Abstract

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 μm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm.

© 2017 Optical Society of America

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    [Crossref] [PubMed]

2017 (1)

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

2016 (1)

2015 (1)

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

2014 (1)

2013 (3)

S. M. S. Kazmi, A. J. Salvaggio, A. D. Estrada, M. A. Hemati, N. K. Shaydyuk, E. Roussakis, T. A. Jones, S. A. Vinogradov, and A. K. Dunn, “Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion,” Biomed. Opt. Express 4(7), 1061–1073 (2013).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

S. Khondee and T. D. Wang, “Progress in molecular imaging in endoscopy and endomicroscopy for cancer imaging,” J. Healthc. Eng. 4(1), 1–22 (2013).
[Crossref] [PubMed]

2012 (2)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

2011 (5)

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

2009 (2)

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

2008 (1)

2005 (1)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

2003 (1)

2001 (1)

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

1995 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Achilefu, S.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Adkins, D. L.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Ahrens, J.

J. Ahrens, B. Geveci, and C. Law, ParaView: An End-User Tool for Large Data Visualization (Elsevier, 2005).

Akers, W.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Asay, A. L.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Beaurepaire, E.

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

Berezin, M.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Betzig, E.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Blinder, P.

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Brown, C. M.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

Brunette, I.

Cardona, A.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Chen, S. J.

Cheng, L. C.

Cheng, Y.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Clark, C. G.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Cruz-Hernández, J. C.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Denk, W.

Donlan, N. A.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Drew, P. J.

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Dunn, A. K.

Durst, M. E.

Eliceiri, K.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Estrada, A. D.

Feng, D. D.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Fernée, D. C.

Friedman, B.

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Frise, E.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Geveci, B.

J. Ahrens, B. Geveci, and C. Law, ParaView: An End-User Tool for Large Data Visualization (Elsevier, 2005).

Giguére, D.

Goldak, J. R.

Guild, J.

Hartenstein, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Harvey, B. K.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Hasan, M. T.

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

Hemati, M. A.

Horton, N. G.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

L. C. Cheng, N. G. Horton, K. Wang, S. J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

Hughes, T. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Ji, N.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Jones, T. A.

S. M. S. Kazmi, A. J. Salvaggio, A. D. Estrada, M. A. Hemati, N. K. Shaydyuk, E. Roussakis, T. A. Jones, S. A. Vinogradov, and A. K. Dunn, “Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion,” Biomed. Opt. Express 4(7), 1061–1073 (2013).
[Crossref] [PubMed]

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Joo, C.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Karten, H. J.

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Kaufhold, J. P.

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Kaynig, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Kazmi, S. M. S.

Khondee, S.

S. Khondee and T. D. Wang, “Progress in molecular imaging in endoscopy and endomicroscopy for cancer imaging,” J. Healthc. Eng. 4(1), 1–22 (2013).
[Crossref] [PubMed]

Kieffer, J.-C.

Kleim, J. A.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Kleinfeld, D.

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Kobat, D.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

Kubby, J. A.

J. A. Kubby, Adaptive Optics for Biological Imaging (CRC Press, 2013).
[Crossref]

Law, C.

J. Ahrens, B. Geveci, and C. Law, ParaView: An End-User Tool for Large Data Visualization (Elsevier, 2005).

Lee, H.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Longair, M.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Lyden, P. D.

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Makarov, N. S.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

McCracken, J. E.

Medina, F. A.

Mertz, J.

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

Miller, D. R.

Nada, O.

Nishimura, N.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

Oheim, M.

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

Olivié, G.

Ouzounov, D. G.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

Ozaki, T.

Pavlova, I.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

Perillo, E. P.

Pietzsch, T.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Preibisch, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Rebane, A.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Reimer, J.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Richie, C. T.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Rivera, D. R.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

Roussakis, E.

Rueden, C.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Saalfeld, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Salvaggio, A. J.

Schaffer, C. B.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

Schindelin, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Schmid, B.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Shaydyuk, N. K.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Sun, W.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Tennant, K. A.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Theer, P.

Thomas, N.

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

Tillo, S. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Tinevez, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Tolias, A. S.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Tomancak, P.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Tsai, P.

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

Tsai, P. S.

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

Vidal, F.

Vinogradov, S. A.

Wang, K.

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

L. C. Cheng, N. G. Horton, K. Wang, S. J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Wang, M.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Wang, T.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

Wang, T. D.

