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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 10 — Oct. 31, 2007
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Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy

François Tiaho, Gaëlle Recher, and Denis Rouède  »View Author Affiliations


Optics Express, Vol. 15, Issue 19, pp. 12286-12295 (2007)
http://dx.doi.org/10.1364/OE.15.012286


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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

1. Introduction

2. Experimental methods

2.1. Tissue preparation

Muscle tissues were obtained from tadpole tails (stage 32, 37 and 46) of Xenopus laevis [12

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

] animals, gastrocnemius of adult xenopus (national breeding facility of xenopus animals in Rennes, France), gastrocnemius of a four months old Golden retriever dog with Duchenne muscular dystrophy (DMD) (provided by Pr. Y. Cherel, ENV, Nantes, France), gastrocnemius of adult healthy Beagle dog (Biotrial, Rennes, France) and gastrocnemius of 71 years old human female (provided by E. Berton, Department of Pathology, Rennes, France). Collagen-rich tissues were obtained from xenopus tendon and aorta and from epimysium of healthy Beagle dog. Muscles were dissected, fixed over night in PFA 4 % at 4°C, and rinsed at least three times with the appropriate saline buffer. We used either isolated fibers from some adult xenopus or 100 μm-thick slices obtained by slicing horizontally agarose-embedded muscles, glued on the stage of a vibroslicer (LEICA, VT 1200S).

2.2. Imaging system

The setup (PIXEL (http://pixel.univ-rennes1.fr/) platform of EUROPIA, University of Rennes1, France) was based on a Leica TCS SP2 confocal scanning head coupled to a Leica DMIRB inverted microscope with a MAITAI Spectra Physics femtosecond laser. A Leica objective HC-PLAPO 20X (NA=0.7) or an Olympus water immersion objective LUMFL 60X (NA=0.9) were used for applying 10-20 mWof 940 nm excitation at the sample. The SHG signal was collected in a forward direction using either a S23 (NA=0.53) or a multi-immersion S1 (NA=0.90–1.4) Leica condenser. A BG39 bandpass filter and a 470 nm IR filter (10 nm FWHM) were placed before the PMT. The dichroic filter wheel of the microscope was removed and replaced by a computer control PR50CC Newport rotation stage (precision 0.1°) equiped with an achromatic zero-order Quartz-MgF2 half-wave plate in order to adjust the polarization angle of the incident IR electric field without movement of the biological specimen.

3. Experimental results

To determine the effect of myofibrillogenesis on SHG signal, we have studied xenopus tadpoles swimming muscles from 1 (stage 32), 2 (stage 37) and 4 (stage 46) days post fertilization (dpf). These developmental stages coincide with the intense period (stage 26 to stage 36) of myofibrillogenesis that has been shown to be under the control of intracellular calcium in this species [13

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

]. In Fig. 1, we present SHG forward images from thin specimens of xenopus muscles (Fig. 1(a–d)). All the specimens were positionned on the fixed stage of the microscope (YZ plane) of the laboratory coordinate systems (X, Y, Z) with light propagating in the X direction (see the inset of Fig. 1(a)). At embryonic stage 32 (1 dpf) muscle fibers have not fully developed their sarcomeres as indicated by large regions of cells without SHG signal (Fig. 1(a)), and the average myocyte diameter was about 15 µm. At stage 37 (Fig. 1(b)) full development of sarcomers was achieved. At this stage, corresponding also to hatching, no significant modification of the diameter of the myocyte was observed. At 4 dpf, in vivo SHG images reported a significant growth of the myocytes with an average measured diameter of 25 µm (Fig. 1(c)). Overall SHG images from body wall swimming muscles at these developmental stages revealed an increase in the number of sarcomeres and in the size of the striated myocytes. During the developmental stages 32 to 46, this growth is in relation with the size of the animal which increases from 5 mm at stage 32 (1dpf) to 10 mm at stage 46 (4 dpf). For adult xenopus we have chosen the gastrocnemius muscle that is also implicated in locomotion since the swimming tail muscles are lost during metamorphosis of the tadpole at stage 66 (58 dpf). Adult gastrocnemius muscle of xenopus was characterized by bundles of muscle fibers emitting SHG signal whose fibers form a syncytium (with average size 150 µm) in which individual myocyte could not be distinguished (Fig. 1(d)). This syncytial organization of muscle fibers deduced from SHG images was also seen with the gastrocnemius muscles of adult human (Fig. 1(e)) and with a four months old Golden retriever dog with Duchenne muscular dystrophy (DMD) (Fig. 1(f,g)). DMD is a muscle pathology related to a defect of the dystrophin gene with a great reduction of the cytoskeletal dystrophin protein that ultimately leads to portion or total muscle fibers degeneration [14

14. 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]

] and appearance of ectopic collagen (probably type I) in the endomysium (collagen IV rich tissue wrapping each musle fiber) [15

15. 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]

