Abstract

We present a global analysis of experimental factors affecting polarization responses in two-photon inverted microscopy. The role of reflection optics and high numerical aperture focusing is investigated in two-photon fluorescence, which can be extended to other nonlinear processes. We show that both effects strongly distort polarization responses and can lead to misleading extraction of molecular order information from polarimetric measurements. We describe a model accounting for these effects and develop a calibration technique for the determination of polarization parameters in the sample plane using two-photon fluorescence polarimetry in liquids.

© 2008 Optical Society of America

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  1. J. A. Dix and A. S. Verkman, "Mapping of fluorescence anisotropy in living cells by ratio imaging," Biophys. J. 57, 231-240 (1990).
  2. T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
    [CrossRef] [PubMed]
  3. J. Borejdo and S. Burlacu, "Measuring Orientation of Actin Filaments within a Cell: orientation of Actin in Intestinal Microvilli," Biophys. J. 65300-309 (1993).
    [CrossRef] [PubMed]
  4. R. K. P. Benninger, Björn önfelt, M. A. A. Neil, D. M. Davis, and P. M. W. French, "Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes," Biophys. J. 88609-622 (2005).
    [CrossRef]
  5. R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
    [CrossRef]
  6. A. M. Vrabioiu and T. J. Mitchison, "Structural insights into yeast septin organization from polarized fluorescence microscopy," Nature 443, 466-468 (2006).
    [CrossRef] [PubMed]
  7. T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
    [CrossRef]
  8. 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. Nat. Acad. Sc. 100, 7081-7086 (2003).
    [CrossRef]
  9. D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
    [CrossRef]
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    [CrossRef]
  12. 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]
  13. V. Le Floc’h, S. Brasselet, J. F. Roch, and J. Zyss, "Monitoring of orientation in molecular ensembles by polarization sensitive nonlinear microscopy," J. Phys. Chem. B 107, 12403-12410 (2003).
    [CrossRef]
  14. C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
    [CrossRef]
  15. S. Brasselet, V. Le Floc’h, F. Treussart, J. F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy," Phys. Rev. Lett. 92, 207401 (2004).
    [CrossRef] [PubMed]
  16. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
    [CrossRef] [PubMed]
  17. H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
    [CrossRef] [PubMed]
  18. D. Lara and C. Dainty, "Axially resolved complete Mueller matrix confocal microscopy," Appl. Opt. 45, 1917-1930 (2006).
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  19. C. E. Bigelow and T.H. Foster, "Confocal fluorescence polarization microscopy in turbid media: Effects of scattering-induced depolarisation," J. Opt. Soc. Am. A 23, 2932 (2006).
    [CrossRef]
  20. T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
    [CrossRef]
  21. D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
    [CrossRef] [PubMed]
  22. E. Y. S. Yew, and ColinJ. R. Sheppard, "Effects of axial field components on second harmonic generation microscopy," Opt. Express 14, 1167-1174 (2006).
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  23. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanetic system," Proc. R. Soc. London Ser. A. 153, 358-579 (1959).
  24. L. Novotny and B. Hecht, Principles of Nano-Optics (Cambridge University Press, Cambridge 2006).
  25. D. Axelrod, "Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization," Biophys. J. 26, 557-574 (1979).
    [CrossRef] [PubMed]
  26. K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
    [CrossRef]

2008 (1)

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]

2007 (2)

S. Brasselet and J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Phys. 8, 165-179 (2007).
[CrossRef]

T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
[CrossRef]

2006 (5)

2005 (5)

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
[CrossRef]

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

R. K. P. Benninger, Björn önfelt, M. A. A. Neil, D. M. Davis, and P. M. W. French, "Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes," Biophys. J. 88609-622 (2005).
[CrossRef]

2004 (2)

S. Brasselet, V. Le Floc’h, F. Treussart, J. F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy," Phys. Rev. Lett. 92, 207401 (2004).
[CrossRef] [PubMed]

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

2003 (3)

V. Le Floc’h, S. Brasselet, J. F. Roch, and J. Zyss, "Monitoring of orientation in molecular ensembles by polarization sensitive nonlinear microscopy," J. Phys. Chem. B 107, 12403-12410 (2003).
[CrossRef]

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
[CrossRef] [PubMed]

2002 (1)

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

1999 (1)

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

1993 (1)

J. Borejdo and S. Burlacu, "Measuring Orientation of Actin Filaments within a Cell: orientation of Actin in Intestinal Microvilli," Biophys. J. 65300-309 (1993).
[CrossRef] [PubMed]

1990 (1)

J. A. Dix and A. S. Verkman, "Mapping of fluorescence anisotropy in living cells by ratio imaging," Biophys. J. 57, 231-240 (1990).

