Abstract

We performed Second Harmonic Microscopy of axonemes obtained from sea urchin sperm. Using polarization analysis and a trade-off between signal and photodamage, we were able to determine, for the first time to our knowledge, the nonlinear susceptibility χzxx/χxzx = 1.1 ± 0.2 and χzzz/χxzx = 4± 0.5 of axonemes.

© 2009 Optical Society of America

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  1. W. F. Marshall,“The cell biological basis of ciliary disease”, J. Cell. Biol. 180, 17–21 (2008).
    [Crossref] [PubMed]
  2. K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
    [Crossref] [PubMed]
  3. A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
    [Crossref]
  4. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic : multiphoton microscopy in the biosciences,” Nat.Biotechnol. 21, 1369–1377 (2003).
    [Crossref] [PubMed]
  5. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
    [Crossref] [PubMed]
  6. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
    [Crossref] [PubMed]
  7. D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
    [Crossref] [PubMed]
  8. P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).
  9. A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. 105, 11370–11375 (2008).
    [Crossref] [PubMed]
  10. A. C. Kwan, K. Duff, G. K. Gouras, and W. W. Webb, “Optical visualization of Alzheimers pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation,” Opt. Express 17, 3679–3689 (2009) http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-17-5-3679.
    [Crossref] [PubMed]
  11. C. M. Waterman-Storer, “Microtubule/organelle motility assays”. In Current Protocols in Cell Biology, J.S. Boni-facino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. (John Wiley, NY.1998), pp Ch.13:Unit 13.1.1-13.1.21.
  12. D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
    [Crossref]
  13. C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
    [Crossref] [PubMed]
  14. C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, “Collagen and myosin characterization by orientation field second harmonic microscopy,” Opt. Express 16, 16151–16165 (2008). http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151
    [Crossref] [PubMed]
  15. D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
    [Crossref] [PubMed]
  16. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
    [Crossref] [PubMed]
  17. M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
    [Crossref]
  18. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
    [Crossref]

2009 (1)

2008 (4)

W. F. Marshall,“The cell biological basis of ciliary disease”, J. Cell. Biol. 180, 17–21 (2008).
[Crossref] [PubMed]

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. 105, 11370–11375 (2008).
[Crossref] [PubMed]

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
[Crossref] [PubMed]

C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, “Collagen and myosin characterization by orientation field second harmonic microscopy,” Opt. Express 16, 16151–16165 (2008). http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151
[Crossref] [PubMed]

2007 (1)

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

2006 (1)

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

2005 (1)

D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
[Crossref] [PubMed]

2003 (4)

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

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
[Crossref] [PubMed]

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

2002 (3)

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
[Crossref]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
[Crossref]

2000 (1)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[Crossref] [PubMed]

1995 (1)

D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
[Crossref]

Alkilani, A.

Andrian, U. H. von

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

Baffet, G.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
[Crossref] [PubMed]

Baumeister, W.

D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
[Crossref] [PubMed]

Boryskina, O. P.

Campagnola, P. J.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
[Crossref] [PubMed]

Celliers, P. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

Chrétien, D.

D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
[Crossref]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Dombeck, D. A.

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. 105, 11370–11375 (2008).
[Crossref] [PubMed]

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

Duff, K.

Fleury, V.

Friedl, P.

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

Fuller, S. D.

D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
[Crossref]

Gailhouste, L.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
[Crossref] [PubMed]

Gonzalez, M. P.

M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
[Crossref]

Gouras, G. K.

Grand, Y. Le

Guilbert, T.

Harms, G.

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

Hyman, B. T.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Ingelsson, M.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

Karsenti, E.

D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
[Crossref]

Kasischke, K. A.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

König, K.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[Crossref] [PubMed]

Kwan, A. C.

Loew, L. M.

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
[Crossref] [PubMed]

Marshall, W. F.

W. F. Marshall,“The cell biological basis of ciliary disease”, J. Cell. Biol. 180, 17–21 (2008).
[Crossref] [PubMed]

McIntosh, J. R.

D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
[Crossref] [PubMed]

Millard, A. C.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

Mohler, W. A.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

Murado, M. A.

M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
[Crossref]

Nicastro, D.

D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
[Crossref] [PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Odin, C.

Plotnikov, S. V.

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

Reiser, K. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

Renault, A.

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
[Crossref] [PubMed]

Rubenchik, A. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

Stoller, P.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
[Crossref]

Vazquez, J. A.

M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
[Crossref]

Vishwasrao, H. D.

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

Waterman-Storer, C. M.

C. M. Waterman-Storer, “Microtubule/organelle motility assays”. In Current Protocols in Cell Biology, J.S. Boni-facino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. (John Wiley, NY.1998), pp Ch.13:Unit 13.1.1-13.1.21.

Webb, W. W.