]. The average size of bundles were 68 µm and 41 µm respectively for human and dog. Concerning Golden retriever with DMD, it was easy to find a field of view with SHG emission from both muscle myosin and supposed extracellular matrix as illustrated in Fig. 1(f, g). The collected signal from myosin was characterized by its striated sarcomeric organization whereas the one from the extracellular matrix was more intense and had a fibrous shape. In order to better characterize the difference between the two SHG signals, collagen-riched tissues like adult xenopus tendon or aorta and adult healthy Beagle dog fresh epimysium (probably type III) (Fig. 1(h–j)) were chosen. According to tissue distribution, we expected type I collagen in tendon, type III in aorta and in epimysium (conjonctive tissue wrapping bundle of muscle fibers). In this later case a fresh specimen was used to probe any artifact that could result from the PFA fixing protocol. Type IV collagen is also expected in muscle endomysium but has been shown to lack SHG signal [16

16. M. Strupler, A. M. Pena, and M. Hernest, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15, 4054–4065 (2007), http://www.opticsexpress.org/abstract.cfm?id=131626. [CrossRef] [PubMed]

] and therefore could not explain the observed signal in Fig. 1(f,g).

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. 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. 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).

4. Theoretical analysis

As a rule, second harmonic electric fields E2ω at 2ω originate from nonlinear polarization P2ω(2) EωEω by mixing of intense electric fields Eω at w in the medium [17

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

]. χ (2) is the macroscopic nonlinear susceptibility tensor and assuming that the distribution of myosin and collagen undergoes cylindrical symmetry [7

7. 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, 328–339 (2006). [CrossRef]

, 9

9. 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]

, 10

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

, 18

18. 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]

, 19

19. 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]

] along the main axis Z of the filament, the only nonvanishing components of χ (2), described by a C6 tensor, reduces to χ(2)ZZZ (=χ33), χ(2)iiZ(2)iZi(=χ15) and χ(2)Zii (=χ31) for i=X or Y in the laboratory coordinates (X, Y, Z) [20

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

]. Considering that light is propagating along direction X, one obtains the following equation in the tranverse wave approximation (EωX=0)

PX2ω=0
PY2ω=2χ15EYωEZω
PZ2ω=χ31(EYω)2+χ33(EZω)2.
(1)

The SHG intensity I~[(PY)2+(P2ωZ)2] can be derived from Eq. 1 using EωY=sinα and EωZ=cosα so as to obtain the standard polarization dependence of SHG intensity for polar filaments [9

9. 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]

, 18

18. 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]

]

I2ω[sin22α+(χ31χ15sin2α+χ33χ15cos2α)2].
(2)

We have used a nonlinear least-squares fit with the Levenberg-Marquardt method [21

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

] in order to adjust the fitting parameters χ31/χ15 and χ33/χ15 with the experimental data of Fig. 2(b) and Fig. 3(b) and the values of these parameters for both myosin and collagen are reported in Table 1. One should notice that χ31/χ15 is close to unity fo all myosin- and collagen-rich

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.

table-icon
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χ33=Nsβ<cos3θ>
χ15=χ31=12Nsβ<cosθsin2θ>.
(3)

In this equation, θ is the polar angle of the nonlinear molecule with axis Z, Ns is the number density of active harmonophores and the brackets <> indicates an orientational average over the distribution of molecular orientation. It is useful to notice that Eq. 3 assumes χ15=χ31 and as this equality is verified for almost all of our results, the assumption of a dominant hyperpolarizability coefficient β seems reasonable for both myosin and collagen tissues. Under this assumption, the meaningful orientation parameter D can also be defined from Eq. 3 [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

, 25

25. 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]

]

D=<cos3θ><cosθ>=χ33χ152+χ33χ15=cos2θe.
(4)

D was initially introduced to describe orientation and disorder of molecules at surfaces and interfaces. Regarding θe, this angle was defined in reference [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

] as an effective (or apparent) angle corresponding to the most probable orientation of the active molecules when the distribution of molecular orientation is very narrow [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

, 25

25. 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]

]. These parameters can be used here to evaluate the organization of the harmonophores in myosin- and collagen-rich tissues. The values of D and θe derived from Eq. 4 are thus reported in Table 1 and one should notice a weak dispersion of the data around mean values D≈0.22, θe≈62° and D≈0.42, θe≈49° for respectively myosin-rich and collagen-rich tissues (the result for xenopus aorta was not taken into account since in that case the approximation χ31/χ15 close to unity was not fullfilled). The difference in the values of obtained for the two proteins underlines a distinct harmonophore organization as it will be discussed in the following.

5. Discussion

Mean values of the orientation parameter D≈0.22 and D≈0.42 for respectivelymyosin-rich and collagen-rich tissues were obtained within the analyzed ROIs. For each analyzed specimen, the error on D value was about 5% and this precision was achieved for analyzed ROIs where the average fibril misalignment was less than 5°. The misalignment of fibrils (>5°) in sub-ROIs can be corrected during simulation to fullfill the approximation χ15 = χ31 and therefore to yield the same value of D. Overall, the value of D is independent of the angular orientation of the fibers. For each protein, we obtained a dispersion of the value of D less than 25% for all studied specimens which is far less than the 200% variation between the D values of the two proteins. Therefore, the value of D can be considered as an intrinsic property of each protein and the difference could reflect a different state of orientational disorder. Indeed, it is clear from Eq. 4 that the effective angle θe merges into the angle of maximum probability θo as the distribution function of molecular orientation sharpens. Otherwise, the maximum discrepancy between θe and θo depends on the value of D and on the magnitude of harmonophore orientational disorder [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

