## Collagen and myosin characterization by orientation field second harmonic microscopy

Optics Express, Vol. 16, Issue 20, pp. 16151-16165 (2008)

http://dx.doi.org/10.1364/OE.16.016151

Acrobat PDF (2332 KB)

### Abstract

Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor *χ*^{(2)} can be determined for each pixel of the image. The ratio *ρ*=*χ*33/*χ*15 of the principal components of *χ*^{(2)} of collagen and myosin was obtained with the same method, and found within the range 1.6–1.8 and 0.5–0.6 respectively. The orientation of the principal axis of *χ*^{(2)} is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).

© 2008 Optical Society of America

## 1. Introduction

1. K. Kroy, “Elasticity, dynamics and relaxation in biopolymer networks,” Curr. Opin. Colloin. Interface Sci. **11**, 56–84 (2006). [CrossRef]

*in-depth*microstructure of biological tissues, recent attention has been devoted to non-destructive multiphoton microscopy techniques. Nonlinear excitation provides now a large panel of imaging applications [3

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

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

*µm*) to point-scanning microscopy despite of scattering, while drastically reducing out-of-focus photobleaching and phototoxicity [5

5. 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]

*in situ*and

*in vivo*studies. In particular, TPEF is an incoherent process whose contrast is proportional to the concentration of fluorophores, while SHG is a coherent phenomenon that arises from supramolecules deprived of center of inversion (harmonophores) and organized in noncentrosymmetrical mesoscopic structures. Combining TPEF and SHG contrasts, as well as their polarization dependence, give a lot of information about harmonophore organization [7

7. 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]

8. S. Brasselet, V. Le Floch, 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**, 207401–207404 (2004). [CrossRef] [PubMed]

11. S. W. Chu, S. Y. Chen, G. W. Chern, Y. C. Tsai, B. L. Chen, C. K. Lin, and Sun. “Studies of * _{χ}*(2)/

*(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J.*

_{χ}**86**, 3914–3922 (2004). [CrossRef] [PubMed]

*endogenous*TPEF and SHG signals [12

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

5. 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]

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

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

14. T. Boulesteix, E. Beaurepaire, M. 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). http://www.opticsinfobase.org/abstract.cfm?URI=ol-29-17-2031 [CrossRef] [PubMed]

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

*µ*m. The spatial FFT of line sections of length ~65

*µ*m are shown in Fig. 1(h). A sharp peak at ≈0.358

*µ*m

^{-1}(that corresponds to a period of 2.8

*µ*m) is observed for both TPEF and SHG signals, indicating the almost perfect periodicity of the array of sarcomeres along myofibrils. This period is an estimate of the sarcomere length for this sample. For meat, the sarcomere length determines meat tenderness [16].

17. 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]

*χ*

^{(2)}(

*ρ*=

*χ*/

_{zzz}*χ*for axial systems) can be derived from this method by means of histograms. Application to various biological samples containing collagen or myosin emphasizes the versatility of the method.

_{zxx}## 2. Theory of SHG polarization analysis and image analysis

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

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]

17. 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]

*E*

^{2ω}originate from a nonlinear polarization

*P*

^{2ω}induced by mixing of intense electric fields

*E*at frequency

^{ω}*ω*in the medium, as described by the tensorial equality

*P*

^{2ω}

_{α}=

*χ*

^{(2)}

_{αβγ}*E*

^{ω}

_{β}

*E*

^{ω}*, where*

_{γ}*χ*

^{(2)}is the local nonlinear susceptibility tensor, and subscribes α,β,γ refer to the laboratory coordinates (X, Y, Z). The Einstein’s convention for implicit summation of repeated indexes was used. Since the scaffold of a lot of natural soft or hard tissues is composed of fibrils and fibers [20

20. P. Fratzl, “Cellulose and collagen: from fibres to tissues,” Curr. Op. Coll. Int. Sc. **8**, 32–39 (2003). [CrossRef]

