I. Najm, C. Ozkara, C. Raybaud, A. Represa, S. N. Roper, N. Salamon, A. Schulze-Bonhage, L. Tassi, A. Vezzani, and R. Spreafico, “The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission,” Epilepsia 52(1), 158–174 (2011).
[Crossref] [PubMed]

Eur. J. Pharmacol. (1)

A. Holtmaat, J. Randall, and M. Cane, “Optical imaging of structural and functional synaptic plasticity in vivo,” Eur. J. Pharmacol. 719(1-3), 128–136 (2013).
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J. Biomed. Opt. (1)

A. L. Allegra Mascaro, L. Silvestri, L. Sacconi, and F. S. Pavone, “Towards a comprehensive understanding of brain machinery by correlative microscopy,” J. Biomed. Opt. 20(6), 061105 (2015).
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J. Neuropathol. Exp. Neurol. (1)

L. Bø, C. A. Vedeler, H. I. Nyland, B. D. Trapp, and S. J. Mørk, “Subpial demyelination in the cerebral cortex of multiple sclerosis patients,” J. Neuropathol. Exp. Neurol. 62(7), 723–732 (2003).
[PubMed]

J. Neurosci. (1)

C. E. Brown, P. Li, J. D. Boyd, K. R. Delaney, and T. H. Murphy, “Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke,” J. Neurosci. 27(15), 4101–4109 (2007).
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Methods (1)

L. Silvestri, A. L. Allegra Mascaro, I. Costantini, L. Sacconi, and F. S. Pavone, “Correlative two-photon and light sheet microscopy,” Methods 66(2), 268–272 (2014).
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Nat. Commun. (1)

A. J. Canty, L. Huang, J. S. Jackson, G. E. Little, G. Knott, B. Maco, and V. De Paola, “In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits,” Nat. Commun. 4, 2038 (2013).
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Nat. Med. (1)

A. J. Schain, R. A. Hill, and J. Grutzendler, “Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy,” Nat. Med. 20(4), 443–449 (2014).
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Nat. Methods (1)

T. Ragan, L. R. Kadiri, K. U. Venkataraju, K. Bahlmann, J. Sutin, J. Taranda, I. Arganda-Carreras, Y. Kim, H. S. Seung, and P. Osten, “Serial two-photon tomography for automated ex vivo mouse brain imaging,” Nat. Methods 9(3), 255–258 (2012).
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Nat. Neurosci. (2)

R. J. Zatorre, R. D. Fields, and H. Johansen-Berg, “Plasticity in gray and white: neuroimaging changes in brain structure during learning,” Nat. Neurosci. 15(4), 528–536 (2012).
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J. Liu, K. Dietz, J. M. DeLoyht, X. Pedre, D. Kelkar, J. Kaur, V. Vialou, M. K. Lobo, D. M. Dietz, E. J. Nestler, J. Dupree, and P. Casaccia, “Impaired adult myelination in the prefrontal cortex of socially isolated mice,” Nat. Neurosci. 15(12), 1621–1623 (2012).
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R. J. Franklin and C. Ffrench-Constant, “Remyelination in the CNS: from biology to therapy,” Nat. Rev. Neurosci. 9(11), 839–855 (2008).
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A. Holtmaat and K. Svoboda, “Experience-dependent structural synaptic plasticity in the mammalian brain,” Nat. Rev. Neurosci. 10(9), 647–658 (2009).
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T. Knöpfel, “Genetically encoded optical indicators for the analysis of neuronal circuits,” Nat. Rev. Neurosci. 13(10), 687–700 (2012).
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Nature (2)

M. E. Sheffield and D. A. Dombeck, “Calcium transient prevalence across the dendritic arbour predicts place field properties,” Nature 517(7533), 200–204 (2014).
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K. A. Nave, “Myelination and support of axonal integrity by glia,” Nature 468(7321), 244–252 (2010).
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Neuroimage (1)

J. Dammers, L. Breuer, M. Axer, M. Kleiner, B. Eiben, D. Grässel, T. Dickscheid, K. Zilles, K. Amunts, N. J. Shah, and U. Pietrzyk, “Automatic identification of gray and white matter components in polarized light imaging,” Neuroimage 59(2), 1338–1347 (2012).
[Crossref] [PubMed]

Neuron (1)

G. Feng, R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, “Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP,” Neuron 28(1), 41–51 (2000).
[Crossref] [PubMed]

Opt. Express (1)

Proc. Natl. Acad. Sci. U.S.A. (1)

A. L. Allegra Mascaro, P. Cesare, L. Sacconi, G. Grasselli, G. Mandolesi, B. Maco, G. W. Knott, L. Huang, V. De Paola, P. Strata, and F. S. Pavone, “In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex,” Proc. Natl. Acad. Sci. U.S.A. 110(26), 10824–10829 (2013).
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Sci. Rep. (1)

I. Costantini, J. P. Ghobril, A. P. Di Giovanna, A. L. Allegra Mascaro, L. Silvestri, M. C. Müllenbroich, L. Onofri, V. Conti, F. Vanzi, L. Sacconi, R. Guerrini, H. Markram, G. Iannello, and F. S. Pavone, “A versatile clearing agent for multi-modal brain imaging,” Sci. Rep. 5, 9808 (2015).
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Other (2)

D. Margolis, H. Lütcke, F. Helmchen, B. Weber, and F. Haiss, “Chronic Two-Photon Imaging of Neural Activity in the Anesthetized and Awake Behaving Rodent,” in Optical Imaging of Neocortical Dynamics, B. Weber, and F. Helmchen, eds. (Humana Press, 2014), pp. 151–173.