S. Khondee and T. D. Wang, “Progress in molecular imaging in endoscopy and endomicroscopy for cancer imaging,” J. Healthc. Eng. 4(1), 1–22 (2013).
[Crossref] [PubMed]

Webb, W. W.

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

C. Xu, J. Guild, W. W. Webb, and W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 20(23), 2372–2374 (1995).
[Crossref] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

White, D. J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Wise, F. W.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Wong, A. W.

Xu, C.

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

L. C. Cheng, N. G. Horton, K. Wang, S. J. Chen, and C. Xu, “Measurements of multiphoton action cross sections for multiphoton microscopy,” Biomed. Opt. Express 5(10), 3427–3433 (2014).
[Crossref] [PubMed]

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009).
[Crossref] [PubMed]

C. Xu, J. Guild, W. W. Webb, and W. Denk, “Determination of absolute two-photon excitation cross sections by in situ second-order autocorrelation,” Opt. Lett. 20(23), 2372–2374 (1995).
[Crossref] [PubMed]

Yazdanfar, S.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Yeh, H. C.

Zhan, C.

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Biomed. Opt. Express (3)

Cereb. Cortex (1)

K. A. Tennant, D. L. Adkins, N. A. Donlan, A. L. Asay, N. Thomas, J. A. Kleim, and T. A. Jones, “The Organization of the Forelimb Representation of the C57BL/6 Mouse Motor Cortex as Defined by Intracortical Microstimulation and Cytoarchitecture,” Cereb. Cortex 21(4), 865–876 (2011).
[Crossref]

J. Biomed. Opt. (1)

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011).
[Crossref] [PubMed]

J. Healthc. Eng. (1)

S. Khondee and T. D. Wang, “Progress in molecular imaging in endoscopy and endomicroscopy for cancer imaging,” J. Healthc. Eng. 4(1), 1–22 (2013).
[Crossref] [PubMed]

J. Neurosci. (1)

P. Tsai, J. P. Kaufhold, P. Blinder, B. Friedman, P. J. Drew, H. J. Karten, P. D. Lyden, and D. Kleinfeld, “Correlations of Neuronal and Microvascular Densities in Murine Cortex Revealed by Direct Counting and Colocalization of Nuclei and Vessels,” J. Neurosci. 29(46), 14553–14570 (2009).
[Crossref] [PubMed]

J. Phys. Chem. B (1)

M. Berezin, C. Zhan, H. Lee, C. Joo, W. Akers, S. Yazdanfar, and S. Achilefu, “Two-Photon Optical Properties of Near-Infrared Dyes at 1.55 microns Excitation,” J. Phys. Chem. B 115(39), 11530–11535 (2011).
[Crossref] [PubMed]

Med. Image. Anal. (1)

J. P. Kaufhold, P. S. Tsai, P. Blinder, and D. Kleinfeld, “Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments,” Med. Image. Anal. 16(6), 1241–1258 (2012).
[Crossref] [PubMed]

Nat. Commun. (1)

K. Wang, W. Sun, C. T. Richie, B. K. Harvey, E. Betzig, and N. Ji, “Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue,” Nat. Commun. 6, 7276 (2015).
[Crossref] [PubMed]

Nat. Methods (4)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

D. G. Ouzounov, T. Wang, M. Wang, D. D. Feng, N. G. Horton, J. C. Cruz-Hernández, Y. Cheng, J. Reimer, A. S. Tolias, N. Nishimura, and C. Xu, “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 288–390 (2017).
[Crossref]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Nat. Photonics (1)

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7, 205–209 (2013).
[Crossref]

Opt. Commun. (1)

E. Beaurepaire, M. Oheim, and J. Mertz, “Ultra-deep two-photon fluorescence excitation in turbid media,” Opt. Commun. 188, 25–29 (2001).
[Crossref]

Opt. Express (2)

Opt. Lett. (2)

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

D. R. Rivera, C. M. Brown, D. G. Ouzounov, I. Pavlova, D. Kobat, W. W. Webb, and C. Xu, “Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue,” P. Natl. Acad. Sci. USA 108(43), 17598–17603 (2011).
[Crossref]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Other (2)

J. A. Kubby, Adaptive Optics for Biological Imaging (CRC Press, 2013).
[Crossref]

J. Ahrens, B. Geveci, and C. Law, ParaView: An End-User Tool for Large Data Visualization (Elsevier, 2005).

Supplementary Material (4)

NameDescription
» Visualization 1: AVI (8010 KB)      Entire 1.53 mm z-stack of vasculature labeled with Texas Red.
» Visualization 2: AVI (9950 KB)      Entire z-stack of vasculature labeled with ICG.
» Visualization 3: AVI (3081 KB)      Entire z-stack of neurons labeled with tdTomato.
» Visualization 4: AVI (4593 KB)      Entire z-stack of vasculature (red) labeled with Texas Red and neurons (green) labeled with YFP.

