Abstract

We have used a multidimensional non-linear laser imaging approach to visualize ex-vivo samples of basal cell carcinoma (BCC). A combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging has been used to image different skin layers. This approach has elucidated some morphological (supported by histopathological images), biochemical, and physiochemical differences of the healthy samples with respect to BCC ones. In particular, in comparison with normal skin, BCC showed a blue-shifted fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean fluorescence lifetime. Finally, the use of aminolevulinic acid as a contrast agent has been demonstrated to increase the constrast in tumor border detection. The results obtained provide further support for in-vivo non-invasive imaging of Basal Cell Carcinoma.

© 2007 Optical Society of America

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscope,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  2. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
    [CrossRef] [PubMed]
  3. A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
    [CrossRef] [PubMed]
  4. 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]
  5. B. R. Masters, P. T. C. So, and E. Gratton, “Optical biopsy of in vivo human skin: multi-photon excitation microscopy,” Lasers Med. Sci. 13, 196–203 (1998).
    [CrossRef]
  6. P. T. C. So,, H. Kim, and I. E. Kochevar, “Two-Photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Express 3, 339–351 (1998).
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  7. J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
    [CrossRef] [PubMed]
  8. B. R. Masters and P. T. C. So, “Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,” Opt. Express 8, 2–9 (2001).
    [CrossRef] [PubMed]
  9. K. König 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]
  10. L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80(3), 1568–1574 (2001).
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  11. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
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  12. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
    [CrossRef]
  13. S. Roth and I. Freund, “Second harmonic generation in collagen,” J. Chem. Phys. 70, 1637–1643 (1979).
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  14. K. König, K. Schenke-Layland, I. Riemann, and U. A. Stock, “Multiphoton autofluorescence imaging of intratissue elastic fibers,” Biomaterials 26, 495–500 (2005).
    [CrossRef]
  15. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88, 1377–1386 (2005).
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  16. P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82, 3330–3342 (2002).
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  17. T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004).
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  18. Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. C. So, and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging,” Biophys. J. 91, 2620–2625 (2006).
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  19. M. Han, G. Giese, and J. F. Bille, “Second harmonic generation imaging of collagen fibrils in cornea and sclera,” Opt. Express 13, 5791–5797 (2005).
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  20. E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
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    [CrossRef]
  24. M. J. Koehler, K. König, P. Elsner, R. Bückle, and M. Kaatz, “In vivo assessment of human skin aging by multiphoton laser scanning tomography,” Opt. Lett. 19, 2879–2881 (2006).
    [CrossRef]
  25. P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging,” J. Pathol. 191, 229–234 (2000).
    [CrossRef] [PubMed]
  26. P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
    [CrossRef] [PubMed]
  27. Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63(1), 72–80 (2004).
    [CrossRef]
  28. S. Y. Breusegem, M. Levi, and N. P. Barry, “Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy,” Nephron. Exp. Nephrol. 103(2), e41–e49 (2006).
    [CrossRef]
  29. K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg, “Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient,” Biophys. J. 83, 1682–1690 (2002).
    [CrossRef] [PubMed]
  30. A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Shanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13, 6268–6274 (2005).
    [CrossRef] [PubMed]
  31. L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. 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]
  32. J. Chen, S. Zhuo, T. Luo, X. Jiang, and J. Zhao, “Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers,” Scanning 28(6), 319–326 (2006).
  33. S. Gonzalez and Z. Tannous, “Real-time in vivo confocal reflectance microscopy of basal cell carcinoma,” J. Am. Acad. Dermatol. 47, 869–874 (2002).
    [CrossRef] [PubMed]
  34. D. E. Marra, A. Torres, C. F: Schanbacher, and S. Gonzalez, “Detection of residual basal cell carcinoma by in vivo confocal microscopy,” Dermatol. Surg. 31, 538–541 (2005).
    [CrossRef] [PubMed]
  35. M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
    [CrossRef]
  36. S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
    [CrossRef]
  37. J. E. Blume and A. R. Oseroff, “Aminolevulinic acid photodynamic therapy for skin cancers,” Dermatol. Clin. 25(1), 5–14 (2007).
  38. J. C. Kennedy, R. H. Pottier, and D. C. Pross, “Photodynamic therapy with endogenous protoporphyrine IX: Basic principles and present clinical experience,” J. Photochem. Photobiol. B 6, 143–148 (1990).
  39. M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
    [CrossRef]
  40. J. R. Lackowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Ann. Biochem. 202: 316–330 (1992).
    [CrossRef]
  41. M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
    [CrossRef]
  42. T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R. M. Maas, B. C. Gärtner, J. Kamradt, E. Meese, W. Tilgen, and J. Reichrath, “Analysis of the vitamin D system in basal cell carcinomas (BCCs),” Lab. Invest. 84, 693–702 (2004).
    [CrossRef] [PubMed]
  43. D. D. Bikle, Y. Oda, and Z. Xie, “Vitamin D and skin cancer: A problem in gene regulation,” J. Steroid. Biochem. Mol. Biol. 97, 83–91 (2005).
    [CrossRef] [PubMed]

