Abstract

We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

© 2005 Optical Society of America

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Corrections

A. Pena, M. Strupler, T. Boulesteix, G. Godeau, and Marie-Claire Schanne-Klein, "Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy: erratum," Opt. Express 13, 6667-6667 (2005)
https://www.osapublishing.org/oe/abstract.cfm?uri=oe-13-17-6667

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Annu. Rev. Phys. Chem. (1)

R. Richards-Kortum and E. Sevick-Muraca, "Quantitative optical spectroscopy for tissue diagnosis," Annu. Rev. Phys. Chem. 47, 555-606 (1996)
[CrossRef] [PubMed]

Biochim. Biophys. Acta (1)

Z. Deyl, K. Macek, M. Adam and Vancikova, "Studies on the chemical nature of elastin fluorescence," Biochim. Biophys. Acta 625, 248-254 (1980)
[PubMed]

Biophys. J. (1)

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)
[CrossRef] [PubMed]

in Advances in Laser and Light Spectrosc (1)

R. A. Davis, H. E. Savage, P. G. Sacks, R. R. Alfano and S. P. Schantz, "The influence of keratin on native cellular fluorescence of human skin," in Advances in Laser and Light Spectroscopy to Diagnose Cancer and Other Diseases III: Optical Biopsy, R. R. Alfano and A. Katzir, Eds., Proc. SPIE 2679, 216-226 (1996)
[CrossRef]

J. Am. Chem. Soc. (1)

T. G. Scott, R. D. Spencer, N. J. Leonard and G. Weber, "Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH and simplified synthetic models," J. Am. Chem. Soc. 92, 687-695 (1969)
[CrossRef]

J. Biomed. Opt. (2)

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[CrossRef] [PubMed]

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[CrossRef] [PubMed]

J. Cell Biol. (1)

R. Eichner and M. Kahn, "Differential extraction of keratin subunits and filaments from normal human epidermis," J. Cell Biol. 110, 1149-1158 (1990)
[CrossRef] [PubMed]

J. Fluorescence (1)

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[CrossRef]

J. Opt. Soc. Am. B (1)

J. Struct. Biol. (1)

G. Cox, E. Kable, A. Jones, I. Fraser, K. Marconi and M. D. Gorrell, "3-dimensional imaging of collagen using second harmonic generation," J. Struct. Biol. 141, 53-62 (2003)
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (4)

Photochem. Photobiol. (1)

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon and N. Kollias, "In vivo fluorescence spectroscopy of nonmelanoma skin cancer," Photochem. Photobiol. 73, 178-183 (2001)
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. (1)

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. USA 100, 7075-7080 (2003)
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning microscopy," Science 248, 73-76 (1990)
[CrossRef] [PubMed]

Trends in Genetics (1)

R. M. Porter and E. B. Lane, "Phenotypes, genotypes and their contributions to understanding keratin function," Trends in Genetics 19, 278-285 (2003)
[CrossRef] [PubMed]

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Figures (3)

Fig. 1.
Fig. 1.

2-photon spectrofluorimeter. WP: rotating half waveplate; L1, L2, L3: lenses; M1, M2: silver mirrors; DM: dichroic mirror; F: optical filters; PMT: photomultiplier tube.

Fig. 2.
Fig. 2.

spectroscopic analysis of the keratin solution in a 100μm optical path fused silica microcell (a): absorption and fluorescence spectra. (b): intensity dependence of the fluorescence signal upon Ti-Sa excitation at 800 nm; the red line correspond to an exponential fit of the experimental data (solid circles). (c) 2PEF spectra recorded at various excitation wavelengths, normalized to the same excitation intensity. (d) 2PEF excitation spectrum; error bar are typically ± 20%, and the absolute scale is determined within a 37% uncertainty.

Fig. 3.
Fig. 3.

skin multiphoton imaging and spectroscopic analysis (a) combined 2PEF (red) and SHG (green) image of a thin histological cut from human normal skin biopsy (b) spectra recorded in relevant areas with the tunable interferential filter (see table 1).

Tables (1)

Tables Icon

Table 1. 2PEF spectral positions in various skin areas

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