1979 (1)

D. Axelrod, "Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization," Biophys. J. 26, 557-574 (1979).
[CrossRef] [PubMed]

1959 (1)

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanetic system," Proc. R. Soc. London Ser. A. 153, 358-579 (1959).

an der Heide, U. A.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

Anceau, C.

C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
[CrossRef]

Axelrod, D.

D. Axelrod, "Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization," Biophys. J. 26, 557-574 (1979).
[CrossRef] [PubMed]

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]

Beaurepaire, E.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Benninger, R. K. P.

R. K. P. Benninger, Björn önfelt, M. A. A. Neil, D. M. Davis, and P. M. W. French, "Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes," Biophys. J. 88609-622 (2005).
[CrossRef]

Bigelow, C. E.

C. E. Bigelow and T.H. Foster, "Confocal fluorescence polarization microscopy in turbid media: Effects of scattering-induced depolarisation," J. Opt. Soc. Am. A 23, 2932 (2006).
[CrossRef]

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

Borejdo, J.

J. Borejdo and S. Burlacu, "Measuring Orientation of Actin Filaments within a Cell: orientation of Actin in Intestinal Microvilli," Biophys. J. 65300-309 (1993).
[CrossRef] [PubMed]

Borisy, G. G.

T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
[CrossRef]

Brasselet, S.

S. Brasselet and J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Phys. 8, 165-179 (2007).
[CrossRef]

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
[CrossRef]

S. Brasselet, V. Le Floc’h, F. Treussart, J. F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy," Phys. Rev. Lett. 92, 207401 (2004).
[CrossRef] [PubMed]

Brixner, T.

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

Burlacu, S.

J. Borejdo and S. Burlacu, "Measuring Orientation of Actin Filaments within a Cell: orientation of Actin in Intestinal Microvilli," Biophys. J. 65300-309 (1993).
[CrossRef] [PubMed]

Cheng, J.-X.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

Colin, E. Y. S.

Combettes, L.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Dainty, C.

Dale, R. E.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

Débarre, D.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Dix, J. A.

J. A. Dix and A. S. Verkman, "Mapping of fluorescence anisotropy in living cells by ratio imaging," Biophys. J. 57, 231-240 (1990).

Dombeck, D. 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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Dudovich, N.

D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
[CrossRef] [PubMed]

Egelhaaf, H. J.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Fabre, A.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Fachima, R.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

Foster, T. H.

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

Foster, T.H.

Fu, Y.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

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]

Gerber, G.

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

Gierschner, J.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Goldman, Y. E.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

Hanack, M.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Hopkins, S. C.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Irving, M.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

Jazdzyk, M.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Komorowska, K.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Krampert, G.

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

Lara, D.

Le Grand, Y.

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]

Marszalek, T.

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

Mitchison, T. J.

A. M. Vrabioiu and T. J. Mitchison, "Structural insights into yeast septin organization from polarized fluorescence microscopy," Nature 443, 466-468 (2006).
[CrossRef] [PubMed]

Mitra, S.

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

Niklaus, P.

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

Odin, C.

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]

Oron, D.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
[CrossRef] [PubMed]

Pearson, B. D.

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

Pena, A. M.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Pourlsen, L.

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Raz, S.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[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]

Richards, B.

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanetic system," Proc. R. Soc. London Ser. A. 153, 358-579 (1959).

Schanne-Klein, M. C.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Schaus, T. E.

T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
[CrossRef]

Shi, R.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

Silberberg, Y.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
[CrossRef] [PubMed]

Supatto, W.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Tal, E.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

Taylor, E. W.

T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
[CrossRef]

Tordjmann, T.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Verkman, A. S.

J. A. Dix and A. S. Verkman, "Mapping of fluorescence anisotropy in living cells by ratio imaging," Biophys. J. 57, 231-240 (1990).

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Vrabioiu, A. M.

A. M. Vrabioiu and T. J. Mitchison, "Structural insights into yeast septin organization from polarized fluorescence microscopy," Nature 443, 466-468 (2006).
[CrossRef] [PubMed]

Wang, H.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

Webb, W. W.

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Wolf, E.

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanetic system," Proc. R. Soc. London Ser. A. 153, 358-579 (1959).

Yelin, D.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

Yew, E. Y. S.

Zickmund, P.

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

Zyss, J.

