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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 2 — Feb. 1, 2011
  • pp: 365–373

Fast nonlinear spectral microscopy of in vivo human skin

Arjen N. Bader, Ana-Maria Pena, C. Johan van Voskuilen, Jonathan A. Palero, Frédéric Leroy, Anne Colonna, and Hans C. Gerritsen  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 2, pp. 365-373 (2011)

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An optimized system for fast, high-resolution spectral imaging of in vivo human skin is developed and evaluated. The spectrograph is composed of a dispersive prism in combination with an electron multiplying CCD camera. Spectra of autofluorescence and second harmonic generation (SHG) are acquired at a rate of 8 kHz and spectral images within seconds. Image quality is significantly enhanced by the simultaneous recording of background spectra. In vivo spectral images of 224 × 224 pixels were acquired, background corrected and previewed in real RGB color in 6.5 seconds. A clear increase in melanin content in deeper epidermal layers in in vivo human skin was observed.

© 2011 OSA

OCIS Codes
(170.1870) Medical optics and biotechnology : Dermatology
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics
(180.2520) Microscopy : Fluorescence microscopy
(190.1900) Nonlinear optics : Diagnostic applications of nonlinear optics
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:
Dermatological Applications

Original Manuscript: October 8, 2010
Revised Manuscript: December 17, 2010
Manuscript Accepted: January 14, 2100
Published: January 18, 2011

Arjen N. Bader, Ana-Maria Pena, C. Johan van Voskuilen, Jonathan A. Palero, Frédéric Leroy, Anne Colonna, and Hans C. Gerritsen, "Fast nonlinear spectral microscopy of in vivo human skin," Biomed. Opt. Express 2, 365-373 (2011)

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  1. C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996). [CrossRef] [PubMed]
  2. B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997). [CrossRef] [PubMed]
  3. K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000). [CrossRef] [PubMed]
  4. K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003). [CrossRef] [PubMed]
  5. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003). [CrossRef] [PubMed]
  6. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005). [CrossRef] [PubMed]
  7. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006). [CrossRef] [PubMed]
  8. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J. 93(3), 992–1007 (2007). [CrossRef] [PubMed]
  9. A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007). [CrossRef] [PubMed]
  10. M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007). [CrossRef] [PubMed]
  11. M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008). [CrossRef] [PubMed]
  12. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  13. D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995). [PubMed]
  14. A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Schanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13(16), 6268–6274 (2005). [CrossRef] [PubMed]
  15. N. Ramanujam, “Fluorescence spectroscopy in vivo,” in Encyclopedia of Analytical Chemistry (Wiley, 2000), pp. 20–56.
  16. S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002). [CrossRef] [PubMed]
  17. E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol. 18(6), 509–515 (2009). [CrossRef] [PubMed]
  18. K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. S. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol. 70(2), 146–151 (1999). [PubMed]
  19. G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003). [CrossRef] [PubMed]
  20. J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008). [CrossRef] [PubMed]
  21. A. J. Radosevich, M. B. Bouchard, S. A. Burgess, B. R. Chen, and E. M. C. Hillman, “Hyperspectral in vivo two-photon microscopy of intrinsic contrast,” Opt. Lett. 33(18), 2164–2166 (2008). [CrossRef] [PubMed]
  22. J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998). [CrossRef]
  23. L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. So, “Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra,” J. Biomed. Opt. 10(2), 024016 (2005). [CrossRef] [PubMed]
  24. V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75(4), 2015–2024 (1998). [CrossRef] [PubMed]
  25. F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008). [CrossRef] [PubMed]
  26. K. König, “Clinical multiphoton tomography,” J Biophotonics 1(1), 13–23 (2008). [CrossRef] [PubMed]
  27. K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000). [CrossRef]
  28. K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010). [CrossRef] [PubMed]

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