*C*

_{∞}symmetry. When Kleinman symmetries [21

21. D. A. Kleinman, “Nonlinear Dielectric Polarization in Optical Media,” Phys. Rev. **126**, 1977–1979 (1962). [CrossRef]

*χ*

^{(2)}has only two independent nonvanishing components

*χ*and

_{zzz}*χ*. From these assumptions, the resulting SHG intensity for a given pixel of the image can be written as [10]

_{zxx}*ψ*to X axis (Fig. 2a). The symmetry axis

*z*

*χ*of

*χ*

^{(2)}is oriented with azimuthal angle

*ϕ*to X axis. The coefficients

*U*,

*V*and

*W*are functions of

*χ*,

_{zzz}*χ*, and are also proportional to the square of both the input intensity

_{zxx}*I*and the local concentration

_{o}*c*(

*r*) of harmonophores. Explicit expressions for a planar geometry and harmonophores of

*C*

_{∞}symmetry were given in [10]. Defining

*I*

_{⊥}∝[

*χ*

_{zxx}*I*

_{o}*c*(

*r*)]

^{2}as the intensity measured when the laser polarization is perpendicular to

*z*, we obtained

_{χ}*ρ*=

*χ*/

_{zzz}*χ*. This parameter will give us information about the structural organization of the harmonophores (Fig. 2b) within the fibril [15

_{zxx}15. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys J. **90**,693–703 (2006). [CrossRef]

17. 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]

*ϕ*and

*ρ*can be obtained at each image pixel from the combination of only four images acquired with input linear polarization

*ψ*=

_{n}*nπ*/4, where n=0,1,2,3. This is the minimum number of images necessary to derive 2

*ϕ*, and the method is analogue to phase cycling in NMR spectroscopy [24

24. C. Odin, “NMR studies of Phase Transitions,” Ann. Rep. NMR Spectr., G. Webb ed (Elsevier/North-Holland, Amsterdam, 2006) 59, 117–205. [CrossRef]

*I*(

_{n}*ϕ*)=

*I*(

*ϕ*,

*ψ*) be the intensity for the n

_{n}*input polarization state*

^{th}*nπ*/4, and combining these four intensities, it is easy to obtain from Eq.(1) that

*ψ*-4

*ϕ*) contributions cancel.

*I*

_{02}(ϕ) and

*I*

_{31}(

*ϕ*) fully determine 2

*ϕ*within the interval [-

*π*,

*π*]. Letting

*c*=cos(2ϕ) and

*s*=sin(2

*ϕ*), angle 2

*ϕ*=

*atan*2(

*s*,

*c*) within [-

*π*,

*π*] is obtained from the four-quadrant generalized inverse tangent function

*atan*2 defined as

*ϕ*is given by

*I*

_{02}and

*I*

_{31}with the assumption that the sign of

*V*is known. Applying the method to each image pixel leads to an image of the orientation

*ϕ*of the symmetry axis

*Z*of

_{χ}*χ*

^{(2)}. If this symmetry axis coincide with the fibril direction, as will be shown in the next sections, then the image of

*ϕ*directly represents the

*orientation field*of the fibril array within the tissue under study. In the following, the method is abbreviated as OF-SHM, for “Orientation Field-Second Harmonic Microscopy”.

*ϕ*can be determined unambiguously even when the values of

*U*,

*V*,

*W*vary from pixel to pixel, as long as the sign of

*V*remains homogeneous over the image. This property explains the robustness of the estimation of

*ϕ*to slight departures from the hypothesis leading to Eq.(1), such as neglecting birefringence effects. Moreover,

*ϕ*is shifted by

*π*/2 if the wrong sign is assigned to

*V*. Using Eq.(3) and the results of [10, 22

22. F. Tiaho, G. Recher, and D. 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/abstract.cfm?URI=oe-15-19-12286 [CrossRef] [PubMed]

