Abstract

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi-THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.

© 2007 Optical Society of America

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References

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  1. S. M. Mithieux and A. S. Weiss, "Elastin," Adv. Protein Chem. 70, 437-461(2005).
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  3. C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, "Higher harmonic generation microscopy for developmental biology," J. Struct. Biol. 147, 19-30 (2004).
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    [CrossRef]
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  20. T. Boulesteix, A. M. Pena, N. Pages, G. Godeau, M. P. Sauviat, E. Beaurepaire, and M. C. Schanne-Klein, "Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure," Cytometry A 69, 20-26 (2006)
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    [CrossRef] [PubMed]
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    [CrossRef]
  26. I.-J. Wang, F.-R. Hu, P. J. Chen, and C. T. Lin, "Mechanism of abnormal elastin gene express in the piguecular part of pterygia,"Am. J. Pathol. 157, 1269-1276 (2000).
    [CrossRef]
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    [CrossRef]
  29. P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, "Highresolution nonlinear optical imaging of live cells by second harmonic generation," Biophys. J. 77, 3341-3349 (1999).
  30. E. Georgiou, T. Theodossiou, V. Hovhannisyan, K. Politopoulos, G. S. Rapti, and D. Yova, "Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region," Opt. Commun. 176, 253-260(2000).
  31. S. Roth and I. Freund, "Optical second-harmonic scattering in rat-tail tendon," Biopolymers. 20, 1271-1290 (1981).
  32. P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, "Second-harmonic imaging microscopy of living cells," J. Biomed. Optics. 6, 277-286(2001).
  33. M. H. Ross, G. I. Kaye, and W. Pawlina, Histology (Lippincott Williams & Wilkins, 2003), Chap 23.
  34. J. G. Azzopardi, I. Zayid, "Elastic tissue in tumours of salivary glands," J. Pathol. 107, 149-156 (1972).
  35. R. David, A. Buchner, "Elastosis in benign and malignant salivary gland tumors," Cancer 45, 2301-2310 (1980).
  36. C. Isaacson, H. Greeff, J. F. Murray, J. Posen, and A. Schmaman, "Elastosis in malignant tumours," S. Afr. Med. J. 68, 30-32 (1985).
  37. Z. Urban and C. D. Boyd, "Elastic-fiber pathologies: Primary defects in assembly and secondary disorders in transport and delivery," Am. J. Hum. Genet. 67, 4-7 (2000).
  38. M. Fukushima, Y. Fukuda, M. Kawamoto, and N. Yamanaka, "Elastosis in lung carcinoma: Immunohistochemical, ultrasturcture and clinical studies," Pathol. Int. 50, 626-635 (2000).

2006

2005

2004

C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, "Higher harmonic generation microscopy for developmental biology," J. Struct. Biol. 147, 19-30 (2004).

M. Zandvoort, W. Engels, K. Douma, L. Beckers, M. Egbrink, M. Daemen, and D. W. Slaaf, "Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study," J. Vasc. Res. 41, 54-63 (2004).

A. Zoumi, X. Lu, G. S. Kassab, and B. J. Tromberg, "Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy," Biophys J. 87, 2778-2786 (2004).

S.-W. Chu, S.-Y Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, 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

2002

S.-W. Chu, I.-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, "Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy," J. Microsc. 208, 190-200 (2002).

2001

T. Osakabe, M. Hayashi, K. Hasegawa, T. Okuaki, T. M. Ritty, R. P. Mecham, H. Wachi, and Y. Seyama, "Age- and gender-related changes in ligament components," Ann. Clin. Biochem. 38, 527-532 (2001).

T.-M. Liu, S.-W. Chiu, C.-K Sun, B.-L. Lin, P. C. Cheng, and I. Johnson, "Multi-photon scanning microscopy using a femtosecond Cr:forsterite laser," Scanning 23, 249-254 (2001).
[CrossRef]

L. Canioni, S. Rivet, L. Sarger, R. Barille, P. Vacher, and P. Voisin, "Imaging of Ca2+ intracellular dynamics with a third-harmonic generation microscope," Opt. Lett. 26, 515-517 (2001).

