Abstract

We performed Second Harmonic Generation (SHG) imaging microscopy of endogeneous myosin-rich and collagen-rich tissues in amphibian and mammals. We determined the relative components of the macroscopic susceptibility tensor χ(2) from polarization dependence of SHG intensity. The effective orientation angle θe of the harmonophores has been determined for each protein. For myosin we found θe≈62° and this value was unchanged during myofibrillogenesis. It was also independent of the animal species (xenopus, dog and human). For collagen we found θe≈49° for both type I- and type III- rich tissues. From these results we localized the source of SHG along the single helix of both myosin and collagen.

© 2007 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  23. P. F. Brevet, Surface Second Harmonic Generation, (first edition, Presses Polytechniques et Universitaires Romandes, Lausanne, 1996).
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    [CrossRef] [PubMed]
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    [CrossRef]
  26. P. Stoller, B. M. Kim, A. M. Rubenchik, K. M. Reiser and L. B. Da Silva, "Polarization-dependent optical second-harmonic imaging of a rat-tail tendon," J. Biomed. Opt. 7,205-214 (2002).
    [CrossRef] [PubMed]
  27. J. Bella, M. Eaton, B. Brodsky and H. M. Berman, " Crystal and molecular structure of a collagen-like peptide at 1.9 °A resolution," Science 266,75-81 (1994).
    [CrossRef] [PubMed]

2007 (3)

2006 (1)

S. V. Plotnikov, A. C. Millard, P. J. Campagnola andW. A.Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys. J. 90,328-339 (2006).
[CrossRef]

2005 (1)

R. M. Williams, W. R. Zipfel and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88,1377-1386 (2005).
[CrossRef]

2004 (4)

Y. Nakae, P. J. Stoward, T. Kashiyama, M. Shono, A. Akagi, T. Matsuzaki and I. Nonaka, "Early onset of lipofuscin accumulation in dystrophin-deficient skeletal muscles of DMD patients and mdx mice," J.Mol. Histol. 35,489-499, (2004).
[CrossRef] [PubMed]

T. Boulesteix, E. Beaurepaire, M. P. Sauviat and M. C. Schanne-Klein, "Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy," Opt. Lett. 29,2031- 2033 (2004).
[CrossRef] [PubMed]

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vi’e, D. Rou`ede, 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 20,8165-8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf.
[CrossRef] [PubMed]

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

2003 (2)

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]

W. A. Mohler, A. C. Millard and P. J. Campagnola, "Second harmonic generation imaging of endogenous structural proteins," Methods,  29,97-109 (2003).
[CrossRef] [PubMed]

2002 (2)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone and W. A. Mohler, "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues," Biophys. J. 82,493-508 (2002).
[CrossRef]

P. Stoller, B. M. Kim, A. M. Rubenchik, K. M. Reiser and L. B. Da Silva, "Polarization-dependent optical second-harmonic imaging of a rat-tail tendon," J. Biomed. Opt. 7,205-214 (2002).
[CrossRef] [PubMed]

1999 (2)

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. 121,2635-2636 (1999).
[CrossRef]

M. B. Ferrari and N. C. Spitzer, "Calcium signaling in the developing xenopus myotome," Dev. Biol. 213,269- 289 (1999).
[CrossRef] [PubMed]

1998 (1)

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

1994 (1)

J. Bella, M. Eaton, B. Brodsky and H. M. Berman, " Crystal and molecular structure of a collagen-like peptide at 1.9 °A resolution," Science 266,75-81 (1994).
[CrossRef] [PubMed]

1989 (1)

Y. R. Shen, "Surface properties probed by second-harmonic and sum-frequency generation," Nature 337,519- 525 (1989).
[CrossRef]

1986 (1)

I. Freund, M. Deutsch and A. Sprecher, "Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon," Biophys. J. 50,693-712 (1986).
[CrossRef] [PubMed]

1983 (1)

T. F. Heinz, H. W. K. Tom and Y. R. Shen, "Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation," Phys. Rev. A 28,1883-1885 (1983).
[CrossRef]

1979 (1)

S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70,1637-1643 (1979).
[CrossRef]

1970 (1)

I. Freund and L. Kopf, "Long-Range Order in NH4Cl," Phys. Rev. Lett. 24,1017-1021 (1970).
[CrossRef]

1962 (1)

D. A. Kleinman, "Nonlinear dielectric polarization in optical media," Phys. Rev. 126,1977-1979 (1962).
[CrossRef]

Am. J. Physiol. Cell. Physiol. (1)