2007 (2)

J. E. Blume and A. R. Oseroff, “Aminolevulinic acid photodynamic therapy for skin cancers,” Dermatol. Clin. 25(1), 5–14 (2007).

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
[CrossRef]

2006 (6)

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett. 31, 2756–2758 (2006).
[CrossRef] [PubMed]

J. Chen, S. Zhuo, T. Luo, X. Jiang, and J. Zhao, “Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers,” Scanning 28(6), 319–326 (2006).

M. J. Koehler, K. König, P. Elsner, R. Bückle, and M. Kaatz, “In vivo assessment of human skin aging by multiphoton laser scanning tomography,” Opt. Lett. 19, 2879–2881 (2006).
[CrossRef]

S. Y. Breusegem, M. Levi, and N. P. Barry, “Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy,” Nephron. Exp. Nephrol. 103(2), e41–e49 (2006).
[CrossRef]

Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. C. So, and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging,” Biophys. J. 91, 2620–2625 (2006).
[CrossRef] [PubMed]

P. P. Provenzano, K. W. Eliceiri, J. M. Campbell, D. R. Inman, J. G. White, and P. J. Keely, “Collagen reorganization at the tumor-stromal interface facilitates local invasion,” BMC Med. 4(1), 38 (2006).
[CrossRef]

2005 (9)

S. J. Lin, R. J. Wu, H. Y. Tan, W. Lo, W. C. Lin, T. H. Young, C. J. Hsu, J. S. Chen, S. H. Jee, and C. Y. Dong, “Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy,” Opt. Lett. 17, 2275–2277 (2005).
[CrossRef]

K. König, K. Schenke-Layland, I. Riemann, and U. A. Stock, “Multiphoton autofluorescence imaging of intratissue elastic fibers,” Biomaterials 26, 495–500 (2005).
[CrossRef]

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88, 1377–1386 (2005).
[CrossRef]

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. 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]

D. E. Marra, A. Torres, C. F: Schanbacher, and S. Gonzalez, “Detection of residual basal cell carcinoma by in vivo confocal microscopy,” Dermatol. Surg. 31, 538–541 (2005).
[CrossRef] [PubMed]

M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
[CrossRef]

M. Han, G. Giese, and J. F. Bille, “Second harmonic generation imaging of collagen fibrils in cornea and sclera,” Opt. Express 13, 5791–5797 (2005).
[CrossRef] [PubMed]

A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Shanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13, 6268–6274 (2005).
[CrossRef] [PubMed]

D. D. Bikle, Y. Oda, and Z. Xie, “Vitamin D and skin cancer: A problem in gene regulation,” J. Steroid. Biochem. Mol. Biol. 97, 83–91 (2005).
[CrossRef] [PubMed]

2004 (3)