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef] [PubMed]

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, "Second-harmonic imaging microscopy of living cells," J. Biomed. Optics. 6, 277-286(2001).

2000

E. Georgiou, T. Theodossiou, V. Hovhannisyan, K. Politopoulos, G. S. Rapti, and D. Yova, "Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region," Opt. Commun. 176, 253-260(2000).

I.-J. Wang, F.-R. Hu, P. J. Chen, and C. T. Lin, "Mechanism of abnormal elastin gene express in the piguecular part of pterygia,"Am. J. Pathol. 157, 1269-1276 (2000).
[CrossRef]

T. Parasassi, W. Yu, D. Durbin, L. Kuriashkina, E. Gratton, N. Maeda, and F. Ursini, "Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides," Free Radic Biol. Med. 28, 1589-1597 (2000).
[CrossRef]

Z. Urban and C. D. Boyd, "Elastic-fiber pathologies: Primary defects in assembly and secondary disorders in transport and delivery," Am. J. Hum. Genet. 67, 4-7 (2000).

M. Fukushima, Y. Fukuda, M. Kawamoto, and N. Yamanaka, "Elastosis in lung carcinoma: Immunohistochemical, ultrasturcture and clinical studies," Pathol. Int. 50, 626-635 (2000).

1999

D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999).

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, "Highresolution nonlinear optical imaging of live cells by second harmonic generation," Biophys. J. 77, 3341-3349 (1999).

1998

1997

1996

H. F. Carvalho and S. R. Taboga, "Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections," Histochem. Cell Biol. 106, 587-592 (1996).

1994

J. N. Bradbeer, M. Miminucci, and P. Bianco, "Giemsa as a fluorescent stain for mineralized bone," J. Histochem. Cytochem. 42, 677-680 (1994).

1985

C. Isaacson, H. Greeff, J. F. Murray, J. Posen, and A. Schmaman, "Elastosis in malignant tumours," S. Afr. Med. J. 68, 30-32 (1985).

1981

S. Roth and I. Freund, "Optical second-harmonic scattering in rat-tail tendon," Biopolymers. 20, 1271-1290 (1981).

1980

R. David, A. Buchner, "Elastosis in benign and malignant salivary gland tumors," Cancer 45, 2301-2310 (1980).

1974

J. G. Azzopardi, R. N. Laurini, "Elastosis in breast cancer," Cancer 33, 174-183 (1974).
[CrossRef]

1972

J. G. Azzopardi, I. Zayid, "Elastic tissue in tumours of salivary glands," J. Pathol. 107, 149-156 (1972).

Adv. Protein Chem.

S. M. Mithieux and A. S. Weiss, "Elastin," Adv. Protein Chem. 70, 437-461(2005).

Am. J. Hum. Genet.

Z. Urban and C. D. Boyd, "Elastic-fiber pathologies: Primary defects in assembly and secondary disorders in transport and delivery," Am. J. Hum. Genet. 67, 4-7 (2000).

Am. J. Pathol.

I.-J. Wang, F.-R. Hu, P. J. Chen, and C. T. Lin, "Mechanism of abnormal elastin gene express in the piguecular part of pterygia,"Am. J. Pathol. 157, 1269-1276 (2000).
[CrossRef]

Ann. Clin. Biochem.

T. Osakabe, M. Hayashi, K. Hasegawa, T. Okuaki, T. M. Ritty, R. P. Mecham, H. Wachi, and Y. Seyama, "Age- and gender-related changes in ligament components," Ann. Clin. Biochem. 38, 527-532 (2001).

Biophys J.

A. Zoumi, X. Lu, G. S. Kassab, and B. J. Tromberg, "Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy," Biophys J. 87, 2778-2786 (2004).

