Abstract

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.

© 2002 Optical Society of America

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P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

2001

K. M. Meek and A. J. Quantock, Prog. Retinal Res. 20, 95 (2001).
[CrossRef]

K. M. Meek and N. J. Fullwood, Micron 32, 261 (2001).
[CrossRef]

R. M. Williams, W. R. Zipfel, and W. W. Webb, Curr. Opin. Chem. Biol. 5, 603 (2001).
[CrossRef] [PubMed]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

2000

K. Konig, J. Microsc. (Oxford) 200, 83 (2000).
[CrossRef]

1999

M. Bohnke and B. R. Masters, Prog. Retinal Res. 18, 553 (1999).
[CrossRef]

1997

1995

D. W. Piston, B. R. Masters, and W. W. Webb, J. Microsc. (Oxford) 178, 20 (1995).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1971

Agarwal, A.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Alfano, R. R.

Bohnke, M.

M. Bohnke and B. R. Masters, Prog. Retinal Res. 18, 553 (1999).
[CrossRef]

Book, L. D.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

Campagnola, P. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Celliers, P. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

Cheng, J.-X.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

Coleno, M. L.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Da Silva, L. B.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

de Boeij, W. P.

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Fine, S.

Fullwood, N. J.

K. M. Meek and N. J. Fullwood, Micron 32, 261 (2001).
[CrossRef]

George, S. C.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Guo, Y.

Haastert, P. J. M.

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Hansen, W. P.

Harris, D.

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Ho, P. P.

Hoppe, P. E.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Kim, B.-M.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

Konig, K.

K. Konig, J. Microsc. (Oxford) 200, 83 (2000).
[CrossRef]

Liu, F.

Malone, C. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Masters, B. R.

M. Bohnke and B. R. Masters, Prog. Retinal Res. 18, 553 (1999).
[CrossRef]

D. W. Piston, B. R. Masters, and W. W. Webb, J. Microsc. (Oxford) 178, 20 (1995).
[CrossRef]

Maurice, D. M.

D. M. Maurice, in The Eye, H. Davison, ed. (Academic, Orlando, Fla., 1984), pp. 1–158.
[CrossRef]

Meek, K. M.

K. M. Meek and A. J. Quantock, Prog. Retinal Res. 20, 95 (2001).
[CrossRef]

K. M. Meek and N. J. Fullwood, Micron 32, 261 (2001).
[CrossRef]

Millard, A. C.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Mohler, W. A.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Piston, D. W.

D. W. Piston, B. R. Masters, and W. W. Webb, J. Microsc. (Oxford) 178, 20 (1995).
[CrossRef]

Potma, E. O.

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Quantock, A. J.

K. M. Meek and A. J. Quantock, Prog. Retinal Res. 20, 95 (2001).
[CrossRef]

Reiser, K. M.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

Rubenchik, A. M.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

Sacks, P.

Savage, H.

Schantz, S.

Stoller, P.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Sun, C.-H.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Terasaki, M.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Tromberg, B. J.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Volkmer, A.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

Wallace, V. P.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Webb, W. W.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

R. M. Williams, W. R. Zipfel, and W. W. Webb, Curr. Opin. Chem. Biol. 5, 603 (2001).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, J. Microsc. (Oxford) 178, 20 (1995).
[CrossRef]

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wiersma, D. A.

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, Curr. Opin. Chem. Biol. 5, 603 (2001).
[CrossRef] [PubMed]

Wu, W.-Y.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Xie, X. S.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

Zhadin, N.

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, Curr. Opin. Chem. Biol. 5, 603 (2001).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, Biophys. J. 82, 3330 (2002).
[CrossRef] [PubMed]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 81, 493 (2002).
[CrossRef]

Curr. Opin. Chem. Biol.

R. M. Williams, W. R. Zipfel, and W. W. Webb, Curr. Opin. Chem. Biol. 5, 603 (2001).
[CrossRef] [PubMed]

J. Biomed. Opt.

P. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da Silva, J. Biomed. Opt. 7, 1 (2002).
[CrossRef]

J. Microsc. (Oxford)

K. Konig, J. Microsc. (Oxford) 200, 83 (2000).
[CrossRef]

D. W. Piston, B. R. Masters, and W. W. Webb, J. Microsc. (Oxford) 178, 20 (1995).
[CrossRef]

J. Phys. Chem. B

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 105, 1277 (2001).

Micron

K. M. Meek and N. J. Fullwood, Micron 32, 261 (2001).
[CrossRef]

Opt. Lett.

Proc. Nat. Acad. Sci. USA

E. O. Potma, W. P. de Boeij, P. J. M. Haastert, and D. A. Wiersma, Proc. Nat. Acad. Sci. USA 98, 1577 (2001).
[CrossRef]

Prog. Retinal Res.

K. M. Meek and A. J. Quantock, Prog. Retinal Res. 20, 95 (2001).
[CrossRef]

M. Bohnke and B. R. Masters, Prog. Retinal Res. 18, 553 (1999).
[CrossRef]

Science

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Tissue Eng.

A. Agarwal, M. L. Coleno, V. P. Wallace, W.-Y. Wu, C.-H. Sun, B. J. Tromberg, and S. C. George, Tissue Eng. 7, 191 (2001).
[CrossRef] [PubMed]

Other

D. M. Maurice, in The Eye, H. Davison, ed. (Academic, Orlando, Fla., 1984), pp. 1–158.
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Spectra of nonlinear optical signals generated in the cornea by multiphoton microscopy. A, Spectrum of cellular fluorescence following two-photon absorption from the epithelia with an incident laser wavelength of 750 nm. A representative image of the corneal epithelia is shown in the inset. B, Spectrum of SHG in the collagen matrix from an acellular region of the corneal stroma. A representative SHG image of an acellular region of the corneal stroma is shown in the inset. The incident wavelength is 800 nm. The scale bars represent 8 µm.

Fig. 2
Fig. 2

A, Three-dimensional reconstruction of the corneal stroma from a stack of MPM images taken in 200-nm increments. B, H&E stained en face formalin-fixed section of the corneal stroma. C, H&E stained cross section of the corneal stroma. Asterisks demark voids similar to those seen in the MPM image stack of Fig. 2A.

Fig. 3
Fig. 3

Spectral filtering of biological components in the corneal stroma with an excitation wavelength of 800 nm. A, Image of a keratocyte in the corneal stroma from a depth of 180 µm. B, Isolation of the keratocyte from the collagen matrix by use of a bandpass filter at 520 nm. C, Imaging of the collagen matrix without the keratocyte by use of a bandpass filter at 400 nm. D, H&E stained en face section of the corneal stroma for comparison. The keratocytes appear blue in the histology.

Fig. 4
Fig. 4

Polarization dependence of SHG in the collagen matrix proteins of the corneal stroma. A, The relative intensity of the second harmonic is greater for fibers aligned in the vertical direction along the electric field of the laser. B, Multiphoton microscopy image of the same imaging plane by use of an orthogonal incident laser polarization.

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