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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References

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    [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, 2405–2412 (1997)
    [Crossref] [PubMed]
  3. K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8, 432–439 (2003)
    [Crossref] [PubMed]
  4. 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]
  5. C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28, 2488–2490 (2003)
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  8. D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
    [Crossref] [PubMed]
  9. Y. Wu, P. Xi, J. Y. Qu, T. H. Cheung, and M. Y. Yu, “Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue,” Opt. Express 13, 382–388 (2005)
    [Crossref] [PubMed]
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    [Crossref]
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  12. 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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    [Crossref] [PubMed]
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    [Crossref]
  15. T. Boulesteix, E. Beaurepaire, M.-P. Sauviat, and M. C. Schanne-Klein, “Second harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20 nm accuracy,” Opt. Lett. 29, 2031–2033 (2004)
    [Crossref] [PubMed]
  16. D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
    [Crossref]
  17. L. Moreaux, O. Sandre, M. Blanchard-Desce, and J. Mertz, “Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy,” Opt. Lett. 25, 320–322 (2000)
    [Crossref]
  18. 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]
  19. C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
    [Crossref]
  20. Z. Deyl, K. Macek, M. Adam, and Vancikova, “Studies on the chemical nature of elastin fluorescence,” Biochim. Biophys. Acta 625, 248–254 (1980)
    [PubMed]
  21. R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47, 555–606 (1996)
    [Crossref] [PubMed]
  22. 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]

2005 (2)

Y. Wu, P. Xi, J. Y. Qu, T. H. Cheung, and M. Y. Yu, “Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue,” Opt. Express 13, 382–388 (2005)
[Crossref] [PubMed]

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

2004 (4)

Y. Wu, P. Xi, J. Y. Qu, T. H. Cheung, and M. Y. Yu, “Depth-resolved fluorescence spectroscopy reveals layered structure of tissue,” Opt. Express 12, 3218–3223 (2004)
[Crossref] [PubMed]

T. Boulesteix, E. Beaurepaire, M.-P. Sauviat, and M. C. Schanne-Klein, “Second harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20 nm accuracy,” Opt. Lett. 29, 2031–2033 (2004)
[Crossref] [PubMed]

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
[Crossref] [PubMed]

2003 (5)

K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8, 432–439 (2003)
[Crossref] [PubMed]

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]

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28, 2488–2490 (2003)
[Crossref] [PubMed]

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]

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]

2001 (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]

2000 (2)

1997 (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]

1996 (3)

C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B 13, 481–491 (1996)
[Crossref]

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]

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

1990 (2)

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]

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

1980 (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]

1969 (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]

Adam, M.

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

Alfano, R. R.

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]

Beaurepaire, E.

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

T. Boulesteix, E. Beaurepaire, M.-P. Sauviat, and M. C. Schanne-Klein, “Second harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20 nm accuracy,” Opt. Lett. 29, 2031–2033 (2004)
[Crossref] [PubMed]

Blanchard-Desce, M.

Boulesteix, T.

Brancaleon, L.

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]

Brookner, C.

Buehler, C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

Chen, C.-C.

Chen, Y.-C.

Cheung, T. H.

Christie, R.

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]

Chu, S.-W.

Coghlan, L.

Cox, G.

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]

Davis, R. A.

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]

Débarre, D.

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

Denk, W.

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

Deyl, Z.

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

Drezek, R.

Duin, R. P. W.

D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
[Crossref] [PubMed]

Durkin, A. J.

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]

Eichner, R.

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]

Fallon, J. D.

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]

Farge, E.

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

Fraser, I.

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]

Gorrell, M. D.

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]

Gratton, E.

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]

Greuter, U.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

Heintzelman, D.

Hyman, B. T.

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]

Jones, A.

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]

Kable, E.

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]

Kahn, M.

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]

Kim, K. H.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

Kollias, N.

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]

König, K.

K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8, 432–439 (2003)
[Crossref] [PubMed]

Lane, E. B.

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]

Leonard, N. J.

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]

Lin, B.-L.

Macek, K.

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

Marconi, K.

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]

Masters, B. R.

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]

Menaker, G.

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]

Mertz, J.

Moreaux, L.

Moulia, B.

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

Nikitin, A. Y.

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]

Porter, R. M.

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]

Qu, J. Y.

Richards-Kortum, R.

Riemann, I.

K. König and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8, 432–439 (2003)
[Crossref] [PubMed]

Roodenburg, J. L. N.

D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
[Crossref] [PubMed]

Sacks, P. G.

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]

Sandre, O.

Sauviat, M.-P.

Savage, H. E.

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]

Schanne-Klein, M. C.

D. Débarre, W. Suppato, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 28881–2884 (2004)
[Crossref]

T. Boulesteix, E. Beaurepaire, M.-P. Sauviat, and M. C. Schanne-Klein, “Second harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20 nm accuracy,” Opt. Lett. 29, 2031–2033 (2004)
[Crossref] [PubMed]

Schantz, S. P.

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]

Schlumpf, N.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

Scott, T. G.

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]

Sevick-Muraca, E.

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

Skurichina, M.

D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
[Crossref] [PubMed]

So, P. T. C.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, “Single-photon counting multicolor multiphoton fluorescence microscope,” J. Fluorescence 15, 41–51 (2005)
[Crossref]

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]

Spencer, R. D.

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]

Sterenborg, H. J. C. M.

D. C. G. D. Veld, M. Skurichina, M. J. H. Witjes, R. P. W. Duin, H. J. C. M. Sterenborg, and J. L. N. Roodenburg, “Clinical study for classification of benign, dysplastic, and malignant oral lesions using autofluorescence spectroscopy,” J. Biomed. Opt. 9, 940–950 (2004)
[Crossref] [PubMed]

Strickler, J. H.

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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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