23. X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, “Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma,” Opt. Express **16**, 1846–1859 (2008). http://www.opticsinfobase.org/abstract.cfm?URI=oe-16-3-1846 [CrossRef] [PubMed]

*ρ*>1, that is

*V*>0 for type I collagen, while

*ρ*<1 and

*V*<0 for myosin. Thus, in the following, we use the convention that the orientation angle is given by

*V*in our method is equivalent to the choice of the orientation reference in a classical SHG polarization analysis : the orientation of the fibrils is determined from the images to fix a reference angle

*before*performing the polarization analysis. The corresponding hypothesis in our method is to fix the sign of

*V*in order to align the orientation field with the apparent orientation of the fibrils deduced from the contrast of the isotropic image. This hypothesis can be applied to determine the sign of

*V*a posteriori if it is not known a priori.

*V*| is estimated by

*U*can be obtained by averaging pixel by pixel the four previous intensities

*U*=[

*I*

_{0}(

*ϕ*)+

*I*

_{1}(

*ϕ*)+

*I*

_{2}(

*ϕ*)+

*I*

_{3}(

*ϕ*)]/4. Such an isotropic SHG image was already obtained by Gao et al. [25

25. L. Gao, L. Jin, P. Xue, J. Xu, Y. Wang, H. Ma, and D. Chen, “Reconstruction of complementary images in second harmonic generation microscopy,” Opt. Express **14**, 4727–4735 (2006). http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-11-4727 [CrossRef] [PubMed]

*π*/3 and 2

*π*/3. However, at least four polarizations are needed to recover

*ϕ*. Note that

*U*and |

*V*| depend mainly on the laser power, the local harmonophore concentration and on the

*χ*

^{(2)}tensor components, that is on

*ρ*. Therefore, the ratio |

*V*|/

*U*, which is only a function of

*ρ*, leads to estimates of

*ρ*, as will be explained in the last section.

*I*

*(*

_{n}*ϕ*), three operations were performed : (i) the saturated pixels and pixels of zero values of each of the four polarization images were detected and indexed, which generated four sets of indexes. Therefore, the union with no repetition of these four sets of indexes defines the set of indexes of non relevant pixels, that is where any of the four images is zero or saturated. All these pixels were discarded for the calculation of orientation fields; (ii) the typical salt and pepper noise of SHG images is attenuated with a 3×3 uniform or gaussian filter (note that both filters give the same result) ; (iii) Using the same method as in (i), the pixels where

*I*

_{02}or

*I*

_{31}are zero or equal to the maximum value are discarded. After calculation of

*U*,

*V*and ϕ, the pixels where the polarization contrast

*V*or mean intensity

*U*are too small with respect to noise can be assigned a special color in the colormap coding of the contrast of the images to highlight doubtful regions of interest.

## 3. Experimental methods

### 3.1. Experimental setup and imaging conditions

*µs*per pixel for 512×512 images).

#### Sample preparation

*µ*m-thick cover-glass. To avoid dehydration, the edges of the cover-glass were sealed with nail polish.

26. H. Hamburger and H. L. Hamilton “A series of normal stages in the development of the chick embryo,” J.Morphol. **88**, 49–92 (1951). [CrossRef]

## 4. Collagen and myosin fibril orientations

### 4.1. Sheep tendon collagen

*w*and axial

_{xy}*w*extents of the two-photon Point Spread Function (PSF) [4

_{z}4. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic : multiphoton microscopy in the biosciences,” Nat. Biotechnol. **21**, 1369–1377 (2003). [CrossRef] [PubMed]

*µ*m and 2

*µ*m respectively. The value of

*w*matches approximately the pixel size. The four images in Fig. 3(a1–a4) show how the SHG contrast varies with laser polarizations. The polarization contrast is rather high, as expected for a sample with a high degree of order. The isotropic image

_{xy}*U*obtained by averaging the four images is presented in Fig. 3(b). From the value of

