Abstract

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.

© 2014 Optical Society of America

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  1. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  17. W. Zhou and F. Hildebrandt, “Inducible podocyte injury and proteinuria in transgenic zebrafish,” J. Am. Soc. Nephrol.23(6), 1039–1047 (2012).
    [CrossRef] [PubMed]

2013 (1)

2012 (5)

W. Zhou and F. Hildebrandt, “Inducible podocyte injury and proteinuria in transgenic zebrafish,” J. Am. Soc. Nephrol.23(6), 1039–1047 (2012).
[CrossRef] [PubMed]

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012).
[CrossRef] [PubMed]

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

A. Kaufmann, M. Mickoleit, M. Weber, and J. Huisken, “Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope,” Development139(17), 3242–3247 (2012).
[CrossRef] [PubMed]

2011 (2)

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

H. Zhang, M. Zhao, and L. Peng, “Nonlinear structured illumination microscopy by surface plasmon enhanced stimulated emission depletion,” Opt. Express19(24), 24783–24794 (2011).
[CrossRef] [PubMed]

2010 (2)

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

2009 (1)

2008 (2)

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

2005 (1)

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2003 (1)

R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
[CrossRef] [PubMed]

1997 (1)

Alessandri, K.

Amat, F.

R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012).
[CrossRef] [PubMed]

Bao, Z.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Betzig, E.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Booth, M. J.

Botcherby, E. J.

Bourque, C.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Bowman, T.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Burke, C.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Burns, C. E.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Ceol, C.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Choi, J. M.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Davidson, M. W.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Débarre, D.

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Dovey, M.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Fahrbach, F. O.

Fraser, S. E.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Gao, L.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Goessling, W.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Goldstein, B.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Gunther, S.

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

Gurchenkov, V.

Gustafsson, M. G.

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Heintzmann, R.

R. Heintzmann, “Saturated patterned excitation microscopy with two-dimensional excitation patterns,” Micron34(6-7), 283–291 (2003).
[CrossRef] [PubMed]

Higgins, C. D.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Hildebrandt, F.

W. Zhou and F. Hildebrandt, “Inducible podocyte injury and proteinuria in transgenic zebrafish,” J. Am. Soc. Nephrol.23(6), 1039–1047 (2012).
[CrossRef] [PubMed]

Hufnagel, L.

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

Huisken, J.

A. Kaufmann, M. Mickoleit, M. Weber, and J. Huisken, “Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope,” Development139(17), 3242–3247 (2012).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Juskaitis, R.

Kaufmann, A.

A. Kaufmann, M. Mickoleit, M. Weber, and J. Huisken, “Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope,” Development139(17), 3242–3247 (2012).
[CrossRef] [PubMed]

Keller, P. J.

R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Khairy, K.

R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Koos, D. S.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Krzic, U.

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

LeBlanc, J.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Mickoleit, M.

A. Kaufmann, M. Mickoleit, M. Weber, and J. Huisken, “Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope,” Development139(17), 3242–3247 (2012).
[CrossRef] [PubMed]

Nassoy, P.

Neil, M. A.

Peifer, M.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Peng, L.

Poulton, J. S.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Rohrbach, A.

Santella, A.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Saunders, T. E.

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Sessa, A.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Shao, L.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Simon, P.

F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics4(11), 780–785 (2010).
[CrossRef]

Srinivas, S.

Stelzer, E. H.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Streichan, S. J.

U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods9(7), 730–733 (2012).
[CrossRef] [PubMed]

Supatto, W.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Tomer, R.

R. Tomer, K. Khairy, F. Amat, and P. J. Keller, “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat. Methods9(7), 755–763 (2012).
[CrossRef] [PubMed]

Truong, T. V.

T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, and S. E. Fraser, “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat. Methods8(9), 757–760 (2011).
[CrossRef] [PubMed]

Watanabe, T.

Weber, M.

A. Kaufmann, M. Mickoleit, M. Weber, and J. Huisken, “Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope,” Development139(17), 3242–3247 (2012).
[CrossRef] [PubMed]

White, R. M.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Wilson, T.

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Wu, X.

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Zhang, H.

Zhao, M.

Zhou, W.

W. Zhou and F. Hildebrandt, “Inducible podocyte injury and proteinuria in transgenic zebrafish,” J. Am. Soc. Nephrol.23(6), 1039–1047 (2012).
[CrossRef] [PubMed]

Zon, L. I.

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Cell (1)

L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell151(6), 1370–1385 (2012).
[CrossRef] [PubMed]

Cell Stem Cell (1)

R. M. White, A. Sessa, C. Burke, T. Bowman, J. LeBlanc, C. Ceol, C. Bourque, M. Dovey, W. Goessling, C. E. Burns, and L. I. Zon, “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell2(2), 183–189 (2008).
[CrossRef] [PubMed]

Development (1)

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Supplementary Material (7)

» Media 1: MP4 (9852 KB)     
» Media 2: MP4 (7976 KB)     
» Media 3: MP4 (3744 KB)     
» Media 4: MP4 (423 KB)     
» Media 5: PDF (16 KB)     
» Media 6: MP4 (3185 KB)     
» Media 7: MP4 (2942 KB)     

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Figures (10)

Fig. 1
Fig. 1

Schematic of the Bessel two-photon light-sheet microscope. (a) Optical setup. The beam path illustrated in the figure is for the case of a uniform phase pattern on the SLM. (b) Two-photon emission from a cuvette of fluorescein solution under a focused Gaussian beam, and (c) under a Bessel beam.