S. Brasselet and J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Phys. 8, 165-179 (2007).
[CrossRef]

C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
[CrossRef]

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Appl. Opt. (1)

Appl. Phys. B (1)

T. Brixner, G. Krampert, P. Niklaus, and G. Gerber, "Generation and characterization of polarization-shaped femtosecond laser pulses," Appl. Phys. B 74, S133-S144 (2002).
[CrossRef]

Biophys. J. (6)

J. Borejdo and S. Burlacu, "Measuring Orientation of Actin Filaments within a Cell: orientation of Actin in Intestinal Microvilli," Biophys. J. 65300-309 (1993).
[CrossRef] [PubMed]

R. K. P. Benninger, Björn önfelt, M. A. A. Neil, D. M. Davis, and P. M. W. French, "Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes," Biophys. J. 88609-622 (2005).
[CrossRef]

R. E. Dale, S. C. Hopkins,U. A. an der Heide, T. Marszałek, M. Irving, and Y. E. Goldman, "Model-Independent Analysis of the Orientation of Fluorescent Probes with Restricted Mobility in Muscle Fibers," Biophys. J. 781606-1618 (1999).
[CrossRef]

J. A. Dix and A. S. Verkman, "Mapping of fluorescence anisotropy in living cells by ratio imaging," Biophys. J. 57, 231-240 (1990).

H. Wang, Y. Fu, P. Zickmund, R. Shi, and J.-X. Cheng, "Coherent Anti-Stokes Raman Scattering Imaging of Axonal Myelin in Live Spinal Tissues," Biophys. J. 89, 581-591 (2005).
[CrossRef] [PubMed]

D. Axelrod, "Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization," Biophys. J. 26, 557-574 (1979).
[CrossRef] [PubMed]

C. R. Phys. (1)

S. Brasselet and J. Zyss, "Nonlinear polarimetry of molecular crystals down to the nanoscale," C. R. Phys. 8, 165-179 (2007).
[CrossRef]

Chem. Phys. (1)

K. Komorowska, S. Brasselet, J. Zyss, L. Pourlsen, M. Jazdzyk, H. J. Egelhaaf, J. Gierschner, and M. Hanack, " Nanometric scale investigation of the nonlinear efficiency of perhydrotriphynylene inclusion compounds," Chem. Phys. 318, 12-20 (2005).
[CrossRef]

Chem. Phys. Lett. (1)

C. Anceau, S. Brasselet, and J. Zyss, "Local orientational distribution of molecular monolayers probed by nonlinear microscopy," Chem. Phys. Lett. 411, 98-102 (2005).
[CrossRef]

J. Microsc. (1)

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]

J. Opt. Soc. Am. A (1)

J. Phys. Chem. B (1)

V. Le Floc’h, S. Brasselet, J. F. Roch, and J. Zyss, "Monitoring of orientation in molecular ensembles by polarization sensitive nonlinear microscopy," J. Phys. Chem. B 107, 12403-12410 (2003).
[CrossRef]

J. Struct. Biol. (1)

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, "Depth-resolved structural imaging by third-harmonic generation microscopy," J. Struct. Biol. 147, 3-11 (2004).
[CrossRef] [PubMed]

Nature (1)

A. M. Vrabioiu and T. J. Mitchison, "Structural insights into yeast septin organization from polarized fluorescence microscopy," Nature 443, 466-468 (2006).
[CrossRef] [PubMed]

Nature Methods (1)

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, " Imaging lipid bodies in cells and tissues using third harmonic generation microscopy," Nature Methods 3, 47-53 (2006).
[CrossRef]

Opt. Express (1)

Photochem. Photobiol. (1)

T. H. Foster, B. D. Pearson, S. Mitra, and C. E. Bigelow, "Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope," Photochem. Photobiol. 81, 1544-1547 (2005).
[CrossRef] [PubMed]

Phys. Rev. Lett. (2)

S. Brasselet, V. Le Floc’h, F. Treussart, J. F. Roch, J. Zyss, E. Botzung-Appert, and A. Ibanez, "In situ diagnostics of the crystalline nature of single organic nanocrystals by nonlinear microscopy," Phys. Rev. Lett. 92, 207401 (2004).
[CrossRef] [PubMed]

D. Oron, N. Dudovich, and Y. Silberberg, "Femtosecond Phase-and-Polarization Control for Background-Free Coherent Anti-Stokes Raman Spectroscopy," Phys. Rev. Lett. 90, 213902 (2003).
[CrossRef] [PubMed]

Proc. Nat. Acad. Sc. (2)

T. E. Schaus, E. W. Taylor, and G. G. Borisy, "Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model," Proc. Nat. Acad. Sc. 104, 7086-7091 (2007).
[CrossRef]

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. Nat. Acad. Sc. 100, 7081-7086 (2003).
[CrossRef]

Proc. R. Soc. London Ser. A. (1)

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanetic system," Proc. R. Soc. London Ser. A. 153, 358-579 (1959).