*w*of our high-NA objective, it can be deduced that the observed fibrils are mainly in the image plane : the typical length of the fibrils being larger than 40

_{z}*µ*m, the maximum out-of-plane angle is ≈1/20 rad <3 °. Thus, we mainly studied the orientation of the symmetry axis of the nonlinear susceptibility of fibrils lying in the focal plane. The corresponding orientation field derived from Eqs.(5,6,7) is shown in Fig. 3(c), where a small bar centered on the Region of Interest (ROI) represents the local orientation

*ϕ*. This orientation has to be compared with the apparent orientation of the fibrils as they appear in Fig. 3(b).

*U*. Under this assumption, apparent fibrils are manually selected by rectangular ROIs to create elongated masks

*M*, that are presented in Fig. 3(d). Then, for each mask, we calculated : (i) the mean direction of the rectangular mask; (ii) the mean orientation angle

*ϕ*of the SHG nonlinear susceptibility axis within the mask. Circular Statistics [27] gives the following formula

_{M}*R*

^{2}≈0.97. This high

*R*

^{2}value demonstrates that the OF-SHM method provides fibril orientation with a good reliability. These results are fully consistent with our previous study, performed at the same scale, on isolated collagen fibrils in rat liver [17

**229**, 32–38 (2008). [CrossRef] [PubMed]

*w*≈1

_{xy}*µ*m and

*w*≈10

_{z}*µ*m).

*w*(

*ψ*) ~ exp(

*κ*cos(

*ψ*-

*ψ*)), where large concentration parameter

_{m}*κ*indicate small orientation disorder around the mean direction

*ψ*. When

_{m}*κ*≫1, it is is well approximated by a gaussian ~ exp[(

*ψ*-

*ψ*)

_{m}^{2}/2

*σ*

^{2}

*] with*

_{ψ}*κ*=1/

*σ*

^{2}

*ψ*. Both distributions were used to fit the data by a leastsquare method, and give almost indistinguishable results with a square of the correlation coefficient

*R*

^{2}≈0.992 for the two orientations. The best fitting parameters were found to be

*ψ*=41.3°±0.1,

_{m}*σ*=6° and

_{ψ}*ψ*=60.1°±0.1,

_{m}*σ*=6.3° for respectively the reference orientation and the one rotated by 20°. For both orientations, the angular dispersions are identical, while the mean angle is modified by the rotation angle, as expected.

_{ψ}*σ*≈6° is due to at least two contributions : (i) disorientation of the fibrils due to the natural crimp of collagen fibrils ; (ii) angular noise coming from the method. As indicated by Fig. 4(b) and (d), regions of different orientations can be distinguished in the whole image. To try to unravel the different contributions, we also select ROIs of 80×80 pixels. We expect the first contribution to decrease as the distribution of the orientation of the fibrils becomes more peaked while the second one should not vary. The corresponding histograms are presented in Fig. 4(e). Again, the mean angles of both ROIs are shifted by 20°. Since the lowest dispersion value was

_{ψ}*ψ*≈3°, we can infer that the contribution of the OF-SHM method to the angular dispersion is around 3°. These results confirm the potentiality of OF-SHM to measure orientation fields when the fibrils cannot be resolved.

_{m}#### 4.2. Myosin in veal muscle and chicken embryo

*V*<0 gives an orientation field (Fig. 5(c)) that fits the orientation of the apparent fibrils as seen on the isotropic image of Fig. 5(b). The important point shown by Fig. 5(b) is that each bundle contains a series of myofibrils arranged in an almost parallel fashion. The myofibrils can be well resolved, and the mean interfibril distance was estimated to be ~15

*µ*m. This is consistent with the fact that at this stage of the development, myofibrils are not fused yet between them in order to form muscular cells.

*R*

^{2}≈0.93. This example demonstrates that OF-SHM also provides reliable orientation fields for myosin.