Biophys. J.

S.-W. Chu, S.-Y Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, 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]

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, "Highresolution nonlinear optical imaging of live cells by second harmonic generation," Biophys. J. 77, 3341-3349 (1999).

Biopolymers.

S. Roth and I. Freund, "Optical second-harmonic scattering in rat-tail tendon," Biopolymers. 20, 1271-1290 (1981).

Cancer

R. David, A. Buchner, "Elastosis in benign and malignant salivary gland tumors," Cancer 45, 2301-2310 (1980).

J. G. Azzopardi, R. N. Laurini, "Elastosis in breast cancer," Cancer 33, 174-183 (1974).
[CrossRef]

Chem. Phys. Lett.

G. A. Blab, P. H. M. Lommerse, L. Cognet, G. S. Harms, and T. Schmidt, "Two-photon excitation action cross-sections of the autofluorescent proteins," Chem. Phys. Lett. 350, 71-77 (2001)
[CrossRef] [PubMed]

Cytometry A

T. Boulesteix, A. M. Pena, N. Pages, G. Godeau, M. P. Sauviat, E. Beaurepaire, and M. C. Schanne-Klein, "Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure," Cytometry A 69, 20-26 (2006)
[CrossRef]

Free Radic Biol. Med.

T. Parasassi, W. Yu, D. Durbin, L. Kuriashkina, E. Gratton, N. Maeda, and F. Ursini, "Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides," Free Radic Biol. Med. 28, 1589-1597 (2000).
[CrossRef]

Histochem. Cell Biol.

H. F. Carvalho and S. R. Taboga, "Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections," Histochem. Cell Biol. 106, 587-592 (1996).

J. Biomed. Optics.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, "Second-harmonic imaging microscopy of living cells," J. Biomed. Optics. 6, 277-286(2001).

J. Histochem. Cytochem.

J. N. Bradbeer, M. Miminucci, and P. Bianco, "Giemsa as a fluorescent stain for mineralized bone," J. Histochem. Cytochem. 42, 677-680 (1994).

J. Microsc.

S.-W. Chu, I.-S. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, "Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy," J. Microsc. 208, 190-200 (2002).

J. Opt. Soc. Am. B

J. Pathol.

J. G. Azzopardi, I. Zayid, "Elastic tissue in tumours of salivary glands," J. Pathol. 107, 149-156 (1972).

J. Struct. Biol.

C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, "Higher harmonic generation microscopy for developmental biology," J. Struct. Biol. 147, 19-30 (2004).

J. Vasc. Res.

M. Zandvoort, W. Engels, K. Douma, L. Beckers, M. Egbrink, M. Daemen, and D. W. Slaaf, "Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study," J. Vasc. Res. 41, 54-63 (2004).

Nat. Methods

D. Debarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, "Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy," Nat. Methods 3, 47-53 (2006).

Opt. Commun.

E. Georgiou, T. Theodossiou, V. Hovhannisyan, K. Politopoulos, G. S. Rapti, and D. Yova, "Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region," Opt. Commun. 176, 253-260(2000).

Opt. Express

Opt. Lett.

Pathol. Int.

M. Fukushima, Y. Fukuda, M. Kawamoto, and N. Yamanaka, "Elastosis in lung carcinoma: Immunohistochemical, ultrasturcture and clinical studies," Pathol. Int. 50, 626-635 (2000).

S. Afr. Med. J.

C. Isaacson, H. Greeff, J. F. Murray, J. Posen, and A. Schmaman, "Elastosis in malignant tumours," S. Afr. Med. J. 68, 30-32 (1985).

Scanning

T.-M. Liu, S.-W. Chiu, C.-K Sun, B.-L. Lin, P. C. Cheng, and I. Johnson, "Multi-photon scanning microscopy using a femtosecond Cr:forsterite laser," Scanning 23, 249-254 (2001).
[CrossRef]

Other

S.-Y. Chen, S.-P. Tai, T.-H. Tsai and C.-K. Sun, "Direct backward-emitted third-harmonic generation and its application to clinical microscopy," in Technical Digest of Conference on Laser and Electro-optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2005), Baltimore, MD, USA, paper QMI3 (2005).