Other (2)

L. Novotny and B. Hecht, Principles of Nano-Optics (Cambridge University Press, Cambridge 2006).

A. M. Pena, T. Boulesteix, T. Dartigalongue, M. Strupler, E. Beaurepaire, and M. C. Schanne-Klein, "Chiroptical effects in the second harmonic generation from collagens I and IV: applications in nonlinear microscopy," Nonlinear Opt. Quantum Opt. 35 (2006).

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Figures (7)

Fig. 1.
Fig. 1.

Geometry of the polarimetric two-photon fluorescence microscopy, also corresponding to the set-up used : O : objective (NA 1.2, x 60, Water Immersion), D : Dichroic mirror, M : mirror, APD1,2 : avalanche photodiodes detecting respectively the x 1 and x 2 components of the signal. P : polarizer, λ/2 : rotatable half waveplate. µ : fluorescence excitation dipole vector at position r and orientation Ω=(θ,ϕ).

Fig. 2.
Fig. 2.

Effect of the dichroic mirror parameters γ and δ (δ in rad) on the TPF polarization response represented as a polar graph in a solution (a,b) and a 1D fluorescent sample oriented at (θ, ϕ)=(π/2, 2π/9) in the sample plane (c) (see Fig. 1 for angle definitions). (a,c) : influence of the ellipticity δ for an amplitude factor γ=0. (b) : influence of γ for δ=0. Continuous lines : Ix 1, dashed lines : Ix 2. The polarization responses are normalized to a maximum value of 1.

Fig. 3.
Fig. 3.

Cartographic map of the fluorescence intensity ratios (a) I i (π/2) I i (0) and (b) I i (π/4) I i (0) as functions of the dichroic parameters (γ,δ) (δ in °). (c) : Typical solution (red space) including experimental error margins for a three-point-fit.

Fig. 4.
Fig. 4.

TPF polarization responses of a 1D fluorescence sample accounting for both reflection optics (using the parameters δ=π/4, γ=0.01) and high numerical aperture focussing (a,c) : NA=0.1, (b,d) : NA=1.2. Sample orientation (see Fig. 1 for angle definitions) (a,b) : (θ, ϕ)=(π/3, π/6), (c,d) : (θ, ϕ)=(π/6, π/6). Continuous line : Ix 1, dashed line : Ix 2. The polarization responses are normalized to a maximum value of 1.

Fig. 5.
Fig. 5.

Experimental measurement of a polarimetric response from a Rh6G solution excited at 900nm : (a) χ 2 parameter represented for a range of (δ, γ) (δ in °), (b) : experimental points (dots) and corresponding fit (continuous line).

Fig. 6.
Fig. 6.

δ (a) and γ (b) parameters deduced from the TPF polarization responses (dots) and ellipsometry data (continuous line) on the dichroic mirror used for two-photon fluorescence, at various incident wavelengths. Each experimental point results from a 6 measurements. The green line in (b) corresponds to the δ ellipsometry data while the red one is wrapped in the [0-π/2] range for comparison with polarimetric measurements. (c–e) TPF polarization responses and fits (red curves) at three different wavelengths : (c) : 780nm, (d) :900nm, (e) : 990nm.

Fig. 7.
Fig. 7.

TPF polarization response of a 1D crystalline fluorescence sample made of DANS molecules in a PHTP crystalline host : (a) 2D scan (by a piezoelectric stage) of the sample showing the macroscopic crystal orientation, (b,c) : experimental data (dots), and corresponding fits (continuous line) at (b) 825nm excitation and (c) 975nm excitation wavelengths. The fits account for the measured dichroic parameters and for the molecular orientation (θ 0 , ϕ 0 )=(90°, 29°).

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

I i ( α ) = μ abs ( Ω , r ) · E ( α , r ) 4 ¯ E em ( Ω , r , k ) · u i 2 f ( Ω ) d Ω d r d k
I i ( α ) = μ abs ( Ω , r ) · E ( α , r ) 4 ¯ d Ω d r E em ( Ω , r , k ) · u i 2 d Ω d r d k
I i ( α ) = C i μ abs ( Ω , r ) · E ( α , r ) 4 ¯ d Ω d r
E ( α , δ , γ ) = E 1 + ( 1 γ ) 2 [ cos α ( 1 γ ) sin α e i δ 0 ]

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