*z*obtained by OF-SHM. This observation raises the question of the extrinsic or intrinsic origin of this ~30° misorientation. Extrinsic origins may come from the physical and optical sectioning of the myofibril bundles. For the intrinsic origin, we propose the following interpretation. If the myofibrils are all packed parallel to a given direction with the same starting point, then the sarcomeres will be in phase with each other, and the dark bands will appear perpendicular to the packing direction. However, if the fibrils are shifted by the same increment from each other, the fibrils staying still parallel, the dark bands will be no longer perpendicular to the myofibril orientations. This example points out the interest in measuring the orientation field to interpret images provided by second harmonic microscopy.

_{χ}## 5. Ratios ρ of the nonlinear susceptibility components for collagen and myosin

22. F. Tiaho, G. Recher, and D. 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/abstract.cfm?URI=oe-15-19-12286 [CrossRef] [PubMed]

30. 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). [CrossRef] [PubMed]

*V*/

*U*is independent of the laser intensity, harmonophore concentration, and reads

*ρ*varies from 0 to +∞,

*V*/

*U*(

*ρ*) is a increasing function varying between -4/7 and 4/3. Eq.(11) can be inverted to give

*ρ*>0 as

*r*=|

*V*|/

*U*under the hypothesis that

*ρ*>1 and

*r*=-|

*V*|/

*U*if 0<

*ρ*<1, since only the absolute value |

*V*|/

*U*can be measured from our 4-polarizations analysis.

*ρ*as a function of |

*V*|/

*U*is presented in Fig. 7(b). When

*ρ*>1 (or |

*V*|/

*U*becoming closer to 4/3),

*ρ*is a very strongly increasing function of |

*V*|/

*U*. The relationship is almost linear for small values of |

*V*|/

*U*. In calculating

*ρ*from Eq.(12), the values of |

*r*| that are not in the range 0<|

*r*|<4/7 when

*ρ*<1 and 0<|

*r*|<4/3 when

*ρ*>1 should be discarded. The discrepancy between the measured |

*V*|/

*U*values and these limits are generally due to noise, simplifying assumptions (for instance the hypothesis

*χ*

_{31}/

*χ*

_{15}=1 may not be exactly verified) and also limitations of the model that does not take into account of sample birefringence and axial field components in the vicinity of the geometrical focal point. Note that these assumptions are more compelling for the estimate of

*ρ*than for the estimation of

*ϕ*that does not depend on the value of the coefficients

*U*,

*V*, and

*W*, but only on the sign of

*V*. The transformation of the histogram of |

*V*|/

*U*in Fig. 7(a) into the histogram of

*ρ*in Fig. 7(c) using Eq.(12) is also illustrated using the data of a SHG image of collagen from sheep tendon.

*ρ*over several samples of collagen and myosin. We made the hypothesis that

*ρ*>1 and

*ρ*<1 for collagen and myosin respectively.

*ρ*value, denoted as <

*ρ*>, and standard deviation

*σ*can be calculated from raw data, and the results are summarized in Table 1. We used Eq.(10) to calculate the polar angles corresponding to the values of

_{ρ}*ρ*. However, in order to obtain symmetric confidence

*θ*, we defined

*θ*̄=(

*θ*++

*θ*-)/2 and Δ

*θ*=|

*θ*+-

*θ*-|/2. These results are also reported in Table 1 as

*θ*̄±Δ

*θ*.