T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R. M. Maas, B. C. Gärtner, J. Kamradt, E. Meese, W. Tilgen, and J. Reichrath, “Analysis of the vitamin D system in basal cell carcinomas (BCCs),” Lab. Invest. 84, 693–702 (2004).
[CrossRef] [PubMed]

Y. Chen and A. Periasamy, “Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization,” Microsc. Res. Tech. 63(1), 72–80 (2004).
[CrossRef]

T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004).
[CrossRef]

2003 (7)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (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]

K. König 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]

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003).
[CrossRef] [PubMed]

E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
[CrossRef] [PubMed]

P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
[CrossRef] [PubMed]

M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
[CrossRef]

2002 (6)

S. Gonzalez and Z. Tannous, “Real-time in vivo confocal reflectance microscopy of basal cell carcinoma,” J. Am. Acad. Dermatol. 47, 869–874 (2002).
[CrossRef] [PubMed]

K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg, “Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient,” Biophys. J. 83, 1682–1690 (2002).
[CrossRef] [PubMed]

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82, 3330–3342 (2002).
[CrossRef] [PubMed]

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
[CrossRef] [PubMed]

2001 (2)

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80(3), 1568–1574 (2001).
[CrossRef]

B. R. Masters and P. T. C. So, “Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,” Opt. Express 8, 2–9 (2001).
[CrossRef] [PubMed]

2000 (2)

P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging,” J. Pathol. 191, 229–234 (2000).
[CrossRef] [PubMed]

S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

1998 (2)

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

P. T. C. So,, H. Kim, and I. E. Kochevar, “Two-Photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,” Opt. Express 3, 339–351 (1998).
[CrossRef]

1992 (1)

J. R. Lackowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Ann. Biochem. 202: 316–330 (1992).
[CrossRef]

1990 (2)

J. C. Kennedy, R. H. Pottier, and D. C. Pross, “Photodynamic therapy with endogenous protoporphyrine IX: Basic principles and present clinical experience,” J. Photochem. Photobiol. B 6, 143–148 (1990).

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

1979 (1)

S. Roth and I. Freund, “Second harmonic generation in collagen,” J. Chem. Phys. 70, 1637–1643 (1979).
[CrossRef]

Andersson-Engels, S.

S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

Araki, T.

T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004).
[CrossRef]

Baldridge, L. A.

J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
[CrossRef] [PubMed]

Barry, N. P.

S. Y. Breusegem, M. Levi, and N. P. Barry, “Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy,” Nephron. Exp. Nephrol. 103(2), e41–e49 (2006).
[CrossRef]

K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg, “Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient,” Biophys. J. 83, 1682–1690 (2002).
[CrossRef] [PubMed]

Behne, M. J.

K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg, “Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient,” Biophys. J. 83, 1682–1690 (2002).
[CrossRef] [PubMed]

Berndt, K. W.

J. R. Lackowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Ann. Biochem. 202: 316–330 (1992).
[CrossRef]

Bikle, D. D.

D. D. Bikle, Y. Oda, and Z. Xie, “Vitamin D and skin cancer: A problem in gene regulation,” J. Steroid. Biochem. Mol. Biol. 97, 83–91 (2005).
[CrossRef] [PubMed]

Bille, J. F.

Bird, D. K.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
[CrossRef]

Blanchard-Desce, M.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80(3), 1568–1574 (2001).
[CrossRef]

Blume, J. E.

J. E. Blume and A. R. Oseroff, “Aminolevulinic acid photodynamic therapy for skin cancers,” Dermatol. Clin. 25(1), 5–14 (2007).

Boucher, Y.

E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
[CrossRef] [PubMed]

Boulesteix, T.

Breusegem, S. Y.

S. Y. Breusegem, M. Levi, and N. P. Barry, “Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy,” Nephron. Exp. Nephrol. 103(2), e41–e49 (2006).
[CrossRef]

Brown, E.

E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
[CrossRef] [PubMed]

Bückle, R.

M. J. Koehler, K. König, P. Elsner, R. Bückle, and M. Kaatz, “In vivo assessment of human skin aging by multiphoton laser scanning tomography,” Opt. Lett. 19, 2879–2881 (2006).
[CrossRef]

Campagnola, P. J.

