Abstract

We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed.

© 2011 OSA

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2011 (5)

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

J. A. Palero, A. N. Bader, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy,” Biomed. Opt. Express2(5), 1030–1039 (2011).
[CrossRef] [PubMed]

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

2010 (1)

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

2009 (3)

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

2008 (3)

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

K. König, “Clinical multiphoton tomography,” J Biophotonics1(1), 13–23 (2008).
[CrossRef] [PubMed]

2007 (3)

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

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] [PubMed]

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

2005 (2)

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

2003 (1)

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt.8(3), 432–439 (2003).
[CrossRef] [PubMed]

2001 (1)

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

1999 (1)

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

1997 (2)

B. R. Masters, P. T. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J.72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

P. K. Gupta, S. K. Majumder, and A. Uppal, “Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy,” Lasers Surg. Med.21(5), 417–422 (1997).
[CrossRef] [PubMed]

1994 (1)

K. Koenig and H. Schneckenburger, “Laser-induced autofluorescence for medical diagnosis,” J. Fluoresc.4(1), 17–40 (1994).
[CrossRef]

1992 (1)

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Altmeyer, P.

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Ameer-Beg, S. M.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Bader, A. N.

Barber, P. R.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Bauer, J.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

Bellini, V.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Benati, E.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Birch, D. J.

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

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] [PubMed]

Blossey, R.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Borsari, S.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Breunig, H. G.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

Buder, S.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

Cicchi, R.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Compton, C. C.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

de Bruijn, H. S.

J. A. Palero, A. N. Bader, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy,” Biomed. Opt. Express2(5), 1030–1039 (2011).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

De Giorgi, V.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Deutsch, T. F.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Dimitrow, E.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

Dunsby, C.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Edens, R. J.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Ehlers, A.

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

Eichhorn, R.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

Eickhoff, J.

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] [PubMed]

Eliceiri, K. W.

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] [PubMed]

Elsner, P.

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Ericson, M. B.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

Ezike, I.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Ferrari, C.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Flotte, T. J.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

French, P.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Freyer, W.

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Frisoli, J. K.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Garbe, C.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

Gendron-Fitzpatrick, A.

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] [PubMed]

Gerritsen, H. C.

J. A. Palero, A. N. Bader, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy,” Biomed. Opt. Express2(5), 1030–1039 (2011).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Gilbey, J. D.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Gratton, E.

B. R. Masters, P. T. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J.72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Grice, J. E.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

Guanti, M.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Guardoli, D.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Gupta, P. K.

P. K. Gupta, S. K. Majumder, and A. Uppal, “Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy,” Lasers Surg. Med.21(5), 417–422 (1997).
[CrossRef] [PubMed]

Heikal, A. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

Héliot, L.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Hoffmann, K.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Kaatz, M.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

Kasischke, K. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

Keely, P. J.

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] [PubMed]

Koehler, M. J.

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Koenig, K.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt.8(3), 432–439 (2003).
[CrossRef] [PubMed]

K. Koenig and H. Schneckenburger, “Laser-induced autofluorescence for medical diagnosis,” J. Fluoresc.4(1), 17–40 (1994).
[CrossRef]

König, K.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

K. König, “Clinical multiphoton tomography,” J Biophotonics1(1), 13–23 (2008).
[CrossRef] [PubMed]

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

Leray, A.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Leupold, D.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Lin, L.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

Lotti, T.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Majumder, S. K.

P. K. Gupta, S. K. Majumder, and A. Uppal, “Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy,” Lasers Surg. Med.21(5), 417–422 (1997).
[CrossRef] [PubMed]

Massi, D.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters, P. T. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J.72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Nishioka, N. S.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Norgauer, J.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

Palero, J. A.

J. A. Palero, A. N. Bader, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy,” Biomed. Opt. Express2(5), 1030–1039 (2011).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Paoli, J.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

Pavone, F. S.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Pellacani, G.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Ponti, G.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Prow, T. W.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

Ramanujam, N.

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] [PubMed]

Raphael, A. P.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

Reda, J.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

Riching, K. M.

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] [PubMed]

Richter, J. M.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Riemann, I.

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt.8(3), 432–439 (2003).
[CrossRef] [PubMed]

Roberts, M. S.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

Sanchez, W. H.