C. Alexakis, T. Partridge and G. Bou-Gharios, "Implication of the satellite cell in dystrophic muscle fibrosis: a self perpetuating mechanism of collagen over-production," Am. J. Physiol. Cell. Physiol. (2007) (to be published).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (5)

I. Freund, M. Deutsch and A. Sprecher, "Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon," Biophys. J. 50,693-712 (1986).
[CrossRef] [PubMed]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone and W. A. Mohler, "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues," Biophys. J. 82,493-508 (2002).
[CrossRef]

S. V. Plotnikov, A. C. Millard, P. J. Campagnola andW. A.Mohler, "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys. J. 90,328-339 (2006).
[CrossRef]

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

R. M. Williams, W. R. Zipfel and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88,1377-1386 (2005).
[CrossRef]

Dev. Biol. (1)

M. B. Ferrari and N. C. Spitzer, "Calcium signaling in the developing xenopus myotome," Dev. Biol. 213,269- 289 (1999).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (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. 121,2635-2636 (1999).
[CrossRef]

J. Biomed. Opt. (1)

P. Stoller, B. M. Kim, A. M. Rubenchik, K. M. Reiser and L. B. Da Silva, "Polarization-dependent optical second-harmonic imaging of a rat-tail tendon," J. Biomed. Opt. 7,205-214 (2002).
[CrossRef] [PubMed]

J. Chem. Phys. (1)

S. Roth and I. Freund, "Second harmonic generation in collagen," J. Chem. Phys. 70,1637-1643 (1979).
[CrossRef]

J. Struct. Biol. (1)

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

J.Mol. Histol. (1)

Y. Nakae, P. J. Stoward, T. Kashiyama, M. Shono, A. Akagi, T. Matsuzaki and I. Nonaka, "Early onset of lipofuscin accumulation in dystrophin-deficient skeletal muscles of DMD patients and mdx mice," J.Mol. Histol. 35,489-499, (2004).
[CrossRef] [PubMed]

Langmuir (1)

A. Leray, L. Leroy, Y. Le Grand, C. Odin, A. Renault, V. Vi’e, D. Rou`ede, 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 20,8165-8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf.
[CrossRef] [PubMed]

Methods (1)

W. A. Mohler, A. C. Millard and P. J. Campagnola, "Second harmonic generation imaging of endogenous structural proteins," Methods,  29,97-109 (2003).
[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]

Nature (1)

Y. R. Shen, "Surface properties probed by second-harmonic and sum-frequency generation," Nature 337,519- 525 (1989).
[CrossRef]

Opt. Express (1)

Opt. Lett. (1)

Phys. Rev. (1)

D. A. Kleinman, "Nonlinear dielectric polarization in optical media," Phys. Rev. 126,1977-1979 (1962).
[CrossRef]

Phys. Rev. A (1)

T. F. Heinz, H. W. K. Tom and Y. R. Shen, "Determination of molecular orientation of monolayer adsorbates by optical second-harmonic generation," Phys. Rev. A 28,1883-1885 (1983).
[CrossRef]

Phys. Rev. Lett. (1)

I. Freund and L. Kopf, "Long-Range Order in NH4Cl," Phys. Rev. Lett. 24,1017-1021 (1970).
[CrossRef]

Science (1)

J. Bella, M. Eaton, B. Brodsky and H. M. Berman, " Crystal and molecular structure of a collagen-like peptide at 1.9 °A resolution," Science 266,75-81 (1994).
[CrossRef] [PubMed]

Other (4)

P. F. Brevet, Surface Second Harmonic Generation, (first edition, Presses Polytechniques et Universitaires Romandes, Lausanne, 1996).

J. F. Nye, Physical Properties of Crystals, (Oxford University Press, Oxford, 1985).

W. H. Press, B. P. Flannery, S. A. Teukolsky and W. T. Veterlin, Numerical Recipe (Section 14.4), (Cambridge, 1986).

P. D. Nieuwkoop and J. Faber, Table of Xenopus laevis (Daudin), (Garland Publishing Inc, New York, 1967).

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Figures (4)

Fig. 1.
Fig. 1.

