Abstract

Harmonics-based optical microscopy has been widely applied in biomedical researches due to its noninvasiveness to the studied biomaterials. Due to momentum conservation consideration, most previous studies collect harmonics generation signals in a forward geometry, especially for third harmonic generation signals. However, the adopted forward transmission type geometry is not feasible for future clinical diagnosis. In this paper, first virtual biopsy based on backward propagating optical higher harmonics, combining second harmonic and third harmonic, is demonstrated in the fixed human skin specimens. In our study, third harmonic generation can provide morphologic information including the distribution of basal cells while second harmonic generation can provide distribution of collagen fibers in dermis. Therefore, this technique is ideal for future noninvasive in vivo skin disease examination without dye.

© 2005 Optical Society of America

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References

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  1. <a href= "http://www.skincancerinfo.com/sectionb/biopsy.html">http://www.skincancerinfo.com/sectionb/biopsy.html</a>
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  7. G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, and J. G. Fujimoto, �??In vivo endoscopic optical biopsy with optical coherent tomography,�?? Science 276, 2037-2039 (1997).
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    [CrossRef]
  11. E. H. K. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, �??Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume,�?? Opt. Commun. 104, 223-228 (1994).
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  25. A. T. Yeh, N. Nassif, A. Zoumi, and B. Tromberg, �??Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,�?? Opt. Lett. 27, 2082-2084 (2002).
    [CrossRef]
  26. A. Zoumi, A. T. Yeh, and B. Tromberg, �??Imaging cells and extracellular matrix in vivo by using second harmonic generation and two photon excited fluorescence,�?? Proc. Natl. Acad. Sci. 99, 11014-11019 (2002).
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  37. D. Yelin and Y. Silberberg, �??Laser scanning third-harmonic-generation microscopy in biology,�?? Opt. Express 5, 169-175 (1999). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169</a>
    [CrossRef] [PubMed]
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  40. 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, Part 3, 190-200 (2002).
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Appl. Phys. Lett. (1)

Y. Barad, H. Eizenberg, M. Horowitz, and Y. Silberberg, �??Nonlinear scanning laser microscopy by third harmonic generation,�?? Appl. Phys. Lett. 70, 922-924 (1997).
[CrossRef]

Biophy. J. (1)

P. J. Caspers, G. W. Lucassen, and G. J. Puppels, �??Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin,�?? Biophy. J. 85, 572-580 (2003).
[CrossRef]

Biophys. J. (5)

P. J. Campagnola, M. D. Wei, A. Lewis, L. M. Loew, �??High resolution non-linear optical microscopy of living cells by second harmonic generation,�?? Biophys. J. 77, 3341-3349 (1999).
[CrossRef] [PubMed]

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]

T.-H. Tsai, S.-P. Tai, W.-J. Lee, H.-Y. Huang, Y.-H. Liao and C.-K. Sun, �??Signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,�?? submitted to Biophys. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, �??Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,�?? Biophys. J. 81, 493-508 (2002).
[CrossRef]

B. R. Masters, P. T. C. So, and E. Gratton, �??Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin�?? Biophys. J. 72, 2405-2412 (1997)

CLEO/QELS 2005 (2)

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).

C.-K Sun, S.-W Chu, S.-P. Tai, M. -C. Chan, I - C. Hsiao, C. -H. Lin, Y. -C. Chen, and B. -L. Lin, �??Origin of backward second harmonic generation emission in a biological sample examined by laser scanning microscopes�??, in Technical Digest of Conference on Laser and Electro-optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2005), Baltimore, MD, USA, paper CWP7 (2005).

Exp. Cell Research (1)

U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and J. J. Halbhuber, �??Femtosecond near infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death,�?? Exp. Cell Research 263, 88-97 (2001)
[CrossRef]

J Invest. Dermatol. (1)

L. D. Swindle, S. G. Thomas, M. Freeman, and P. M. Delaney, �??View of normal human skin in vivo as observed using fluorescent fiber-optic confocal microscopic imaging,�?? J Invest. Dermatol. 121, 706-712 (2003).
[CrossRef] [PubMed]

