Abstract

This paper reports a facile and effortless method to realize three-dimensional (3D) molecular third-harmonic-generation (THG) microscopy through the technique of resonance enhancement with absorbing dye. Hematoxylin, a popular absorbing stain, is applied as an example to verify the multiphoton resonant enhancement based on the 1230nm excitation light and can selectively enhance THG yield at cell nuclear sites in the studied specimens, serving as a cell nucleus contrast agent. It is concluded that combining THG microscopy with the mature staining technique can readily achieve 3D molecular imaging without using fluorescence.

© 2008 Optical Society of America

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References

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2007

S.-P. Tai, Y. Wu, D.-B. Shieh, L.-J. Chen, K.-J. Lin, C.-H. Yu, S.-W. Chu, C.-H. Chang, X.-Y. Shi, Y.-C. Wen, K.-H. Lin, T.-M. Liu, and C.-K. Sun, Adv. Mater. (Weinheim, Ger.) 19, 4520 (2007).
[CrossRef]

C.-H. Yu, S.-P. Tai, C.-T. Kung, I.-J. Wang, H.-C. Yu, H.-J. Huang, W.-J. Lee, Y.-F. Chan, and C.-K. Sun, Opt. Express 15, 11167 (2007).
[CrossRef] [PubMed]

2006

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, Opt. Express 14, 6178 (2006).
[CrossRef] [PubMed]

D. Debarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, Nat. Methods 3, 47 (2006).
[CrossRef]

2004

C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, J. Struct. Biol. 147, 19 (2004).
[CrossRef] [PubMed]

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, Biophys. J. 86, 3914 (2004).
[CrossRef] [PubMed]

2003

2002

G. Veres, S. Matsumoto, Y. Nabekawa, and K. Midorikawa, Appl. Phys. Lett. 81, 3714 (2002).
[CrossRef]

2000

R. D. Schaller, J. C. Johnson, and R. J. Saykally, Anal. Chem. 72, 5361 (2000).
[CrossRef] [PubMed]

1999

Adv. Mater. (Weinheim, Ger.)

S.-P. Tai, Y. Wu, D.-B. Shieh, L.-J. Chen, K.-J. Lin, C.-H. Yu, S.-W. Chu, C.-H. Chang, X.-Y. Shi, Y.-C. Wen, K.-H. Lin, T.-M. Liu, and C.-K. Sun, Adv. Mater. (Weinheim, Ger.) 19, 4520 (2007).
[CrossRef]

Anal. Chem.

R. D. Schaller, J. C. Johnson, and R. J. Saykally, Anal. Chem. 72, 5361 (2000).
[CrossRef] [PubMed]

Appl. Phys. Lett.

G. Veres, S. Matsumoto, Y. Nabekawa, and K. Midorikawa, Appl. Phys. Lett. 81, 3714 (2002).
[CrossRef]

Biophys. J.

S.-W. Chu, S.-Y. Chen, G.-W. Chern, T.-H. Tsai, Y.-C. Chen, B.-L. Lin, and C.-K. Sun, Biophys. J. 86, 3914 (2004).
[CrossRef] [PubMed]

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, J. Struct. Biol. 147, 19 (2004).
[CrossRef] [PubMed]

Nat. Methods

D. Debarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, Nat. Methods 3, 47 (2006).
[CrossRef]

Opt. Express

Opt. Lett.

Other

R. B. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

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Figures (4)

Fig. 1
Fig. 1

(a) Absorption spectrum of hematoxylin dissolved in water. (b) Schematic diagram showing the two- and three-photon resonant enhancement of the THG process.

Fig. 2
Fig. 2

(a) Combined epi-THG (blue)/epi-SHG (green) image of the nomal human lung before staining. THG signals from curvilinear elastic fibers (arrow) and dotlike lipid bodies are identified. SHG signals come from collage fibers in alveolar walls. Photomultiplier voltage for THG: 1200 V . (b) Combined epi-THG (red)/epi-SHG (green) image after 1 min hematoxylin staining. Multiple ovoid-shaped cell nuclei (circle) are observed. Photomultiplier voltage for THG: 1000 V . (c) Overlaped image of (a) and (b). (d) and (e) Corresponding conventional images of (a) and (b). The color of the cell nuclei after hematoxylin stain is blue-purple in conventional microscopy. Scale bar: 50 μ m .

Fig. 3
Fig. 3

Depth-resolved epi-SHG/epi-THG images of the normal human lung tissue at depths of (a) 8 μ m , (b) 16 μ m , and (c) 24 μ m . Epi-SHG/epi-THG signals before hematoxylin staining are denoted by green/blue. Epi-THG signals after hematoxylin staining are denoted by red. Scale bar: 50 μ m .

Fig. 4
Fig. 4

Hematoxylin-stained epi-THG images of two surgical resected human lung cancer (adenocarcinoma) specimens. (a) and (c) Normal and cancerous parts of the first specimen, respectively. (b) and (d) Normal and cancerous parts of the second specimen, respectively. The size of the cell nuclei is enlarged in the cancerous part, and the cancerous cells are arranged in a circular pattern (acinar pattern), typical for adenocarcinoma. Scale bar: 50 μ m .

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