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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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  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]
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  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).
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    [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)

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).

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]

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]

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]

2005 (9)

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]

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]

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]

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]

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]

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)

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. 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]

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. 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)

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]

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]

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]

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]

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]

2001 (2)

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]

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]

2000 (2)

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]

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]

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)

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

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).

1979 (1)

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

Ann. Biochem. (1)

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

Biomaterials (1)

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

Biophys. J. (6)

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]

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]

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]

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]

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]

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]

BMC Med. (1)

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]

Dermatol. Clin. (1)

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

Dermatol. Surg. (1)

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]

J. Am. Acad. Dermatol. (1)

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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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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. 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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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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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).
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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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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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J. Pathol. (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).
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J. Steroid. Biochem. Mol. Biol. (1)

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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Lab. Invest. (1)

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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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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Microsc. Res. Tech. (1)

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).
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Nat. Biotechnol. (2)

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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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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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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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).
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Opt. Express (4)

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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).
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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).
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Proc. Natl. Acad. Sci. USA (2)

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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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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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).

Science (1)

W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscope," Science 248, 73-76 (1990).
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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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