J. Invest. Dermatol. (2)

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, �??In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,�?? J. Invest. Dermatol. 104, 946-952 (1995).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Aderson, R. H. Webb RH, and R. R. Anderson, �??In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,�?? J. Invest. Dermatol. 113, 293-303 (1999).
[CrossRef] [PubMed]

J. Microsc. (1)

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, Part 3, 190-200 (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (2)

J. Phys. Chem. (1)

J. X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, �??An epi-detected coherent anti-stokes Raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,�?? J. Phys. Chem. B 105, 1277-1280 (2001).
[CrossRef]

J. Phys. D: Appl. Phys. (1)

A. Volkmer, �??Vibrational imaging and microspectroscopies based on coherent anti-Stoke Raman scattering microscopy,�?? J. Phys. D: Appl. Phys. 38, R59-R81 (2005).
[CrossRef]

J. Struct. Biol. (1)

C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin and H.-J. Tsai, �??Harmonics generation microscopy for developmental biology,�?? J. Struct. Biol. 147, 19-30 (2004).
[CrossRef] [PubMed]

Nat. Med. (1)

E. Brown, T. Mckee, E. Ditomaso, E. Pluen, B. Seed, Y. Boucher, and R. K. Jain, �??Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,�?? Nat. Med. 9, 796-800 (2003)
[CrossRef] [PubMed]

Opt. Commun. (1)

E. H. K. Stelzer, S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter, and N. Salmon, �??Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume,�?? Opt. Commun. 104, 223-228 (1994).
[CrossRef]

Opt. Express (6)

J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, �??Third harmonic generation microscopy,�?? Opt. Express 3, 315-324 (1998). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-315">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-315</a>
[CrossRef] [PubMed]

P. T. C. So, H. Kim, and I. E. Kochevar, �??Two photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,�?? Opt. Express 3, 339-350 (1998). <a href= "http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-339">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-3-9-339</a>
[CrossRef] [PubMed]

D. Yelin and Y. Silberberg, �??Laser scanning third-harmonic-generation microscopy in biology,�?? Opt. Express 5, 169-175 (1999). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-8-169</a>
[CrossRef] [PubMed]

B. R. Masters, and P. T. C. So, �??Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,�?? Opt. Express 8, 2-10 (2001). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-8-1-2">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-8-1-2</a>
[CrossRef] [PubMed]

S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai and C.-K. Sun, �??In vivo developmental biology study using invasive multi-harmonic generation microscopy,�?? Opt. Express 11, 3093-3099 (2003). <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-23-3093">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-23-3093</a>
[CrossRef] [PubMed]

M. Han, G. Giese, and J. F. Bille, �??Second harmonic generation imaging of collagen fibrils in cornea and sclera,�?? Opt. Express 13, 5791-5797 (2005) <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-13-15-5791">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-13-15-5791</a>
[CrossRef] [PubMed]

Opt. Lett. (7)

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen and B.-L. Lin, �??Multi-harmonic-generation biopsy of skin,�?? Opt. Lett. 28, 2488-2490 (2003).
[CrossRef] [PubMed]

S.-W. Chu, I.-H. Chen, T.-M. Liu, P.-C. Cheng, C.-K.Sun, and B.-L. Lin, �??Multimodal nonlinear spectral microscopy based on femtosecond Cr:foristerite laser,�?? Opt. Lett. 26, 1909-1911 (2001).
[CrossRef]

B. Povazay, K. Bizheva, A. Unterhuber, B. Hermann, H. Sattmann, A. F. Fercher, W. Drexler, A. Apolonski, W. J. Wadsworth, J. C. Knight, P. S. J. Russell, M. Vetterlein, and E. Scherzer, �??Submicron axial resolution optical coherence tomography,�?? Opt. Lett. 27, 1800-1802 (2002).
[CrossRef]

Y. Yasuno, S. Makita, Y. Sutoh, M. Itoh, and T. Yatagai, �??Birefringence imaging of human skin by polarization-sensitive spectral interferometric optical coherent tomography,�?? Opt. Lett. 27, 1803-1805 (2002).
[CrossRef]

A. T. Yeh, N. Nassif, A. Zoumi, and B. Tromberg, �??Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,�?? Opt. Lett. 27, 2082-2084 (2002).
[CrossRef]

