Abstract

Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation (SHG) imaging. SHG imaging revealed that corneal collagen fibrils are regularly packed as a polycrystalline lattice, accounting for the transparency of cornea. In contrast, scleral fibrils possess inhomogeneous, tubelike structures with thin hard shells, maintaining the high stiffness and elasticity of the sclera.

© 2005 Optical Society of America

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References

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Appl. Opt.

Biophy. J.

R.M. Williams, W.R. Zipfel and W.W. Webb, �??Interpreting Second-Harmonic Generation Images of Collagen I Fibrils," Biophy. J. 88, 1377-1386 (2005)
[CrossRef]

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,�?? Biophy. J. 50, 693-712 (1986)
[CrossRef]

Biophys. J.

T. Gutsmann, G.E. Fantner, M. Venturoni, et al. �??Evidence that Collagen Fibrils in Tendons Are Inhomogeneously Structured in a Tubelike Manner," Biophys. J. 84, 2593-2598 (2003).
[CrossRef] [PubMed]

Invest. Ophthalmol. Vis. Sci.

Y. Komai, T. Ushiki, �??The three-dimensional organization of collagen fibrils in the human cornea and sclera,�??, Invest. Ophthalmol. Vis. Sci. 32, 2244-2258 (1991).
[PubMed]

C. Boote, S. Dennis, R.H. Newton, H. Puri, and K.M. Meek, �??Collagen fibrils appear more closely packed in the prepupillary cornea: optical and biomechanical implications,�?? Invest. Ophthalmol. Vis. Sci. 44, 2941-2948 (2003).
[CrossRef] [PubMed]

J. Biomed. Opt.

P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis and L.M. Loew, �??Second-harmonic Imaging Microscopy of Living Cells," J. Biomed. Opt. 6, 277-286 (2001)
[CrossRef] [PubMed]

M. Han, L. Zickler, G. Giese, F. Loesel, M. Walter and J. Bille, "Second Harmonic Corneal Imaging after femtosecond laser surgery�??, J. Biomed. Opt. 9, 760-766 (2004)
[CrossRef]

J. Opt. Soc. Am B

L. Moreaux, O. Sandre, and J. Mertz, �??Membrane imaging by second-harmonic generation microscopy,�?? J. Opt. Soc. Am B 17, 1685-1694 (2000)
[CrossRef]

J. Cheng, A. Volkmer, and X.S. Xie, �??Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy," J. Opt. Soc. Am B 19, 1363-1375 (2002)
[CrossRef]

J. Opt. Soc. Am.

J. Physiol.

D.M. Maurice, �??The structure and transparency of the cornea," J. Physiol. 136, 263-286 (1957).
[PubMed]

J. Struct. Bio.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi and M. D. Gorrell, �??3-Dimensional Imaging of Collagen Using Second Harmonic Generation,�?? J. Struct. Bio., 141, 53-62 (2003)
[CrossRef]

Nature Biotech

W.R. Zipfel, R.M. Williams, and W.W. Webb, �??Nonlinear magic: multiphoton microscopy in the biosciences,�?? Nature Biotech. 21, 1369-1377 (2003)
[CrossRef]

Nature Biotech.

P. J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis and L.M. Loew, �??Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,�?? Nature Biotech. 21, 1356-1360 (2003)
[CrossRef]

Opt. Commun.

S. Roth and I. Freund, �??Coherent Optical Harmonic Generation in Rat-tail,�?? Opt. Commun. 33, 292-296 (1980)
[CrossRef]

J. Mertz, and L. Moreaux, �??Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Commun. 196, 325-330 (2001)
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

A. Zumbusch, G.R. Holtom, & X.S. Xie, �??Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering,�?? Phys. Rev. Lett. 82, 4142-4145 (1999).
[CrossRef]

Science

W. Denk, J.H. Strickler, and W.W. Webb, �??Two-Photon Laser Scanning Fluoresence Microscopy,�?? Science 248, 73-76 (1990)
[CrossRef] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

Schematic drawing of the SHG imaging experimental setup

Fig. 2.
Fig. 2.

SHG imaging of collagen fibrils in (a)cornea and (b)sclera. The femtosecond Ti:Sapphire laser was focused by a 40×(N.A. 0.8) water immersion objective. SHG signals were collected in the forward direction for both cornea and sclera. The image plan is parallel to the cornea/sclera surface. Bars: 20 μm

Fig. 3.
Fig. 3.

SHG imaging of corneal collagen fibrils in (a)forward and (b)backward directions. The fibrillar structures resolved in (a) correspond to collagen bundles which are composed of regularly packed collagen fibrils. Objective: 63×/1.0W, Bars: 10 μm

Fig. 4.
Fig. 4.

SHG imaging of scleral collagen fibrils in (a)forward and (b)backward directions. In contrast to cornea, the backward SHG signals from sclera are significant. Identical structures are revealed by forward and backward SHG imaging. Objective: 63×/1.0W, Bars: 10 μm

Fig. 5.
Fig. 5.

SHG imaging of single scleral collagen fibril in the scleral slice which was manually dissociated from the scleral substrate. (a) and (b) were recorded at different locations. The sharp bends of the collagen fibrils are commonly observed and are indicated by the solid triangles. Objective: 63×/1.0W, Bars: 10 μm

Equations (2)

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Δ k · l 2 Δ ϕ g π
j = 1 n E 2 ω 0 e i Δ k · r j

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