]. In order to take into account this disorder, the average in Eq. 4 has to be performed and the distribution function has to be specified. We can use the results of reference [24] to evaluate the possible expanse of this orientational disorder. In this work, it was shown that the use of a uniform distribution of molecular orientation could account for most of the significant results. This distribution is defined for equal probability of orientation angles around a mean value θo and has a full width δ of angular disorder. Using this distribution, the average in Eq. 4 can be performed and a simple analytical relation can be derived [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

]

D(δ,θo)=12(1+cosδcos2θo).
(5)

This equation clearly shows that different values of θo and δ, representing a different situation of orientational disorder, can give the same value of D. Moreover, for any value of δ, qe always underestimates θo (cos2θe=cosδ cos2θo). Equation 5 was defined for an angular variation limited to [0 π/2] what corresponds in our case to a fully stretched (θo=0°) or compressed (θo=90°) helix. The graphical solution of Eq. 5 showing all possible values of θo and δ for fixed value of D is given in Fig. 9 of reference [24

24. 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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

] but it is easy to verify that mean orientation angle θo is ranging from 62° to 69° for myosin (D≈0.22) and 49° to 57° for collagen (D≈0.42) according to the limited angular interval chosen [0, p/2]. The associated disorder is ranging from δ=0° to δ=41° for myosin and δ=0° to δ=67° for collagen. A schematic view of single helix of both myosin and collagen showing mean angle θo and maximum possible angular disorder δ is given in Fig. 4. It is of interest to notice that the mean value of D≈0.42 found for collagen is close to the limit value of 0.5 (θe=45°) which allows maximum possible disorder δ=90° within the model. This possible disorder could originate from the superhelical structure of the two fibrillar proteins. Superhelical assembly requires stabilization forces, and the number and the type of interactions between chains of myosin and collagen superstructures are different [11

11. 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]

]. In the case of collagen triple helix, stabilization is mainly due to hydrogen bonding and there is only one bond per (Gly-X-Y) triplet. Concerning myosin, the interactions between chains are more numerous and more cohesive since there is one hydrogen bond per amino-acid (corresponding to three times more than in collagen). Moreover, the assembly of myosin superhelix is “locked” by hydrophobic and electrostatic interactions. Taken together, these observations suggest that collagen seems to be more flexible than myosin and this reinforces the possibility to have more orientational disorder for this protein.

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].

6. Conclusion

In this report we show from extensive studies performed on amphibian and mammals that myosin and collagen are efficiently discriminated by different signatures of polarization dependence of SHG signal. The deduced orientation angles of harmonophores in myosin-rich and collagen-rich tissues, using conventional model of SHG in fibrillar proteins, match the pitch angles of the single helices of these two proteins. Overall the polarization dependence study of SHG signal appears to be a non-invasive and efficient method for probing molecular organization of endogeneous fibrillar proteins and could be a valuable tool to diagnose diseases involving either myosin or collagen structural disorders.

Acknowledgments

We thanks Région Bretagne, Rennes Métropole, Conseil Général d’Ille-et-Villaine for their financial support and Hélène Mereau, Florence Billon, Amandine Rojo for their technical help.

References and links

1.

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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]

7.

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, 328–339 (2006). [CrossRef]

8.

C. Greenhalgh, N. Prent, C. Green, R. Cisek, A. Major, B. Stewart, and V. Barzda, “Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes,” Appl. Opt. 46, 1852–1859 (2007). [CrossRef] [PubMed]

9.

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]

10.

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

11.

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]

12.

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

13.

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

14.

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]

15.

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]

16.

M. Strupler, A. M. Pena, and M. Hernest, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15, 4054–4065 (2007), http://www.opticsexpress.org/abstract.cfm?id=131626. [CrossRef] [PubMed]

17.

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

18.

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]

19.

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]

20.

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

21.

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

22.

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]

23.

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

24.

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, 8165–8171 (2004), http://www.perso.univ-rennes1.fr/denis.rouede/research/la0491706.pdf. [CrossRef] [PubMed]

25.

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]

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 Å resolution,” Science 266, 75–81 (1994). [CrossRef] [PubMed]

OCIS Codes
(170.0180) Medical optics and biotechnology : Microscopy
(180.0180) Microscopy : Microscopy
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: July 26, 2007
Revised Manuscript: August 27, 2007
Manuscript Accepted: August 30, 2007
Published: September 12, 2007

Virtual Issues
Vol. 2, Iss. 10 Virtual Journal for Biomedical Optics

Citation
François Tiaho, Gaëlle Recher, and Denis Rouède, "Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy," Opt. Express 15, 12286-12295 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-19-12286


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References

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  22. 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]
  23. P. F. Brevet, Surface Second Harmonic Generation, (first edition, Presses Polytechniques et Universitaires Romandes, Lausanne, 1996).
  24. 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]
  25. 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]
  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]

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