K. König K, P. T. C. So, W. W. Mantulin, and E. Gratton, �??Cellular response to near-infrared femtosecond laser pulses in two photon microscope,�?? Opt. Lett. 22, 135-136 (1997).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, �??Second-harmonic tomography of tissues,�?? Opt. Lett. 22, 1323-1325 (1997).
[CrossRef]

Opt. Quantum. Electron. (1)

I-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin, �??Wavelength dependent cell damages in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources,�?? Opt. Quantum. Electron. 34, 1251-1266 (2002).
[CrossRef]

Phys. Rev. Lett. (1)

A. Volkmer, J. X. Cheng, and X. S. Xie, �??Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy,�?? Phys. Rev. Lett. 87, 023901 (2001).
[CrossRef]

Proc. Natl. Acad. Sci. (2)

A. Zoumi, A. T. Yeh, and B. Tromberg, �??Imaging cells and extracellular matrix in vivo by using second harmonic generation and two photon excited fluorescence,�?? Proc. Natl. Acad. Sci. 99, 11014-11019 (2002).
[CrossRef] [PubMed]

G. Peleg, A. Lewis, M. Linial, and L. M. Loew �??Nonlinear optical measurement of membrane potential around single molecules at selected celluar sites,�?? Proc. Natl. Acad. Sci. 96, 6700-6704 (1999).
[CrossRef] [PubMed]

Science (2)

G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, and J. G. Fujimoto, �??In vivo endoscopic optical biopsy with optical coherent tomography,�?? Science 276, 2037-2039 (1997).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, �??Two-photon laser scanning fluorescence microscopy,�?? Science 249, 73-76 (1990).
[CrossRef]

Other (1)

<a href= "http://www.skincancerinfo.com/sectionb/biopsy.html">http://www.skincancerinfo.com/sectionb/biopsy.html</a>

Supplementary Material (2)

» Media 1: MOV (1653 KB)     
» Media 2: MOV (13821 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

Harmonics optical biopsy system setup.

Fig. 2.
Fig. 2.

Vertically sectioned (a) B-SHG, (b) B-THG, and (c) combined HOB images of fixed human skin. B-SHG and B-THG signals are denoted by green and blue colors respectively. From epidermis to dermis of the studied fixed human skin, we can specify coverglass-formalin interface (d), stratum corneum (e), stratum granulosum & spinosum (f) and the basal layer (g) from the B-THG image. In the B-SHG image, the dermal papilla (h) and dermal capillaries (i) are clearly resolved. Image size: 240μm×360μm

Fig. 3.
Fig. 3.

Horizontally sectioned HOB images of fixed human skin at depths of (a) 29μm, (b) 54μm, (c) 90μm, and (d) 156μm from the specimen surface. B-SHG and B-THG signals are denoted by green and blue colors respectively. Using B-HOB, we can identify the morphology of (a) stratum corneum, (b) stratum spinosum, and (c) the basal layer in epidermis where distinctive morphological and optical properties displayed during keratinocyte maturation process could be appreciated. Besides, the capillary networks (e) and the Meissner’s corpuscle (f) located within the dermal papilla could be clearly observed by HOB. These observations indicate that B-HOB could be an ideal platform of noninvasive skin diagnostic tool without staining. Image size: 120μm× 120μm.

Fig. 4.
Fig. 4.

(1.62 MB) Movie of stack of horizontal sections in the fixed human skin (13.5 MB version). Using B-HOB, a series of horizontal sections from epidermis to dermis are demonstrated. The movie is composed of 100 horizontal images. The optical depth difference between adjacent images is 1.5 μm. Image size: 120μm×120μm

Fig. 5.
Fig. 5.

Horizontally sectioned (a-d) B-THG and (e-f) F-THG images taken at different depths inside the epidermis of the fixed human skin specimen. (i-l) are the combined images for comparison. B-THG and F-THG signals are denoted by blue and red colors respectively. Image size: 80μm×80μm

Fig. 6.
Fig. 6.

Horizontally sectioned (a-d) B-SHG and (e-f) F-SHG images taken at different depths inside the dermis of the fixed human skin specimen. (i-l) are the combined images for comparison. B-SHG and F-SHG signals are denoted by green and red colors respectively. Image size: 80μm×80μm

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