M. H. Ross, G. I. Kaye, and W. Pawlina, Histology (Lippincott Williams & Wilkins, 2003), Chap 23.

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Figures (10)

Fig. 1.
Fig. 1.

System setup. (a) Experimental diagram of THG and multiphoton microscopes. (b) Fixation of the nude mouse ear. (c) Acquire images of the test animal under the microscope with a thermal blanket.

Fig. 2.
Fig. 2.

Normalized emission spectra of elastin powder under 1230 nm nonlinear excitation. All emission spectra are with a central wavelength around 655nm. The emission spectra from elastin powder of aorta and lung are denoted in black and red colors respectively; while the emission spectra from elastin solution of aorta and lung are denoted in green and blue colors respectively.

Fig. 3.
Fig. 3.

Intra-tissue emission spectrum measured from the area of the elastic fiber in a rat aorta. SHG signal comes from the collagen closely adjacent to the elastic fiber in the artery media.

Fig. 4.
Fig. 4.

THG and multi-photon fluorescence imaging of human lung tissue and rat aorta. (a) and (b) are simultaneous THG (blue) and endogenous 2PF (magenta) images of a human lung tissue and a rat aorta, respectively. (c) and (d) are simultaneous THG (blue) and eosin-stained exogenous fluorescence (orange) images of a human lung tissue and a rat aorta, respectively. The co-localization of blue and orange colors shows white color. All yellow arrows within four images indicate the location of elastic fibers. Scale bar: 20µm.

Fig. 5.
Fig. 5.

Normalized spectrum of eosin. The emission maximum is around 555nm.

Fig. 6.
Fig. 6.

In vivo horizontal sections of nude mouse elastic cartilage using THG (blue) and endogenous 2PF (magenta) microscopes. (a) Endogenous 2PF image of elastic cartilage adjusted at the same contrast level with Fig. 6(c). PMT acquisition voltage: 3000V. (b)Endogenous 2PF image adjusted at a very low contrast level. PMT acquisition voltage: 3000V. (c) THG image. PMT acquisition voltage: 1200V. (d) Simultaneous THG and endogenous 2PF image. Yellow arrows indicate one of the locations of the elastic fibers. Elastic fibers in the elastic cartilage shows high contrast, high spatial resolution, and distinguishable intensity using in vivo THG microscopy. Scale bar: 20µm.

Fig. 7.
Fig. 7.

(690 KB) A movie of in vivo depth-resolved horizontal sections in elastic cartilage of the nude mouse ear. This movie is composed of 15 horizontal images. The optical depth difference between adjacent images is 2.1 µm. Image size: 80µm×80µm. [Media 1]

Fig. 8.
Fig. 8.

(2.19 MB) A movie of 3-D reconstruction of a sequential set of horizontally sectioned in vivo images from the elastic cartilage of the nude mouse ear. Green arrows indicate one of the locations of the elastic fibers. This movie is composed of 25 horizontal images. The optical depth difference between adjacent images is 2.1 µm. Image size: 120µm×120µm. [Media 2]

Fig. 9.
Fig. 9.

Normalized THG intensity after a linear polarization as a function of the polarizer angle θ relative to the linear polarization angle of the incident excitation. The square data points represent the case that the excitation polarization is parallel (0°) to the elastic fiber. The triangular data points represent the case that the excitation polarization is 45° to the elastic fiber.

Fig. 10.
Fig. 10.

Simultaneous THG (blue) and SHG (green) images of human conjunctiva (a) Image of normal conjunctiva shows distribution of collagen without elastosis. (b) Image of diseased conjunctiva, pterygium, shows elastin proliferation revealed by THG signals. Scale bar: 20µm.

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