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J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
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D. E. Marra, A. Torres, C. F: Schanbacher, and S. Gonzalez, “Detection of residual basal cell carcinoma by in vivo confocal microscopy,” Dermatol. Surg. 31, 538–541 (2005).
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B. R. Masters and P. T. C. So, “Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,” Opt. Express 8, 2–9 (2001).
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T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R. M. Maas, B. C. Gärtner, J. Kamradt, E. Meese, W. Tilgen, and J. Reichrath, “Analysis of the vitamin D system in basal cell carcinomas (BCCs),” Lab. Invest. 84, 693–702 (2004).
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M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
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P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
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J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
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D. D. Bikle, Y. Oda, and Z. Xie, “Vitamin D and skin cancer: A problem in gene regulation,” J. Steroid. Biochem. Mol. Biol. 97, 83–91 (2005).
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M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
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L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. 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).
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J. C. Malone, A. F. Hood, T. Conley, J. Nürnberger, L. A. Baldridge, J. L. Clendenon, K. W. Dunn, and C. L. Phillips, “Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy,” J. Cutan. Pathol. 29, 453–458 (2002).
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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
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E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
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P. P. Provenzano, K. W. Eliceiri, J. M. Campbell, D. R. Inman, J. G. White, and P. J. Keely, “Collagen reorganization at the tumor-stromal interface facilitates local invasion,” BMC Med. 4(1), 38 (2006).
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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
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T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R. M. Maas, B. C. Gärtner, J. Kamradt, E. Meese, W. Tilgen, and J. Reichrath, “Analysis of the vitamin D system in basal cell carcinomas (BCCs),” Lab. Invest. 84, 693–702 (2004).
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P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82, 3330–3342 (2002).
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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
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M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
[CrossRef]

Sandre, O.

L. Moreaux, O. Sandre, S. Charpak, M. Blanchard-Desce, and J. Mertz, “Coherent scattering in multi-harmonic light microscopy,” Biophys. J. 80(3), 1568–1574 (2001).
[CrossRef]

Schanbacher, C. F:

D. E. Marra, A. Torres, C. F: Schanbacher, and S. Gonzalez, “Detection of residual basal cell carcinoma by in vivo confocal microscopy,” Dermatol. Surg. 31, 538–541 (2005).
[CrossRef] [PubMed]

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K. König, K. Schenke-Layland, I. Riemann, and U. A. Stock, “Multiphoton autofluorescence imaging of intratissue elastic fibers,” Biomaterials 26, 495–500 (2005).
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E. Brown, T. McKee, E. DiTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003).
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Shousha, S.

P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
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Siegel, J.

P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
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Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. C. So, and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging,” Biophys. J. 91, 2620–2625 (2006).
[CrossRef] [PubMed]

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. 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]

B. R. Masters and P. T. C. So, “Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,” Opt. Express 8, 2–9 (2001).
[CrossRef] [PubMed]

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

So,, P. T. C.

Stamp, G. W. H.

P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
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Steiner, R.

M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J. Biomed. Opt. 8(1), 26–32 (2003).
[CrossRef]

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M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
[CrossRef]

Stock, U. A.

K. König, K. Schenke-Layland, I. Riemann, and U. A. Stock, “Multiphoton autofluorescence imaging of intratissue elastic fibers,” Biomaterials 26, 495–500 (2005).
[CrossRef]

Stoller, P.

P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik, “Polarization-modulated second harmonic generation in collagen,” Biophys. J. 82, 3330–3342 (2002).
[CrossRef] [PubMed]

Strandeberg, C.

M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
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W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscope,” Science 248, 73–76 (1990).
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Sun, Y.

Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. C. So, and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging,” Biophys. J. 91, 2620–2625 (2006).
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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

Szmacinski, H.

J. R. Lackowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Ann. Biochem. 202: 316–330 (1992).
[CrossRef]

Tadrous, P. J.

