Abstract

Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin’s dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.

© 2014 Optical Society of America

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References

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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
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    [Crossref] [PubMed]
  23. Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
    [Crossref] [PubMed]
  24. G. J. Simpson and K. L. Rowlen, “An SHG magic angle: dependence of second harmonic generation orientation measurements on the width of the orientation distribution,” J. Am. Chem. Soc. 121(11), 2635–2636 (1999).
    [Crossref]
  25. S. Brenner, “The Genetics of Caenorhabditis elegans,” Genetics 77(1), 71–94 (1974).
    [PubMed]
  26. K. Beck and B. Brodsky, “Supercoiled protein motifs: the collagen triple-helix and the α-helical coiled coil,” J. Struct. Biol. 122(1-2), 17–29 (1998).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  29. E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
    [Crossref]
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    [Crossref] [PubMed]

2013 (4)

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J. 104(9), 1959–1968 (2013).
[Crossref] [PubMed]

N. Mazumder, J. Qiu, M. R. Foreman, C. M. Romero, P. Török, and F. J. Kao, “Stokes vector based polarization resolved second harmonic microscopy of starch granules,” Biomed. Opt. Express 4(4), 538–547 (2013).
[Crossref] [PubMed]

C.-H. Lien, K. Tilbury, S.-J. Chen, and P. J. Campagnola, “Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space,” Biomed. Opt. Express 4(10), 1991–2002 (2013).
[PubMed]

2012 (8)

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M. C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).
[Crossref] [PubMed]

X. Chen, C. Raggio, and P. J. Campagnola, “Second-harmonic generation circular dichroism studies of osteogenesis imperfecta,” Opt. Lett. 37(18), 3837–3839 (2012).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, I. Amat-Roldan, P. Loza-Alvarez, and D. Artigas, “Effect of molecular organization on the image histograms of polarization SHG microscopy,” Biomed. Opt. Express 3(10), 2681–2693 (2012).
[Crossref] [PubMed]

I. Gusachenko, V. Tran, Y. Goulam Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).
[Crossref] [PubMed]

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012).
[Crossref] [PubMed]

2011 (2)

P. Campagnola, “Second harmonic generation imaging microscopy: applications to diseases diagnostics,” Anal. Chem. 83(9), 3224–3231 (2011).
[Crossref] [PubMed]

S. Brasselet, “Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging,” Adv. Opt. Photonics 3(3), 205–271 (2011).
[Crossref]

2010 (1)

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

2009 (2)

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

2008 (2)

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(1), 32–38 (2008).
[Crossref] [PubMed]

M. E. Llewellyn, R. P. J. Barretto, S. L. Delp, and M. J. Schnitzer, “Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans,” Nature 454(7205), 784–788 (2008).
[PubMed]

2007 (1)

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(2), 693–703 (2006).
[Crossref] [PubMed]

2005 (2)

D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005).
[Crossref] [PubMed]

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (2005).
[Crossref] [PubMed]

2004 (2)

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

2003 (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

2002 (1)

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002).
[Crossref] [PubMed]

1999 (1)

G. J. Simpson and K. L. Rowlen, “An SHG magic angle: dependence of second harmonic generation orientation measurements on the width of the orientation distribution,” J. Am. Chem. Soc. 121(11), 2635–2636 (1999).
[Crossref]

1998 (1)

K. Beck and B. Brodsky, “Supercoiled protein motifs: the collagen triple-helix and the α-helical coiled coil,” J. Struct. Biol. 122(1-2), 17–29 (1998).
[Crossref] [PubMed]

1974 (1)

S. Brenner, “The Genetics of Caenorhabditis elegans,” Genetics 77(1), 71–94 (1974).
[PubMed]

Aït-Belkacem, D.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Allain, J. M.

I. Gusachenko, V. Tran, Y. Goulam Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).
[Crossref] [PubMed]

Amat-Roldan, I.

S. Psilodimitrakopoulos, I. Amat-Roldan, P. Loza-Alvarez, and D. Artigas, “Effect of molecular organization on the image histograms of polarization SHG microscopy,” Biomed. Opt. Express 3(10), 2681–2693 (2012).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

Ameloot, M.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

Artigas, D.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, I. Amat-Roldan, P. Loza-Alvarez, and D. Artigas, “Effect of molecular organization on the image histograms of polarization SHG microscopy,” Biomed. Opt. Express 3(10), 2681–2693 (2012).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

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(1), 32–38 (2008).
[Crossref] [PubMed]

Barretto, R. P. J.

