The confocal laser scanning microscopy and two-photon microscopy was implemented based on a single laser source and an objective lens. We imaged and compared the morphology of identical sites of ex vivo human skin using both microscopes. The back-scattering emission from the sample provided the contrast for the confocal microscopy. The intrinsic autofluorescence and the second harmonic generation were used as the luminescence source for the two-photon microscopy. The wavelength of the Ti:Sapphire laser was tuned at 710 nm, which corresponds to the excitation peak of NADH and FAD in skin tissue. The various cell layers in the epidermis and the papillary dermis were clearly distinguished by both imaging modalities. The two-photon microscopy more clearly visualized the intercellular region and the nucleus of the cell compared to the confocal microscopy. The fibrous structures in the dermis were more clearly resolved by the confocal microscopy. Numerous cells in papillary dermal layer, as deep as 100 µm, were observed in both CLSM and two-photon microscopy. While most previous studies focused on fibrous structure imaging (collagen and elastin fiber) in the dermis, we demonstrated that the combined imaging with the CLSM and two-photon microscopy can be applied for the non-invasive study of the population, distribution and metabolism of papillary dermal cells in skin.

© 2011 Optical Society of Korea

1. M. Minsky, “Microscopy apparatus,” U.S. Patent 3013467(1961).
2. T. Wilson, Confocal Microscopy (Academic Press, San Diego,USA, 1990).
3. J. B. Pawley, Handbook of Biological Confocal Microscopy(Plenum Press, New York, USA, 1990).
4. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photonlaser scanning fluorescence microscopy,” Science 248, 73-76(1990). [CrossRef]
5. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinearmagic: multiphoton microscopy in the biosciences,” Nat.Biotechnol. 21, 1369-1377 (2003). [CrossRef]
6. W. Kim, C. Kim, S. Lee, S. Lim, C. Park, H. Lee, and M.Park, “Particle image velocimetry of the blood flow in amicro-channel using the confocal laser scanning microscope,”J. Opt. Soc. Korea 14, 42-48 (2010). [CrossRef]
7. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin,B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emissionmicroscopy using multiphoton-excited native fluorescenceand second harmonic generation,” Proc. Natl. Acad. Sci.USA 100, 7075-7080 (2003). [CrossRef]
8. M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R.Anderson, and R. H. Webb, “In vivo confocal scanning lasermicroscopy of human skin II: advances in instrumentation and comparison with histology,” J. Investig. Dermatol. 113,293-303 (1999). [CrossRef]
9. B. R. Masters, P. T. C. So, and E. Gratton, “Optical biopsyof in vivo human skin: multiphoton excitation microscopy,”Lasers Med. Sci. 13, 196-203 (1998). [CrossRef]
10. B. Masters and P. So, “Confocal microscopy and multiphotonexcitation microscopy of human skin in vivo,” Opt.Express 8, 2-10 (2001). [CrossRef]
11. K. Konig and I. Riemann, “High-resolution multiphotontomography of human skin with subcellular spatial resolutionand picosecond time resolution,” J. Biomed. Opt. 8, 432 (2003). [CrossRef]
12. M. G. Lin, T. L. Yang, C. T. Chiang, H. C. Kao, J. N.Lee, W. Lo, S. H. Jee, Y. F. Chen, C. Y. Dong, and S. J.Lin, “Evaluation of dermal thermal damage by multiphotonautofluorescence and second-harmonic-generation microscopy,”J. Biomed. Opt. 11, 064006 (2006). [CrossRef]
13. M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, andS. Gonzalez, “Confocal examination of nonmelanoma cancersin thick skin excisions to potentially guide mohs micrographicsurgery without frozen histopathology,” J. Investig.Dermatol. 117, 1137-1143 (2001). [CrossRef]
14. W. L. Chen, C. K. Chou, M. G. Lin, Y. F. Chen, S. W.Jee, H. Y. Tan, T. H. Tsai, K. H. Kim, D. Kim. P. T. C.So, S. J. Lin, and C. Y. Dong, “Single-wavelengthreflected confocal and multiphoton microscopy for tissueimaging,” J. Biomed. Opt. 14, 054026 (2009). [CrossRef]

J. Biomed. Opt.

• K. Konig and I. Riemann, “High-resolution multiphotontomography of human skin with subcellular spatial resolutionand picosecond time resolution,” J. Biomed. Opt. 8, 432 (2003). [CrossRef]
• M. G. Lin, T. L. Yang, C. T. Chiang, H. C. Kao, J. N.Lee, W. Lo, S. H. Jee, Y. F. Chen, C. Y. Dong, and S. J.Lin, “Evaluation of dermal thermal damage by multiphotonautofluorescence and second-harmonic-generation microscopy,”J. Biomed. Opt. 11, 064006 (2006). [CrossRef]
• W. L. Chen, C. K. Chou, M. G. Lin, Y. F. Chen, S. W.Jee, H. Y. Tan, T. H. Tsai, K. H. Kim, D. Kim. P. T. C.So, S. J. Lin, and C. Y. Dong, “Single-wavelengthreflected confocal and multiphoton microscopy for tissueimaging,” J. Biomed. Opt. 14, 054026 (2009). [CrossRef]

J. Investig. Dermatol.

• M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, andS. Gonzalez, “Confocal examination of nonmelanoma cancersin thick skin excisions to potentially guide mohs micrographicsurgery without frozen histopathology,” J. Investig.Dermatol. 117, 1137-1143 (2001). [CrossRef]
• M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R.Anderson, and R. H. Webb, “In vivo confocal scanning lasermicroscopy of human skin II: advances in instrumentation and comparison with histology,” J. Investig. Dermatol. 113,293-303 (1999). [CrossRef]

Journal of the Optical Society of Korea

• W. Kim, C. Kim, S. Lee, S. Lim, C. Park, H. Lee, and M.Park, “Particle image velocimetry of the blood flow in amicro-channel using the confocal laser scanning microscope,”J. Opt. Soc. Korea 14, 42-48 (2010). [CrossRef]

Lasers Med. Sci.

• B. R. Masters, P. T. C. So, and E. Gratton, “Optical biopsyof in vivo human skin: multiphoton excitation microscopy,”Lasers Med. Sci. 13, 196-203 (1998). [CrossRef]

Nat. Biotechnol.

• W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinearmagic: multiphoton microscopy in the biosciences,” Nat.Biotechnol. 21, 1369-1377 (2003). [CrossRef]

Opt. Express

• B. Masters and P. So, “Confocal microscopy and multiphotonexcitation microscopy of human skin in vivo,” Opt.Express 8, 2-10 (2001). [CrossRef]

Proc. Natl. Acad. Sci. USA

• W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin,B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emissionmicroscopy using multiphoton-excited native fluorescenceand second harmonic generation,” Proc. Natl. Acad. Sci.USA 100, 7075-7080 (2003). [CrossRef]

Science

• W. Denk, J. H. Strickler, and W. W. Webb, “Two-photonlaser scanning fluorescence microscopy,” Science 248, 73-76(1990). [CrossRef]

Other

• M. Minsky, “Microscopy apparatus,” U.S. Patent 3013467(1961).
• T. Wilson, Confocal Microscopy (Academic Press, San Diego,USA, 1990).
• J. B. Pawley, Handbook of Biological Confocal Microscopy(Plenum Press, New York, USA, 1990).

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