P. J. Tadrous, J. Siegel, P. M. W. French, S. Shousha, E. N. Lalani, and G. W. H. Stamp, “Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer,” J. Pathol. 199, 309–317 (2003).
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P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging,” J. Pathol. 191, 229–234 (2000).
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S. J. Lin, R. J. Wu, H. Y. Tan, W. Lo, W. C. Lin, T. H. Young, C. J. Hsu, J. S. Chen, S. H. Jee, and C. Y. Dong, “Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy,” Opt. Lett. 17, 2275–2277 (2005).
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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

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P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002).
[CrossRef]

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T. Mitschele, B. Diesel, M. Friedrich, V. Meineke, R. M. Maas, B. C. Gärtner, J. Kamradt, E. Meese, W. Tilgen, and J. Reichrath, “Analysis of the vitamin D system in basal cell carcinomas (BCCs),” Lab. Invest. 84, 693–702 (2004).
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T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004).
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D. E. Marra, A. Torres, C. F: Schanbacher, and S. Gonzalez, “Detection of residual basal cell carcinoma by in vivo confocal microscopy,” Dermatol. Surg. 31, 538–541 (2005).
[CrossRef] [PubMed]

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A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
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Tsai, T. F.

Uhre, J.

M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
[CrossRef]

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S. Andersson-Engels, G. Canti, R. Cubeddu, C. Eker, C. af Klinteberg, A. Pifferi, K. Svanberg, S. Svanberg, P. Taroni, G. Valentini, and I. Wang, “Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin,” Lasers Surg. Med. 26(1), 76–82 (2000).
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R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88, 1377–1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003).
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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]

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

Wennberg, A. M.

M. B. Ericson, J. Uhre, C. Strandeberg, B. Stenquist, O. Larko, A. M. Wennberg, and A. Rosen, “Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma,” J. Biomed. Opt. 10(3), 034009 (2005).
[CrossRef]

White, J. G.

P. P. Provenzano, K. W. Eliceiri, J. M. Campbell, D. R. Inman, J. G. White, and P. J. Keely, “Collagen reorganization at the tumor-stromal interface facilitates local invasion,” BMC Med. 4(1), 38 (2006).
[CrossRef]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88, 1377–1386 (2005).
[CrossRef]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (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).
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S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett. 31, 2756–2758 (2006).
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S. J. Lin, R. J. Wu, H. Y. Tan, W. Lo, W. C. Lin, T. H. Young, C. J. Hsu, J. S. Chen, S. H. Jee, and C. Y. Dong, “Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy,” Opt. Lett. 17, 2275–2277 (2005).
[CrossRef]

Xie, Z.

D. D. Bikle, Y. Oda, and Z. Xie, “Vitamin D and skin cancer: A problem in gene regulation,” J. Steroid. Biochem. Mol. Biol. 97, 83–91 (2005).
[CrossRef] [PubMed]

Yasui, T.

T. Yasui, Y. Tohno, and T. Araki, “Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry,” J. Biomed. Opt. 9(2), 259–264 (2004).
[CrossRef]

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
[CrossRef] [PubMed]

Young, T. H.

S. J. Lin, R. J. Wu, H. Y. Tan, W. Lo, W. C. Lin, T. H. Young, C. J. Hsu, J. S. Chen, S. H. Jee, and C. Y. Dong, “Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy,” Opt. Lett. 17, 2275–2277 (2005).
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Zhuo, S.

J. Chen, S. Zhuo, T. Luo, X. Jiang, and J. Zhao, “Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers,” Scanning 28(6), 319–326 (2006).

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88, 1377–1386 (2005).
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Figures (5)

Fig. 1.
Fig. 1.

