Confocal and nonlinear optical microscopies have been applied for dermatological studies because of their capability to provide sub-surface three-dimensional images with sub-µm spatial resolutions. Optical signal degradation as the imaging plane being moved toward deeper regions in skin specimens is the key factor that limits the observation depth for the laser scanning based linear or nonlinear imaging modalities. In this article, we studied the signal degradation in fixed human skin specimens using reflection confocal microscopy and higher-harmonic optical microscopy based on a Cr:forsterite femtosecond laser centered at 1230-nm. By analyzing the optical properties through these linear and nonlinear imaging modalities, we found that the optical signal degradation in the studied human skin specimen is dominated by the distortion of the point spread function.

© 2006 Optical Society of America

1. 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 coherent tomography," Science 276, 2037-2039 (1997). [CrossRef] [PubMed]
2. 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]
3. B. Povazay, K. Bizheva, A. Unterhuber, B. Hermann, H. Sattmann, A. F. Fercher, W. Drexler, A. Apolonski, W. J. Wadsworth, J. C. Knight, P. St. J. Russell, M. Vetterlein, and E. Scherzer, "Submicron axial resolution optical coherence tomography," Opt. Lett. 27, 1800-1802 (2002). [CrossRef]
4. M. Minsky, Microscopy apparatus. U.S. Patent 03013467 (1957).
5. C. J. R. Sheppard and D. M. Shotton, Confocal Laser Scanning Microscopy (BIOS Scientific Publisher, Oxford, 1997).
6. 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). [CrossRef] [PubMed]
7. 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). [CrossRef] [PubMed]
8. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, and S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997). [CrossRef]
9. 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]
10. S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Y.-J. Zhang, H.-L. Liu, and C.-K. Sun, "Optical biopsy of fixed human skin with backward-collected optical harmonics signals," Opt. Express 13, 8231-8242 (2005). [CrossRef] [PubMed]
11. B. Selkin, M. Rajadhyaksha, S. González, and R. G. Langley RG, "In vivo confocal microscopy in dermatology," Dermatol. Clin. 19369-77, ix-x (2001).
12. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, 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]
13. J. A. Veiro, and P. G. Cummins, "Imaging of skin epidermis from various origins using confocal laser scanning microscopy," Dermatology 189: 16-22 (1994). [CrossRef] [PubMed]
14. W. Denk, J. H. Strickler, W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). [CrossRef] [PubMed]
15. V. R. Daria, C. Saloma, and S. Kawata, "Excitation with a focused, pulsed optical beam in scattering media: diffraction effects," Appl. Opt. 39, 5244-5255 (2000). [CrossRef]
16. M. Gu, X. Gan, A. Kisteman, and M. G. Xu, "Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media," Appl. Phys. Lett. 77, 1551-1553 (2000). [CrossRef]
17. A. Egner and S. Hell, "Equivalence of the Huygens-Fresnel and Debye approach for the calculation of high aperture pointspread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999). [CrossRef]
18. R. R. Anderson, and J. A. Parrish, "The optics of human skin," J. Invest. Dermatol. 77, 13-19 (1981). [CrossRef] [PubMed]
19. 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. Bio. 147, 19-30 (2004). [CrossRef]
20. 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 noninvasive multi-harmonic generation microscopy," Opt. Express 11, 3093-3099 (2003). [CrossRef] [PubMed]
21. P. N. Prasad, Introduction to Biophotonics (John Wiley & Sons, Hoboken, 2003).
22. 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 a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1921 (2001). [CrossRef]
23. D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999). [CrossRef] [PubMed]
24. J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, "Third harmonic generation microscopy," Opt. Express 3, 315-324 (1999). [CrossRef]
25. 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," Biophy. J. 81, 493-508 (2002). [CrossRef]
26. 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]
27. H. Elias, J. E. Pauly, and E. R. Burns, Histology and Human Microanatomy, 4th Edition, Piccin Medical Books (Wiley, New York, 1978).
28. M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, "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]
29. S. González and Z. Tannous, "Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma," J. Am. Acad. Dermatol. 47, 869-874 (2002). [CrossRef] [PubMed]
30. T. Yamashita, T. Kuwahara, S. González, and M. Takahashi, "Non-invasive visualization of melanin and melanocytes by reflectance-mode confocal microscopy," J. Invest. Dermotal. 124, 235-240 (2005). [CrossRef]
31. A. Dunn and R. Richards-Kortum, "Three-dimensional computation of light scattering from cells," IEEE J. Sel. Top. Quantum Electron. 2, 898-905 (1996). [CrossRef]
32. H. Beyer, Handbuch der Mikroskopie, 2nd Edition (VEB Verlag Technik, Berlin, 1985).
33. J. Squier, and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001). [CrossRef]
34. J. Mertz and L. Moreaux, "Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Comm. 196, 325-330 (2001). [CrossRef]
35. W.H. Press, S.A. Teukolsky, W.T. Vetterling, and B.P. Flannery, Numerical Recipes in C (Cambridge Univ. Press, Cambridge, 1992).