Optical sections illustrating SHG images from different muscles and collagen-rich tissues. (a–c): swimming body muscles of developing xenopus tadpoles of respectively 1 day (stage 32), 2 days (stage 37) and 4 days (stage 46) post fertilization. (d): gastrocnemius muscle of adult xenopus. (e): gastrocnemius muscle of a 71 years old human female. (f,g): gastrocnemius muscle and collagen of 4 months old Golden retriever dog with DMD. (h–j): collagen from respectively adult xenopus tendon and aorta and adult healthy Beagle dog muscle. Images (a–e, g) are optical section images of 50×50mm2 while image (f) is a 90×90µm2 XY crossed-section. Note the presence of ectopic collagen in the endomysium (star). Arrowhead indicated a transsected muscle fiber. (g): Arrowhead and star indicated respectively muscle and ectopic collagen fibers. (h): Projection of 100mm thick stack of a 500×500µm2 image. (i,j): Projection of 17mm thick stack of a 500×500µm2 image. Note that image (c) was obtained from in vivo 46 stage xenopus larva and image (j) was obtained from fresh slice of Beagle dog muscle whereas all other images were from PFA-fixed tissues.

Fig. 2.
Fig. 2.

Polarization dependence of the SHG signal of different muscles. (a): SHG optical sections of adult xenopus gastrocnemius muscle illustrating the effect of four different incident polarization angles α(0°, 45°, 90°, 135°) on the emitted signal from the same field of view. Scale bar: 20 µm. Arrows represent the polarization of the incident electric field (0 degree is vertical). (b): normalized SHG signal as a function of the incident polarization angle α for different muscles of different species. Experimental data are represented with different symbols. ♦, ◦ and ▾ from xenopus tadpole body wall muscles of respectively 1 day (stage 32), 2 days (stage 37) and 4 days (stage 46). ▪, ▴ and ● from adult gastrocnemius muscles of respectively xenopus, 71 years old human female and DMD Golden retriever dog. The full lines are drawn using the best fit obtained from Eq. 2. On the inset, a schematic top view is shown. The long axis of myosin filaments for each specimen was oriented along the Z axis of the laboratory coordinates (X, Y, Z).

Fig. 3.
Fig. 3.

Polarization dependence of the SHG signal of different collagen-rich tissues. (a): SHG optical sections of adult xenopus tendon illustrating the effect of four different incident polarization angles α(0°, 45°, 90°, 135°) on the emitted signal from the same field of view. Scale bar: 10 µm. Arrows represent the incident polarization angles α. (b): normalized SHG signal as a function of the incident polarization anglea for different collagen-rich tissues of xenopus and dog. Experimental data are represented with different symbols. ●, ◦ respectively for xenopus tendon and aorta. ▾ and ▪ respectively for epimysium of healthy dog and muscles of DMD dog. Note that data from healthy dog muscle epimysium was from fresh slice whereas all other data were from PFA-fixed tissues. The lines are drawn from the best fit obtained from Eq. 2. On the inset, a schematic top view is shown. The long axis of collagen filaments for each specimen was oriented along the Z axis of the laboratory coordinates (X, Y, Z).

Fig. 4.
Fig. 4.

Schematic view of single helix of myosin (left) and collagen (right). Mean harmonophore orientation angle θo and disorder width δ are shown. According to the model, θo is ranging from 62° to 69° for myosin and 49° to 57° for collagen with maximum disorder width δ=41° (D=0.22, θo =69°) for myosin and δ=67° (D=0.42, θo =57°) for collagen. P and R are helix pitch and radius. For myosin P=5.5 Å, R=2.2 Å and for collagen P=9.5 Å, R=1.5 Å [11, 27].

Tables (1)

Tables Icon

Table 1. Ratio of coefficients χ31/χ15 and χ33/χ15 for myosin-rich and collagen-rich tissues obtained from fit of Eq. 2 with the experimental data of Fig. 2(b) and Fig. 3(b). The orientation parameter D and the effective orientation angle θe are defined by Eq. 4.

Equations (8)

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P X 2 ω = 0
P Y 2 ω = 2 χ 15 E Y ω E Z ω
P Z 2 ω = χ 31 ( E Y ω ) 2 + χ 33 ( E Z ω ) 2 .
I 2 ω [ sin 2 2 α + ( χ 31 χ 15 sin 2 α + χ 33 χ 15 cos 2 α ) 2 ] .
χ 33 = N s β < cos 3 θ >
χ 15 = χ 31 = 1 2 N s β < cos θ sin 2 θ > .
D = < cos 3 θ > < cos θ > = χ 33 χ 15 2 + χ 33 χ 15 = cos 2 θ e .
D ( δ , θ o ) = 1 2 ( 1 + cos δ cos 2 θ o ) .

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