A. C. Kwan, K. Duff, G. K. Gouras, and W. W. Webb, “Optical visualization of Alzheimers pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation,” Opt. Express 17, 3679–3689 (2009) http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-17-5-3679.
[Crossref] [PubMed]

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. 105, 11370–11375 (2008).
[Crossref] [PubMed]

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

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

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Williams, R. M.

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

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Wolf, K.

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
[Crossref]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

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

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
[Crossref]

Biophys J. (1)

S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. 90,693–703 (2006).
[Crossref]

Biophys. J. (1)

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-Modulated Second Harmonic Generation in llagen,” Biophys. J. 82,3330–3342 (2002).
[Crossref] [PubMed]

Curr. Prot. Cell Biol. (1)

P. Friedl, K. Wolf, U. H. von Andrian, and G. Harms, “Biological second and third harmonic generation microscopy,” Curr. Prot. Cell Biol. 4.15, 1–21 (2007).

Enz. and Micr. Techn. (1)

M. A. Murado, M. P. Gonzalez, and J. A. Vazquez, “Dose-response relationships: an overview, a generative model and its application to the verification of descriptive models”, Enz. and Micr. Techn. 31, 439–455 (2002).
[Crossref]

J. Cell Biol (1)

D. Chrétien, S. D. Fuller, and E. Karsenti, “Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates,” J. Cell Biol 129 , 13111328 (1995).
[Crossref]

J. Cell. Biol. (1)

W. F. Marshall,“The cell biological basis of ciliary disease”, J. Cell. Biol. 180, 17–21 (2008).
[Crossref] [PubMed]

J. Microsc. (2)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200, 83–104 (2000).
[Crossref] [PubMed]

C. Odin, Y. Le Grand, A. Renault, L. Gailhouste, and G. Baffet, “Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy,” J. Microsc. 229, 32–38 (2008).
[Crossref] [PubMed]

Nat. Biotechnol. (1)

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
[Crossref] [PubMed]

Nat.Biotechnol. (1)

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

Opt. Express (2)

PNAS (1)

D. Nicastro, J. R. McIntosh, and W. Baumeister, “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” PNAS 10215889–15894 (2005).
[Crossref] [PubMed]

Proc. Nat. Acad. Sc. (1)

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. 20, 11014–11019 (2002).
[Crossref]

Proc. Natl. Acad. Sci. (2)

D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. 100, 7081–7086 (2003).
[Crossref] [PubMed]

A. C. Kwan, D. A. Dombeck, and W. W. Webb, “Polarized microtubule arrays in apical dendrites and axons,” Proc. Natl. Acad. Sci. 105, 11370–11375 (2008).
[Crossref] [PubMed]

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

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100,7075–7080 (2003).
[Crossref] [PubMed]

Other (1)

C. M. Waterman-Storer, “Microtubule/organelle motility assays”. In Current Protocols in Cell Biology, J.S. Boni-facino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. (John Wiley, NY.1998), pp Ch.13:Unit 13.1.1-13.1.21.

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Figures (4)

Fig. 1.
Fig. 1.

(color online) :(a) DIC images of axonemes; (b) Isotropic SHG images of axonemes ( both images 23.5 × 23.5 μm2); (c) SHG: 2.4 × 5.5μm2 zoom on a vertical axoneme . Right: two examples of horizontal profiles. Bottom : mean profile integrated along all the axoneme. Continuous lines represent the best gaussian fits of the data.

Fig. 2.
Fig. 2.

(color online) : (a) Linear relationship between SHG signal mean and variance ; (b) Histogram of the mean number of photoelectrons < n > per pixel. (c) Axoneme photodamage induced by repetitive laser scanning (23.5×23.5 μm2). The number represents the image scan number. (d) Axoneme mean SHG intensity as a function of scan number. The continuous line represent the best fit with a logistic function.

Fig. 3.
Fig. 3.

(color online) : OF-SHM studies of axonemes (512×512 images, zoom 10X, full scale 23.5μm). (a1–a4) A set of 4 SHG polarization images indicated by the double white arrows; (b) isotropic image U; (c) orientation field represented by bars directed along the symmetry axis of χ (2). For clarity, only a few bars are represented; (d)–(e) Correlation between the orientation ω of the axonemes, and ϕ of the principal axis of χ (2) for 4 (d) or 6 (e) polarizations. Lines represent the bissectrices.

Fig. 4.
Fig. 4.

Determination of ξ and ρ by OF-SHM with 6 polarization /6. (a) Examples of fits of the SHG polarization data derived from 3 axonemes of different orientations. (b) Histograms of the ratios χαβγ /X, where X = χzxx + χxzx + χzzz . Continuous lines represents the best fit with gaussians. (c) Master curve obtained from the intensities as a function of θ for all the axonemes. (d) Mean intensity curve obtained by binning the intensity over bins of 5° width. Continuous lines are best fits with Eq. 1.

Equations (1)

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I2ω(ϕ,ψ)[χxzxsin2θ]2+[χzzzcos2θ+χzxxsin2θ]2

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