M. E. Llewellyn, R. P. J. Barretto, S. L. Delp, and M. J. Schnitzer, “Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans,” Nature 454(7205), 784–788 (2008).
[PubMed]

Beck, K.

K. Beck and B. Brodsky, “Supercoiled protein motifs: the collagen triple-helix and the α-helical coiled coil,” J. Struct. Biol. 122(1-2), 17–29 (1998).
[Crossref] [PubMed]

Behar-Cohen, F.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

Bellanger, J. J.

D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J. 104(9), 1959–1968 (2013).
[Crossref] [PubMed]

Blanchard-Desce, M.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Bourges, J.-L.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

Brasselet, S.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

S. Brasselet, “Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging,” Adv. Opt. Photonics 3(3), 205–271 (2011).
[Crossref]

Brenner, S.

S. Brenner, “The Genetics of Caenorhabditis elegans,” Genetics 77(1), 71–94 (1974).
[PubMed]

Brodsky, B.

K. Beck and B. Brodsky, “Supercoiled protein motifs: the collagen triple-helix and the α-helical coiled coil,” J. Struct. Biol. 122(1-2), 17–29 (1998).
[Crossref] [PubMed]

Campagnola, P.

P. Campagnola, “Second harmonic generation imaging microscopy: applications to diseases diagnostics,” Anal. Chem. 83(9), 3224–3231 (2011).
[Crossref] [PubMed]

Campagnola, P. J.

C.-H. Lien, K. Tilbury, S.-J. Chen, and P. J. Campagnola, “Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space,” Biomed. Opt. Express 4(10), 1991–2002 (2013).
[PubMed]

X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012).
[Crossref] [PubMed]

X. Chen, C. Raggio, and P. J. Campagnola, “Second-harmonic generation circular dichroism studies of osteogenesis imperfecta,” Opt. Lett. 37(18), 3837–3839 (2012).
[Crossref] [PubMed]

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(2), 693–703 (2006).
[Crossref] [PubMed]

Celliers, P. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002).
[Crossref] [PubMed]

Chang, Y.

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

Chen, C.

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

Chen, J.

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

Chen, S.-J.

Chen, S.-Y.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

Chen, W. Q.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

Chen, X.

X. Chen, C. Raggio, and P. J. Campagnola, “Second-harmonic generation circular dichroism studies of osteogenesis imperfecta,” Opt. Lett. 37(18), 3837–3839 (2012).
[Crossref] [PubMed]

X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012).
[Crossref] [PubMed]

Chen, Y.-C.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

Chern, G.-W.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

Chu, S.-W.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

Clays, K.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

De Meulenaere, E.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

de Vera, N.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

Delp, S. L.

M. E. Llewellyn, R. P. J. Barretto, S. L. Delp, and M. J. Schnitzer, “Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans,” Nature 454(7205), 784–788 (2008).
[PubMed]

Deng, X.

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

Duan, X. M.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

Duboisset, J.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Ferrand, P.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Foreman, M. R.

Fusi, L.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[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(1), 32–38 (2008).
[Crossref] [PubMed]

Goulam Houssen, Y.

I. Gusachenko, V. Tran, Y. Goulam Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).
[Crossref] [PubMed]

Guilbert, M.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Gusachenko, I.

I. Gusachenko, V. Tran, Y. Goulam Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).
[Crossref] [PubMed]

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M. C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).
[Crossref] [PubMed]

Hernandez, O.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

Jeannesson, P.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Jin, Y.

Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
[Crossref] [PubMed]

Kao, F. J.

Kowalczuk, L.

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M. C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).
[Crossref] [PubMed]

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

Lamarre, I.

Latour, G.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M. C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).
[Crossref] [PubMed]

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(1), 32–38 (2008).
[Crossref] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Leray, A.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Leroy, L.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Lien, C.-H.

Lin, B.-L.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, “Studies of chi(2)/chi(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86(6), 3914–3922 (2004).
[Crossref] [PubMed]

Linari, M.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Llewellyn, M. E.