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

Sanchez, W. Y.

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

Schianchi, S.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Schneckenburger, H.

K. Koenig and H. Schneckenburger, “Laser-induced autofluorescence for medical diagnosis,” J. Fluoresc.4(1), 17–40 (1994).
[CrossRef]

Scholz, M.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

Schomacker, K. T.

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Seidenari, S.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Sestini, S.

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

Skala, M. C.

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] [PubMed]

Smedh, M.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

So, P. T.

B. R. Masters, P. T. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J.72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Soyer, H. P.

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

Spriet, C.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Stankovic, G.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

Stark, M.

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

Sterenborg, H. J.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Sterenborg, H. J. C. M.

Stücker, M.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Talbot, C.

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Teuchner, K.

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Trinel, D.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Uppal, A.

P. K. Gupta, S. K. Majumder, and A. Uppal, “Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy,” Lasers Surg. Med.21(5), 417–422 (1997).
[CrossRef] [PubMed]

Usson, Y.

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

van der Ploeg van den Heuvel, A.

J. A. Palero, A. N. Bader, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy,” Biomed. Opt. Express2(5), 1030–1039 (2011).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Vishwasrao, H. D.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

Vojnovic, B.

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Volkmer, A.

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Webb, W. W.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

Wennberg, A. M.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

Wessler, G.

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

Ziemer, M.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Adv. Drug Deliv. Rev. (1)

K. König, A. P. Raphael, L. Lin, J. E. Grice, H. P. Soyer, H. G. Breunig, M. S. Roberts, and T. W. Prow, “Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research,” Adv. Drug Deliv. Rev.63(4-5), 388–404 (2011).
[CrossRef] [PubMed]

Biomed. Opt. Express (1)

Biophys. J. (2)

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues,” Biophys. J.93(3), 992–1007 (2007).
[CrossRef] [PubMed]

B. R. Masters, P. T. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J.72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Cytometry A (1)

A. Leray, C. Spriet, D. Trinel, R. Blossey, Y. Usson, and L. Héliot, “Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis,” Cytometry A79A(2), 149–158 (2011).
[CrossRef] [PubMed]

Exp. Dermatol. (1)

E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009).
[CrossRef] [PubMed]

J Biophotonics (2)

K. König, “Clinical multiphoton tomography,” J Biophotonics1(1), 13–23 (2008).
[CrossRef] [PubMed]

R. Cicchi, S. Sestini, V. De Giorgi, D. Massi, T. Lotti, and F. S. Pavone, “Nonlinear laser imaging of skin lesions,” J Biophotonics1(1), 62–73 (2008).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, “Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy,” J. Biol. Chem.280(26), 25119–25126 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt. (4)

R. Eichhorn, G. Wessler, M. Scholz, D. Leupold, G. Stankovic, S. Buder, M. Stücker, and K. Hoffmann, “Early diagnosis of melanotic melanoma based on laser-induced melanin fluorescence,” J. Biomed. Opt.14(3), 034033 (2009).
[CrossRef] [PubMed]

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt.15(4), 046008 (2010).
[CrossRef] [PubMed]

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] [PubMed]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt.8(3), 432–439 (2003).
[CrossRef] [PubMed]

J. Fluoresc. (1)

K. Koenig and H. Schneckenburger, “Laser-induced autofluorescence for medical diagnosis,” J. Fluoresc.4(1), 17–40 (1994).
[CrossRef]

J. Invest. Dermatol. (3)

K. Hoffmann, M. Stücker, P. Altmeyer, K. Teuchner, and D. Leupold, “Selective femtosecond pulse-excitation of melanin fluorescence in tissue,” J. Invest. Dermatol.116(4), 629–630 (2001).
[CrossRef] [PubMed]

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol.128(5), 1248–1255 (2008).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol.129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Lasers Surg. Med. (2)

P. K. Gupta, S. K. Majumder, and A. Uppal, “Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy,” Lasers Surg. Med.21(5), 417–422 (1997).
[CrossRef] [PubMed]

K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med.12(1), 63–78 (1992).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

A. Ehlers, I. Riemann, M. Stark, and K. König, “Multiphoton fluorescence lifetime imaging of human hair,” Microsc. Res. Tech.70(2), 154–161 (2007).
[CrossRef] [PubMed]

Photochem. Photobiol. (1)