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Figures (7)

Fig. 1
Fig. 1

(a) Schematic of laser and microscope design. A Ti:S oscillator seeds a regenerative amplifier, which is tunable up to 511 kHz. The amplified pulse is converted to a longer wavelength by an optical parametric amplifier (OPA). The OPA output pulse is tunable between 1,100 nm and 1,400 nm and has a maximum pulse energy of 400 nJ at 511 kHz. (b) Interferometric autocorrelation trace of the OPA pulse after the microscope objective; red dots indicate peaks of the autocorrelation trace and the blue line is a sech2(t) fit to the autocorrelation envelope. The measured temporal pulse width of the OPA was τ=42 femtoseconds. (c) Spectrum of OPA for imaging tdTomato at 1,140 nm, Texas Red at 1,215 nm, Texas Red and YFP simultaneously at 1,280 nm, and ICG at 1,350 nm.

Fig. 2
Fig. 2

In vivo two-photon microscopy images of vasculature labeled with Texas Red. (a) Three-dimensional reconstruction of a 1,535 μm stack. (b) x–y intensity projections of stack shown in (a). (c) (top) x–y intensity projection at depth of 1200 μm (note: image from different stack than shown in (a)). A line scan was performed at the highlighted red line. (bottom) Line scan at depth of 1200 μm. All scale bars are 50 μm unless otherwise indicated.

Fig. 3
Fig. 3

(a) The SBR as a function of depth for a laser repetition rate of 511 kHz (red) and 255 kHz (blue). The lines serve as guides for the eye. (b) Comparison of the SBR for a vessel at z=940 μm for laser repetition rates of 511 and 255 kHz. The vessel images are a 10-frame average at the respective repetition rate; the images are shown at full scale. The line profile is averaged over a 6 μm line indicated by the yellow lines in the vessel images. (c) Normalized intensity profile for a line through a blood vessel at z=1430 μm, demonstrating a SBR above 2 (the dotted black line indicates the calculated background). The yellow line on the vessel image, which is a 16-frame average, indicates where the line profile was taken. (d) Centerlines of vessels from 1,200 to 1,450 μm depth encoded by color. The centerlines are overlaid on a maximum intensity projection of the raw data from Fig. 2. All scale bars are 50 μm unless otherwise indicated.

Fig. 4
Fig. 4

In vivo two-photon microscopy images of vasculature labeled with ICG. (a) Three-dimensional reconstruction of a 1,000 μm stack. (b) x–y intensity projections from another mouse. All scale bars are 50 μm.

Fig. 5
Fig. 5

In vivo two-photon microscopy images of neurons labeled with tdTomato. (a) Three-dimensional reconstruction of a 1,160 μm stack of neurons within a mouse brain. (b) x–y intensity projections from stack shown in (a). All scale bars are 50 μm.

Fig. 6
Fig. 6

In vivo two-photon microscopy images of vasculature labeled with Texas Red and neurons labeled with YFP. (a) Laser speckle contrast image of mouse craniotomy. The red square indicates the two-photon imaging location. The zoomed view is a 300 × 300 × 400 μm3 two-photon maximum intensity projection. (b) x–z intensity projection of a 1,330 μm stack of vasculature (red) and neurons (green). (c) x–y intensity projections of stack shown in (b) demonstrating neuron cell bodies in layer V and a large blood vessel at a depth of 1,330 μm. All scale bars are 50 μm unless otherwise indicated.

Fig. 7
Fig. 7

Characterization of Texas Red, ICG, and YFP. (a) Logarithmic power dependence plot for ICG, Texas Red, and YFP. ICG undergoes two-photon excitation at 1,350 nm indicated by a slope of 2.03. Texas Red undergoes two-photon excitation at 1,280 nm as indicated by a slope of 1.97. YFP undergoes three-photon excitation at 1,280 nm as indicated by a slope of 3.00. Note that the power was measured after the objective. (b) Two-photon action cross section of Texas Red in units of Goeppert-Mayer (GM). Solid line serves as a guide to the eye. (c) Relative two-photon cross section of ICG. Solid line serves as a guide to the eye.

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