M. E. Llewellyn, R. P. J. Barretto, S. L. Delp, and M. J. Schnitzer, “Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans,” Nature 454(7205), 784–788 (2008).
[PubMed]

Lombardi, V.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Loza-Alvarez, P.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, I. Amat-Roldan, P. Loza-Alvarez, and D. Artigas, “Effect of molecular organization on the image histograms of polarization SHG microscopy,” Biomed. Opt. Express 3(10), 2681–2693 (2012).
[Crossref] [PubMed]

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

Mallegol, T.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Mazumder, N.

McAloney, R. A.

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (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(2), 693–703 (2006).
[Crossref] [PubMed]

Mitchell, S. A.

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (2005).
[Crossref] [PubMed]

Moffatt, D.

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (2005).
[Crossref] [PubMed]

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(2), 693–703 (2006).
[Crossref] [PubMed]

Mongin, O.

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Mora-Diez, N.

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (2005).
[Crossref] [PubMed]

Nadiarynkh, O.

X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012).
[Crossref] [PubMed]

Nucciotti, V.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

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(1), 32–38 (2008).
[Crossref] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Oron, D.

Paesen, R.

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

Pavone, F. S.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Petegnief, V.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

Piazzesi, G.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Plamann, K.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

Planas, A. M.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

Plotnikov, S.

X. Chen, O. Nadiarynkh, S. Plotnikov, and P. J. Campagnola, “Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure,” Nat. Protoc. 7(4), 654–669 (2012).
[Crossref] [PubMed]

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(2), 693–703 (2006).
[Crossref] [PubMed]

Psilodimitrakopoulos, S.

S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons,” Biophys. J. 104(5), 968–975 (2013).
[Crossref] [PubMed]

E. De Meulenaere, W. Q. Chen, S. Van Cleuvenbergen, M. L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J. M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X. M. Duan, and K. Clays, “Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging,” Chem. Sci. 3(4), 984–995 (2012).
[Crossref]

S. Psilodimitrakopoulos, I. Amat-Roldan, P. Loza-Alvarez, and D. Artigas, “Effect of molecular organization on the image histograms of polarization SHG microscopy,” Biomed. Opt. Express 3(10), 2681–2693 (2012).
[Crossref] [PubMed]

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

Qiu, J.

Raggio, C.

Recher, G.

D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J. 104(9), 1959–1968 (2013).
[Crossref] [PubMed]

F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15(19), 12286–12295 (2007).
[Crossref] [PubMed]

Reiser, K. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 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(1), 32–38 (2008).
[Crossref] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Roche, M.

D. Aït-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet, “Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation,” J. Biomed. Opt. 17(8), 080506 (2012).
[Crossref] [PubMed]

Romero, C. M.

Rouède, D.

D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J. 104(9), 1959–1968 (2013).
[Crossref] [PubMed]

F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15(19), 12286–12295 (2007).
[Crossref] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vié, D. Rouède, T. Mallegol, O. Mongin, M. H. V. Werts, and M. Blanchard-Desce, “Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy,” Langmuir 20(19), 8165–8171 (2004).
[Crossref] [PubMed]

Rowlen, K. L.

G. J. Simpson and K. L. Rowlen, “An SHG magic angle: dependence of second harmonic generation orientation measurements on the width of the orientation distribution,” J. Am. Chem. Soc. 121(11), 2635–2636 (1999).
[Crossref]

Rubenchik, A. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82(6), 3330–3342 (2002).
[Crossref] [PubMed]

Sacconi, L.

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Santos, S.

S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

Savoldelli, M.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

Schanne-Klein, M. C.

G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, and M. C. Schanne-Klein, “Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy,” PLoS ONE 7(11), e48388 (2012).
[Crossref] [PubMed]

I. Gusachenko, V. Tran, Y. Goulam Houssen, J. M. Allain, and M. C. Schanne-Klein, “Polarization-resolved second-harmonic generation in tendon upon mechanical stretching,” Biophys. J. 102(9), 2220–2229 (2012).
[Crossref] [PubMed]

G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M. C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012).
[Crossref] [PubMed]

Schaub, E.