K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stücker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol.70(2), 146–151 (1999).
[PubMed]

Pigment Cell Melanoma Res (1)

D. Leupold, M. Scholz, G. Stankovic, J. Reda, S. Buder, R. Eichhorn, G. Wessler, M. Stücker, K. Hoffmann, J. Bauer, and C. Garbe, “The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes,” Pigment Cell Melanoma Res24(3), 438–445 (2011).
[CrossRef] [PubMed]

Proc. SPIE (1)

P. R. Barber, S. M. Ameer-Beg, J. D. Gilbey, R. J. Edens, I. Ezike, and B. Vojnovic, “Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM,” Proc. SPIE5700, 171–181 (2005).
[CrossRef]

Skin Res. Technol. (1)

E. Benati, V. Bellini, S. Borsari, C. Dunsby, C. Ferrari, P. French, M. Guanti, D. Guardoli, K. Koenig, G. Pellacani, G. Ponti, S. Schianchi, C. Talbot, and S. Seidenari, “Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy,” Skin Res. Technol.17(3), 295–303 (2011).
[CrossRef] [PubMed]

Other (2)

W. Becker, Advanced Time-Correlated Single Photon Counting Techniques (Springer, Berlin, 2005).

J. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).

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Figures (5)

Fig. 1
Fig. 1

a) example autofluorescence image obtained from a dysplastic nevus ex vivo. Scale bar 50 µm. b) shows the same data as a) but with the manually defined cellular regions of interest overlaid in green. c-top) shows the spatially integrated fluorescence decay profile obtained in the blue channel from a defined cellular region of interest (black dots) together with the instrument response function (blue) and double exponential fit to the data (green). c-bottom) shows the weighted residuals of the fit to the experimental data.

Fig. 5
Fig. 5

a and b) relative fluorescence emission intensity plotted against mean fluorescence lifetime in each channel respectively for the DN specimens. Different colors are used to represent each patient and each FOV. c and d) shows the same as a and b) but for the nBCC data.

Fig. 2
Fig. 2

a) Schematic of the emission spectra from intracellular fluorophores. Adapted from Palero et al. [4]. b) dermatoscopic image of a dysplastic nevus (scale bar 1 mm). c) corresponding depth resolved fluorescence intensity image stacks for the two spectral channels (scale bar 50 µm). d) macroscopic image of a nodular basal cell carcinoma (scale bar 5 mm). e) corresponding depth resolved fluorescence intensity image stacks (scale bar 50 µm). We note that the feint striping artifact in the deeper images in e) is due to unavoidable stray light from the computer monitor.

Fig. 3
Fig. 3

Histograms of the fluorescence decay parameters calculated on a per-cell basis for the dysplastic nevi (DN) and nodular Basal Cell Carcinoma (nBCC) specimens. a and b) histograms of τ1, c and d) histograms of τ2 and e and f) histograms of τmean. a,c,e) fluorescence lifetimes from the blue channel (left column) and b,d,f) fluorescence lifetimes from the green channel (right column).

Fig. 4
Fig. 4

a) shows f1 correlated against τ1 and b) shows f2 correlated against τ2. c) shows τ1 correlated against τ2. a-c) show data obtained in the green channel. d) shows τmean from the blue and green spectral channels plotted against each other. This plot demonstrates that the distribution of mean lifetimes from cells for each diagnostic group are partially separated, with a tendency for shorter lifetimes in both channels for DN cells. The dashed line is x = y.

Tables (1)

Tables Icon

Table 1 Summary of the 25th, median and 75th percentile for all of the spectroscopic parameters for each spectral channel (blue or green) for each cell. Blue detection channel <500 nm and green detection channel >500 nm.

Equations (5)

Equations on this page are rendered with MathJax. Learn more.

I mod (t)= I tot [ f 1 D(t, τ 1 , T p )+ f 2 D(t, τ 2 , T p ) ] D(t,τ, T p )= e t/τ τ 1 1 e T p /τ
χ 2 = k=1 N bins [ I predicted ( t k ) I measured ( t k )] 2 I predicted ( t k )
I predicted (t)=( I mod (t)+ I mod (t+T) )IRF(t)+H
H= p B t exp (1r)+ r N bins F
R F b = F b F b + F g R F g = F g F b + F g

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