A. Bria, G. Iannello, and H. Peng, “An Open-Source Vaa3D Plugin for Real-Time 3D Visualization of Terabyte-Sized Volumetric Images.,” in Procs. of 2015 IEEE Int.Symp. on Biomedical Imaging.(2015), pp. 520–523.
[Crossref]

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Figures (5)

Fig. 1
Fig. 1 Custom-made NIR confocal reflectance combined with two-photon fluorescence apparatus. The figure shows the NIR laser light (in red) that is first scanned by two galvanometric mirrors (scanning head), then expanded by a telescope (SL and TL), and finally focused by the objective onto the specimen. The emitted light is separated from the exciting beam by a first dichroic mirror (DM1) and then split by a second dichroic mirror (DM2). Two photomultipliers detect the split fluorescence emissions (PMT1 and PMT2). A polarization beamsplitter (PBS) along with a quarter waveplate is used to direct the descanned reflectance confocal signals to an avalanche photodiode (APD) module with a point spread function size pinhole (PH) in front. The black arrows highlight the status of polarization light in forward and backward direction.
Fig. 2
Fig. 2 Ex vivo BS NIR and two-photon fluorescence imaging. Ex vivo maximum intensity projection (MIP) of stacks (depth = 10 µm) showing the fluorescence and BS NIR signals in a Thy1-GFPM mouse hippocampus. In the upper panel there is a non-reflective GFP-labeled dendrite (arrowhead). Cell bodies (in the middle panel) are also non-reflective. Axons (lower panel) can be both fluorescent and reflective. From left to right: GFP fluorescence, reflectance, merge with GFP in green and reflectance in red. Scale bar, 10 μm.
Fig. 3
Fig. 3 Thy1-GFPM mouse hippocampus imaging. Reconstruction of a coronal slide of a Thy1-GFPM mouse hippocampus fixed with PFA: in red there is the reflectance signal, in green the fluorescence signal. The upper panel shows the rendering of maximum intensity projection (MIP, depth = 5 µm) of stacks stitched together to reconstruct the entire coronal slide. Scale bar = 100 µm. (1, 2, 3) High magnification inset corresponding to white boxes in the upper panel highlighting the different organization of axonal fibers in the hippocampal areas DG, EC and CA3.
Fig. 4
Fig. 4 In vivo NIR reflectance and two-photon fluorescence imaging. a) Mosaic of 4 × 4 maximum intensity projections from 60 µm deep stacks (90 × 90 µm wide, 10 µm overlap between the stacks). Colocalization of an axon in the fluorescence and BS channel, as pointed out by the arrowhead. Scale bar, 50 µm. b) Upper panels are MIP of TPF and BS images showing the possible colocalization of the BS signal with Fluoromyelin labeling (as highlighted by the yellow arrowhead). Yellow arrow points at a BS filament not colocalized with Fluoromyeling signal. The magenta arrowhead highlights a Fluoromyelin labeled axon which is not visible in the BS channel. Scale bar, 20 µm. Lower panels show an example of colocalization of NIR reflectance signal (BS) with GFP fluorescence and with Fluoromyelin labeling on the same axons (highlighted by yellow arrowheads). c) MIP of TFP and BS images where a binary of reflectance signal shows the sides of the myelinated tube. d) Blood capillary as visualized by NIR reflectance imaging. By performing line scans along the magenta and yellow line, we could measure blood flow velocity and lumen diameter (respectively) from the NIR reflectance signal. The graph on the right reports the profile of BS signal measured by the line scan (yellow line) on the blood vessel. Unless otherwise stated, scale bar = 10 µm.
Fig. 5
Fig. 5 In vivo imaging of NIR reflectance after stroke. On the left, a cartoon of a mouse head illustrate the relative position of the craniotomy (light pink area), the stroke core (red circle) and the imaging region (blue square). Upper panels show the retention of the BS signal in an axon partially degenerated one hour after producing a photothrombotic stroke. The yellow arrowhead points at a swelled region of a partially degenerated axon. The dashed white lines highlight the presence of a blood vessel. Lower panels show axonal elongations detected by NIR reflectance imaging in the peri-infarct area 72 hours after stroke. Scale bar, 10 µm.

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