Fig. 2
Fig. 2

Experimental resolution calibration. (a) Field uniformity over a 600-μm span and 2 μm axial resolution, measured by imaging a fluorescein-filled FEP tube under a stationary Bessel beam. (b) Diffraction limited 0.5 μm lateral resolution, measured by imaging 20-nm diameter beads at 0.27 μm per pixel.

Fig. 3
Fig. 3

3D two-photon imaging of the entire vasculature of a transgenic Tg(kdrl:GFP) zebrafish head at 3 days post fertilization (dpf). The fish vasculature is labeled with GFP. (a-e) Selected x-y plane slices of two-photon Bessel light-sheet images. (f) x-z plane cross section of the 3D image stacks (g) y-z plane cross section of the 3D image stack. The full image set (512 × 512 × 577) can be viewed in Media 1. 3D images were acquired by moving the sample tube at a Nyquist sampling density of 1-μm per z step, with the imaging plane moving deeper into the fish as z increases (tube moves toward camera). The tube lens focal length was set to 70 mm, resulting in a lateral pixel size of 1.1 μm. The expose time was 1s per step. Scale bar: 100 μm.

Fig. 4
Fig. 4

Cellular 3D imaging by two-photon Bessel light-sheet microscope. (a) 3D rendered image of epithelial cells forming vasculature in a Tg(kdrl:GFP) zebrafish tail. The 3D image is color-coded in z-axis. (b) Cross section in the y-z plane and (c) in the x-z plane, showing vessel structures. Full 3D image (512 × 512 × 88) can be viewed in Media 2. 3D images were acquired in 1-μm z-steps by moving the sample tube, with the imaging plane moving deeper into the fish as z increases. The tube lens focal length was set to 300 mm, resulting in a lateral pixel size of 0.27 μm. The exposure time was 1s per step.

Fig. 5
Fig. 5

Video rate two-photon images of heartbeat and blood cell (green arrows) circulation in live Tg(kdrl:GFP) zebrafish at 2-3dpf. The video was taken with the tube lens at f = 70mm and a lateral pixel size of 1.1 μm. Areas outside of the heart were chopped in the figure. Full size video (512 × 512) is provided as Media 3. Scale bar: 50 μm.

Fig. 6
Fig. 6

Long time time-lapse images of vasculature development in fish tail, starting at 1 dfp. The 3D image is color-coded in z-axis. The full image series are shown in Media 4.

Fig. 7
Fig. 7

Nonlinear SIM Imaging processing steps to remove strong diffused background in light-sheet imaging of deep tissue structures. Matlab script for the non-linear SIM reconstruction algorithm is provided in Media 5.

Fig. 8
Fig. 8

Optimizing structured illumination. (a) two-photon image from a fluorescein solution sample, showing a typical structured excitation pattern. (b) Normalized spatial power spectrum of two-photon excitation patterns showing high order harmonics of the pattern. (c) Normalized spectra powers at different harmonic orders of the excitation pattern with different pattern period. The dashed line is the typical noise level of light-sheet images deep inside zebrafish.

Fig. 9
Fig. 9

Diffusion reduction with a modified SIM reconstruction algorithm. (a) Merged image of 5 SIM raw frames with phase-shifted patterns. The merged image is equivalent to a 10-seconds-exposure image under uniform light-sheet. (b) Raw image frame under structured light-sheet illumination; the expose time was 2s; (c) Reconstructed image by the standard SIM algorithm, showing stripe-shaped artifacts due to residual diffusion background; (d) Reconstructed image from ± 1 order harmonics by the improved SIM algorithm. Stripe-shape artifacts were removed; (e) Reconstructed image from ± 2 order harmonics by the improved SIM algorithm; (f) Weighted merge of ± 1 and ± 2 order reconstructed image. All images were taken from the kidney of a live transgenic Tg(pod:NTR-mCherry) zebrafish at 4 dpf. Podocytes in the fish kidney is labeled with mCherry. Images was taken with the tube lens at f = 135mm and a lateral pixel size of 0.6 μm. The scale bar is 10 μm.

Fig. 10
Fig. 10

3D two-color images of kidney cellular structure of a live double transgenic zebrafish larva at 4dpf. GFP (green) labels endothelial cells in the vasculature. mCherry (red) labels podocytes. (a) Maximum intensity projection of 3D image set taken with a uniform light-sheet (full image set (256 × 256 × 68) in Media 6); (b) Maximum intensity projection of diffusion-free 3D image set obtained with structured light-sheet (full image set (256 × 256 × 68) in Media 7); (c) A x-z plane cross section (anterior view) of the 3D image taken with a uniform light-sheet (d) Same cross section as (c), taken with structured light-sheet; (e) A y-z plane cross section (lateral view) of the 3D image taken with a uniform light-sheet; (f) Same cross section as (e), taken with structured light-sheet. The scale bar is 20 μm.

Equations (8)

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I( x,y )= N N a n exp( in k 0 y )
M( x,y )=O( x,y )I( x,y )PSF
M( x,y )= O b ( x,y )I( x,y )PSF+ O d ( x,y )I( x,y )PS F s PSF
M ˜ ( k x , k y )= O ˜ b I ˜ ×OTF+ O ˜ d I ˜ ×OT F s ×OTF = n=N N a n [ O ˜ b ( k x , k y +n k 0 )+ O ˜ d ( k x , k y +n k 0 )OT F s ]×OTF
O ˜ b ( k x , k y )+ O ˜ b ( k x , k y n k 0 )OT F s ( k x , k y ) =conj[ O ˜ b ( k x , k y )+ O ˜ b ( k x , k y +n k 0 )OT F s ( k x , k y ) ]
δ N 2 n total T
N b a n n b T
SN R SIM m T n b n total

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