D. Rouède, J. J. Bellanger, E. Schaub, G. Recher, and F. Tiaho, “Theoretical and experimental SHG angular intensity patterns from healthy and proteolysed muscles,” Biophys. J. 104(9), 1959–1968 (2013).
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Y. Chang, C. Chen, J. Chen, Y. Jin, and X. Deng, “Theoretical simulation study of linearly polarized light on microscopic second-harmonic generation in collagen type I,” J. Biomed. Opt. 14(4), 044016 (2009).
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S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009).

J. Chem. Phys. (1)

S. A. Mitchell, R. A. McAloney, D. Moffatt, N. Mora-Diez, and M. Z. Zgierski, “Second-harmonic generation optical activity of a polypeptide alpha-helix at the air/water interface,” J. Chem. Phys. 122(11), 114707 (2005).
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Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
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Proc. Natl. Acad. Sci. U.S.A. (1)

V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, L. Fusi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone, “Probing myosin structural conformation in vivo by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 107(17), 7763–7768 (2010).
[Crossref] [PubMed]

Supplementary Material (1)

» Media 1: AVI (21362 KB)     

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Figures (4)

Fig. 1
Fig. 1 Scheme of the experimental setup showing the excitation with circular polarization, the coordinates system used, the 3 PMTs detecting the SHG signal at 0°, 45° and 90° and an example of the generated images using starch.
Fig. 2
Fig. 2 Results for a) starch (SHG active molecule is amylopectin), b) collagen, c) muscle (active molecule is myosin) and d) mitotic spindles (active molecule is α-β tubulin heterodimer). Every panel from a) to d) shows the three acquired images (i.e. from each of the PMTs) required for the SS-PSHG analysis (top row), the retrieved main axis orientation, anisotropy parameter and effective orientation of the nonlinear dipole of the macromolecule (center) and the corresponding image histograms (bottom)..
Fig. 3
Fig. 3 SS-PSHG analysis of moving C. elegans worm. The analyzed region corresponds to the pharynx with the most posterior lobe (terminal bulb) at the bottom of the image, framed by the body walls (the two lateral lines). Eight consecutive frames at 1 frame per second, with a size of 500x500 pixels are shown. a) Total emitted SHG signal. b) Mapping of the thick filaments orientation in every pixel measured with respect the vertical axis. c) Mapping of the anisotropy parameter. Note the change in color in the last frame (red square) due to the contraction of the left worm body wall.
Fig. 4
Fig. 4 Monitoring of muscular contraction and relaxation with SS-PSHG. The images (500x500 pixels) of the posterior lobe of a C. elegans worm waking up after anesthesia are analyzed for 30 seconds. a) The total detected SHG signal (left), the thick filament orientation (center) and the anisotropy parameter (right) with the two region of interest (ROI) are shown (Media 1). b) Five frames for ROI 1 showing the changes on the thick filaments orientation (top) and anisotropy parameter (bottom). Note the change in color at second 4. c) Evolution of average values for the thick filaments orientation (top) and anisotropy parameter (bottom) in ROI 1 (left) and ROI 2 (right). Note that an increase on the anisotropy parameter is associated to a contraction process, while a decrease is associated to a relaxation process. Error-bars corresponds to standard deviation within the ROI.

Equations (8)

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μ ν = κζ β νκζ E κ E ζ
χ ijk =N ( i ^ · ν ^ )( j ^ · ν ^ )( k ^ · ν ^ ) β ννν
d 33 d 15 = 2 tan 2 θ e ,
E ω = E 0 ( z ^ ±i x ^ 2 ) e iωt ,
E 2ω P x 2ω x ^ + P z 2ω z ^ = E 0 2 ( x ^ ±i 1 2 ( d 33 d 15 1) z ^ ) e 2ωt
I α 2ω E 0 2 ( sin 2 (φα)+ 1 4 ( d 33 d 15 1 ) 2 cos 2 (φα) )
φ= 1 2 tan 1 { 2 I 45º 2ω I 0º 2ω I 90º 2ω I 0º 2ω I 90º 2ω },
d 33 d 15 =1±2 I 0º 2ω cos 2 φ I 90º 2ω sin 2 φ I 90º 2ω cos 2 φ I 0º 2ω sin 2 φ ,

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