Appl. Opt.

• V. R. Daria, C. Saloma, and S. Kawata, "Excitation with a focused, pulsed optical beam in scattering media: diffraction effects," Appl. Opt. 39, 5244-5255 (2000). [CrossRef]

Appl. Phys. Lett.

• M. Gu, X. Gan, A. Kisteman, and M. G. Xu, "Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media," Appl. Phys. Lett. 77, 1551-1553 (2000). [CrossRef]

Biophy. 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," Biophy. J. 81, 493-508 (2002). [CrossRef]

Dermatol. Clin.

• B. Selkin, M. Rajadhyaksha, S. González, and R. G. Langley RG, "In vivo confocal microscopy in dermatology," Dermatol. Clin. 19369-77, ix-x (2001).

Dermatology

• J. A. Veiro, and P. G. Cummins, "Imaging of skin epidermis from various origins using confocal laser scanning microscopy," Dermatology 189: 16-22 (1994). [CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron.

• A. Dunn and R. Richards-Kortum, "Three-dimensional computation of light scattering from cells," IEEE J. Sel. Top. Quantum Electron. 2, 898-905 (1996). [CrossRef]

J. Am. Acad. Dermatol.

• S. González and Z. Tannous, "Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma," J. Am. Acad. Dermatol. 47, 869-874 (2002). [CrossRef] [PubMed]

J. Invest. Dermatol.

• M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, "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]
• R. R. Anderson, and J. A. Parrish, "The optics of human skin," J. Invest. Dermatol. 77, 13-19 (1981). [CrossRef] [PubMed]
• M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, 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]

J. Invest. Dermotal.

• T. Yamashita, T. Kuwahara, S. González, and M. Takahashi, "Non-invasive visualization of melanin and melanocytes by reflectance-mode confocal microscopy," J. Invest. Dermotal. 124, 235-240 (2005). [CrossRef]

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, Part 3, 190-200 (2002). [CrossRef] [PubMed]
• A. Egner and S. Hell, "Equivalence of the Huygens-Fresnel and Debye approach for the calculation of high aperture pointspread functions in the presence of refractive index mismatch," J. Microsc. 193, 244-249 (1999). [CrossRef]

J. Struct. Bio.

• 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. Bio. 147, 19-30 (2004). [CrossRef]

Opt. Comm.

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

Opt. Express

• 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 noninvasive multi-harmonic generation microscopy," Opt. Express 11, 3093-3099 (2003). [CrossRef] [PubMed]
• D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999). [CrossRef] [PubMed]
• J. A. Squier, M. Muller, G. J. Brakenhoff, and K. R. Wilson, "Third harmonic generation microscopy," Opt. Express 3, 315-324 (1999). [CrossRef]
• S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Y.-J. Zhang, H.-L. Liu, and C.-K. Sun, "Optical biopsy of fixed human skin with backward-collected optical harmonics signals," Opt. Express 13, 8231-8242 (2005). [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). [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). [CrossRef] [PubMed]

Opt. Lett.

• Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, and S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997). [CrossRef]
• 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]
• 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]
• B. Povazay, K. Bizheva, A. Unterhuber, B. Hermann, H. Sattmann, A. F. Fercher, W. Drexler, A. Apolonski, W. J. Wadsworth, J. C. Knight, P. St. J. Russell, M. Vetterlein, and E. Scherzer, "Submicron axial resolution optical coherence tomography," Opt. Lett. 27, 1800-1802 (2002). [CrossRef]
• 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 a femtosecond Cr:forsterite laser," Opt. Lett. 26, 1909-1921 (2001). [CrossRef]

Piccin Medical Books

• H. Elias, J. E. Pauly, and E. R. Burns, Histology and Human Microanatomy, 4th Edition, Piccin Medical Books (Wiley, New York, 1978).

Rev. Sci. Instrum.

• J. Squier, and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001). [CrossRef]

Science

• 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 coherent tomography," Science 276, 2037-2039 (1997). [CrossRef] [PubMed]
• W. Denk, J. H. Strickler, W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990). [CrossRef] [PubMed]

Other

• M. Minsky, Microscopy apparatus. U.S. Patent 03013467 (1957).
• C. J. R. Sheppard and D. M. Shotton, Confocal Laser Scanning Microscopy (BIOS Scientific Publisher, Oxford, 1997).
• P. N. Prasad, Introduction to Biophotonics (John Wiley & Sons, Hoboken, 2003).
• W.H. Press, S.A. Teukolsky, W.T. Vetterling, and B.P. Flannery, Numerical Recipes in C (Cambridge Univ. Press, Cambridge, 1992).
• H. Beyer, Handbuch der Mikroskopie, 2nd Edition (VEB Verlag Technik, Berlin, 1985).

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