*ρ*, as well as the corresponding polar angles

*θ*, are in good agreement with the values reported in literature [10, 11

11. S. W. Chu, S. Y. Chen, G. W. Chern, Y. C. Tsai, B. L. Chen, C. K. Lin, and Sun. “Studies of * _{χ}*(2)/

*(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J.*

_{χ}**86**, 3914–3922 (2004). [CrossRef] [PubMed]

**90**,693–703 (2006). [CrossRef]

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

22. F. Tiaho, G. Recher, and D. 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/abstract.cfm?URI=oe-15-19-12286 [CrossRef] [PubMed]

23. X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, “Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma,” Opt. Express **16**, 1846–1859 (2008). http://www.opticsinfobase.org/abstract.cfm?URI=oe-16-3-1846 [CrossRef] [PubMed]

## 6. Conclusion and perspectives

32. M. L. Concha and R. J. Adams, “Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis,” Development **125**, 983–994 (1998). [PubMed]

## Acknowledgments

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12. | W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb. “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. |

13. | A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Nat. Acad. Sc. |

14. | T. Boulesteix, E. Beaurepaire, M. Sauviat, and M.-C. Schanne-Klein, “Second-harmonic microscopy of unstained living cardiac myocytes:measurements of sarcomere length with 20-nm accuracy,” Opt. Lett. |

15. | 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. |

16. | S. T. Jiang, “Contribution of Muscle Proteinases to Meat Tenderization,” Proc. Natl. Sci. Council, ROC, Part B |

17. | 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. |

18. | S. Roth and I. Freund, “Second harmonic generation in collagen,” J. Chem. Phys. |

19. | R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting Second-Harmonic Generation Images of Collagen I Fibrils,” Biophys. J. |

20. | P. Fratzl, “Cellulose and collagen: from fibres to tissues,” Curr. Op. Coll. Int. Sc. |

21. | D. A. Kleinman, “Nonlinear Dielectric Polarization in Optical Media,” Phys. Rev. |

22. | F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express |

23. | X. Han, R. M. Burke, M. L. Zettel, P. Tang, and E. B. Brown, “Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma,” Opt. Express |

24. | C. Odin, “NMR studies of Phase Transitions,” Ann. Rep. NMR Spectr., G. Webb ed (Elsevier/North-Holland, Amsterdam, 2006) 59, 117–205. [CrossRef] |

25. | L. Gao, L. Jin, P. Xue, J. Xu, Y. Wang, H. Ma, and D. Chen, “Reconstruction of complementary images in second harmonic generation microscopy,” Opt. Express |

26. | H. Hamburger and H. L. Hamilton “A series of normal stages in the development of the chick embryo,” J.Morphol. |

27. | K. V. Mardia and P. E. Jupp, |

28. | P. J. Elbischger, H. Bischof, P. Regitnig, and G. A. Holzapfel, “Automatic analysis of collagen fiber orientation in the outermost layer of human arteries,” Pattern Anal Applic |

29. | M. H. Stromer, D. E. Goll, R. B. Young, R. M. Robson, and F. C. Parrish, “Ultrastructural features of skeletal muscle differentiation and development,” Jr. J. Anim Sci. |

30. | 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 |

31. | O. P. Boryskina, Y. Le Grand, C. Odin, and V. Fleury, “The role of distribution and orientation of collagen fibers in tissue development: study by means of double imaging by two-photon excited fluorescence and second harmonic generation microscopy”, Proc Europ. Microw. Assoc. |

32. | M. L. Concha and R. J. Adams, “Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis,” Development |

**OCIS Codes**

(170.3880) Medical optics and biotechnology : Medical and biological imaging

(160.1435) Materials : Biomaterials

(180.4315) Microscopy : Nonlinear microscopy

**ToC Category:**

Medical Optics and Biotechnology

**History**

Original Manuscript: June 18, 2008

Revised Manuscript: August 26, 2008

Manuscript Accepted: August 29, 2008

Published: September 26, 2008

**Virtual Issues**

Vol. 3, Iss. 11 *Virtual Journal for Biomedical Optics*

**Citation**

Christophe Odin, Thomas Guilbert, Alia Alkilani, Olena P. Boryskina, Vincent Fleury, and Yann Le Grand, "Collagen and myosin characterization by orientation field second harmonic microscopy," Opt. Express **16**, 16151-16165 (2008)

http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151

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