Journal of the Optical Society of Korea

Optical Society of Korea (한국광학회)
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Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy

  • Choi, Sang-Hoon (School of Mechanical Engineering, Kyungpook National University) ;
  • Kim, Wi-Han (School of Mechanical Engineering, Kyungpook National University) ;
  • Lee, Yong-Joong (School of Mechanical Engineering, Kyungpook National University) ;
  • Lee, Ho (School of Mechanical Engineering, Kyungpook National University) ;
  • Lee, Weon-Ju (Department of Dermatology, Graduate School of Medicine, Kyungpook National University) ;
  • Yang, Jung-Dug (Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyungpook National University) ;
  • Shim, Jong-Won (Skin Research Institute, Amorepacific Corporation R&D) ;
  • Kim, Jin-Woong (Department of Applied Chemistry, Hanyang University)
  • Received : 2010.11.15
  • Accepted : 2011.01.19
  • Published : 2011.03.25
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Abstract

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 $100\;{\mu}m$, 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.

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References

  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). https://doi.org/10.1126/science.2321027
  5. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinearmagic: multiphoton microscopy in the biosciences,” Nat.Biotechnol. 21, 1369-1377 (2003). https://doi.org/10.1038/nbt899
  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). https://doi.org/10.3807/JOSK.2010.14.1.042
  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). https://doi.org/10.1073/pnas.0832308100
  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). https://doi.org/10.1046/j.1523-1747.1999.00690.x
  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). https://doi.org/10.1007/s101030050074
  10. B. Masters and P. So, “Confocal microscopy and multiphotonexcitation microscopy of human skin in vivo,” Opt.Express 8, 2-10 (2001). https://doi.org/10.1364/OE.8.000002
  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). https://doi.org/10.1117/1.1577349
  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). https://doi.org/10.1117/1.2405347
  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). https://doi.org/10.1046/j.0022-202x.2001.01524.x
  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). https://doi.org/10.1117/1.3247157

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