Comparison between en face optically sectioned human skin sample imaged with combined TPE intrinsic fluorescence (green) and SHG (red) microscopy and corresponding microphotographs acquired with optical microscope on 5 µm thick axially sliced human skin samples stained with haematoxylin-eosin. TPE fluorescence image (a) and corresponding histology (b) at 50 µm depth, approximately corresponding to spinous layer. Field of view: 200 µm×200 µm; scale bars: 20 µm. Magnified detail (corresponding to the dashed square in Fig. 1a) of healthy skin cells (c), acquired by TPE fluorescence. Field of view: 90 µm×90 µm; scale bars: 10 µm. BCC cells in human skin at 85 µm depth acquired by TPE fluorescence (d) and corresponding histology (e). Field of view: 90 µm×90 µm; scale bars: 10 µm. Elastic (green) and collagen (red) fibers in human skin at 120 µm depth acquired by combined TPE fluorescence and SHG (f) and corresponding histology stained with Verhoeff-Van Gieson stain (g). Field of view: 250 µm×250 µm; scale bars: 25 µm. The excitation wavelength was 740 nm for TPE fluorescence and 840 nm for SHG.

Fig. 2.
Fig. 2.

Perpendicular optically sectioned human skin tissue image acquired using TPE autofluorescence on fresh sample, excised 4 hours after ALA application (a) and the corresponding microphotograph taken after conventional histological examination and visualization by optical microscope (d). Regions B and D are BCC tissue and healthy dermis, respectively. Field of view: 290 µm×300 µm; scale bars: 30 µm. High magnified TPE autofluorescence images of healthy dermis (b) and BCC tissue (c), approximately corresponding to the regions B and D indicated in (a). Field of view: 90 µm×90 µm; scale bars: 15 µm. Perpendicular optically sectioned human skin tissue imaged using TPE autofluorescence on fresh sample, excised 4 hours after ALA application (f) and corresponding histological microphotograph (e). Inside the sample both normal epidermis tissue E and BCC tissue B are present. Field of view: 300 µm×300 µm; scale bars: 30 µm.

Fig. 3.
Fig. 3.

FLIM images of an en face optically sectioned human healthy skin fresh sample, acquired by photon counting method, using an excitation wavelength of 740 nm. Image pixels lifetimes were obtained after system response de-convolution and single exponential fit and represented in a color-coded scale. Epidermal layers at 10(a), 30(b), 50(c), and 80(d) mm below the skin surface, respectively. Field of view: 130 µm×130 µm; scale bars: 15 µm. The corresponding normalized lifetime distributions and the color-coded scale for 30 µm depth and 50 µm depth layers are plotted in (e) and (f) for healthy skin and BCC, respectively. The lifetime shift for each epidermal layer is plotted versus the depth of recording in (g).

Fig. 4.
Fig. 4.

χ-square color-coded matrixes for an image acquired by photon counting inside ALA-treated BCC tissue and fitted with a single-exponential (a), with a bi-exponential (b), and with a three-exponential (c) decay function. Field of view: 200 µm×200 µm. Scale bars: 20 µm. χ-square distributions and color-coded scale (d) for the single-exponential (black line), the bi-exponential (red line), and the three-exponential (blue line) decay function, corresponding to the matrixes represented in (a), (b), and (c), respectively. En face optically sectioned human skin tissue image acquired using TPE autofluorescence on fresh sample, excised 4 hours after ALA application (e) with the corresponding high contrast FLIM image (f), obtained after system response de-convolution and three-exponential fit of the image pixels fluorescence decay. Inside the sample both normal healthy skin tissue (H) and BCC tissue (B) are present. Field of view: 200 µm×200 µm; scale bars: 20 µm. The color-coded scale and the mean lifetime distribution of the image in (f) are plotted in (g) in a logarithmic scale.

Fig. 5.
Fig. 5.

Normalized TPE fluorescence emission spectra measured at 30 µm depth (a), and at 50 µm depth (b) below the skin surface for human healthy skin (black line) and BCC (red line) using 740 nm excitation wavelength. The “Spectral Shift” (calculated as the enclosed area between healthy skin and BCC fluorescence emission spectra) is plotted versus the depth of recording in (c). The normalized TPE fluorescence response varying the excitation wavelength is represented in (d) for a healthy skin cellular layer (black) and for a BCC cellular layer (red) as well as the difference between the two (blue).

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