Abstract

An optimized system for fast, high-resolution spectral imaging of in vivo human skin is developed and evaluated. The spectrograph is composed of a dispersive prism in combination with an electron multiplying CCD camera. Spectra of autofluorescence and second harmonic generation (SHG) are acquired at a rate of 8 kHz and spectral images within seconds. Image quality is significantly enhanced by the simultaneous recording of background spectra. In vivo spectral images of 224 × 224 pixels were acquired, background corrected and previewed in real RGB color in 6.5 seconds. A clear increase in melanin content in deeper epidermal layers in in vivo human skin was observed.

© 2011 OSA

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2010 (1)

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
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2009 (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]

2008 (5)

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

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

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, B. R. Chen, and E. M. C. Hillman, “Hyperspectral in vivo two-photon microscopy of intrinsic contrast,” Opt. Lett. 33(18), 2164–2166 (2008).
[CrossRef] [PubMed]

2007 (3)

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (3)

A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Schanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13(16), 6268–6274 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

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

2003 (3)

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[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]

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. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

2002 (1)

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

2000 (2)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

1999 (1)

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

1998 (2)

V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[CrossRef] [PubMed]

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

1997 (1)

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

1996 (1)

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

1995 (1)

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Altmeyer, P.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Beaurepaire, E.

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

Birch, D. J. S.

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

Bouchard, M. B.

Boulesteix, T.

Breitbart, W.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Burgess, S. A.

Centonze, V. E.

V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[CrossRef] [PubMed]

Chen, B. R.

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Cox, G.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Crestani, B.

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

de Bruijn, H. S.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
[CrossRef] [PubMed]

De Grauw, C. J.

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

Débarre, D.

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dimitrow, E.

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]

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]

Ehlert, J.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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]

Fabre, A.

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

Fischer, F.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Fligny, C.

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

Fraser, I.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Freyer, W.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Gerritsen, H. C.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
[CrossRef] [PubMed]

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

Gorrell, M. D.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Gratton, E.

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

Greinert, R.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Hancewicz, T. M.

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

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Hernest, M.

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

Hillman, E. M. C.

Hoffmann, K.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Jones, A.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Kaatz, M.

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]

Kable, E.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Kaplan, P. D.

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

Kiefer, J.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Kitahara, T.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[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]

Koenig, K.

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]

König, K.

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]

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

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

Laiho, L. H.

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

Latouche, G.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

Leupold, D.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Manconi, F.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Marchal-Somme, J.

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

Martin, J. L.

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

Masters, B. R.

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

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Norgauer, 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]

Noriaki, N.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[CrossRef] [PubMed]

Osanai, O.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[CrossRef] [PubMed]

Palero, J. A.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
[CrossRef] [PubMed]

Pelet, S.

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

Pena, A. M.

Piston, D. W.

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

Puschmann, S.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Radosevich, A. J.

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]

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]

Sakai, S.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[CrossRef] [PubMed]

Schanne-Klein, M. C.

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Schanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13(16), 6268–6274 (2005).
[CrossRef] [PubMed]

Shear, J. B.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

So, P. T.

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

So, P. T. C.

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

Sterenborg, H. J.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

Sterenborg, H. J. C. M.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Strupler, M.

Stücker, M.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Sugata, K.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[CrossRef] [PubMed]

Sytsma, J.

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

Takema, Y.

K. Sugata, S. Sakai, N. Noriaki, O. Osanai, T. Kitahara, and Y. Takema, “Imaging of melanin distribution using multiphoton autofluorescence decay curves,” Skin Res. Technol. 16(1), 55–59 (2010).
[CrossRef] [PubMed]

Teuchner, K.

K. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

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

Tharaux, P. L.

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[CrossRef] [PubMed]

M. Strupler, A. M. Pena, M. Hernest, P. L. Tharaux, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Second harmonic imaging and scoring of collagen in fibrotic tissues,” Opt. Express 15(7), 4054–4065 (2007).
[CrossRef] [PubMed]

van der Ploeg van den Heuvel, A.

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

van der Ploeg-van den Heuvel, A.

Volkmer, A.

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

Volkmer, B.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

Vroom, J. M.

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

Webb, W. W.

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. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wepf, R.

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

White, J. G.

V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[CrossRef] [PubMed]

Williams, R. M.

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. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Xu, C.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Zipfel, W.

C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, and W. W. Webb, “Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy,” Proc. Natl. Acad. Sci. U.S.A. 93(20), 10763–10768 (1996).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Biophys. J. (4)

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

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. 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]

S. Huang, A. A. Heikal, and W. W. Webb, “Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein,” Biophys. J. 82(5), 2811–2825 (2002).
[CrossRef] [PubMed]

V. E. Centonze and J. G. White, “Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging,” Biophys. J. 75(4), 2015–2024 (1998).
[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 (1)

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

J. Biomed. Opt. (5)

F. Fischer, B. Volkmer, S. Puschmann, R. Greinert, W. Breitbart, J. Kiefer, and R. Wepf, “Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm,” J. Biomed. Opt. 13(4), 041320 (2008).
[CrossRef] [PubMed]

J. A. Palero, G. Latouche, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope,” J. Biomed. Opt. 13(4), 044019 (2008).
[CrossRef] [PubMed]

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

M. Strupler, M. Hernest, C. Fligny, J. L. Martin, P. L. Tharaux, and M. C. Schanne-Klein, “Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling,” J. Biomed. Opt. 13(5), 054041 (2008).
[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. Teuchner, J. Ehlert, W. Freyer, D. Leupold, P. Altmeyer, M. Stücker, and K. Hoffmann, “Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation,” J. Fluoresc. 10(3), 275–281 (2000).
[CrossRef]

J. Microsc. (3)

J. Sytsma, J. M. Vroom, C. J. De Grauw, and H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. 191(1), 39–51 (1998).
[CrossRef]

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, “Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy,” J. Microsc. 178(Pt 1), 20–27 (1995).
[PubMed]

J. Struct. Biol. (1)

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Microsc. Res. Tech. (1)

A. M. Pena, A. Fabre, D. Débarre, J. Marchal-Somme, B. Crestani, J. L. Martin, E. Beaurepaire, and M. C. Schanne-Klein, “Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy,” Microsc. Res. Tech. 70(2), 162–170 (2007).
[CrossRef] [PubMed]

Nat. Methods (1)

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Figures (6)

Fig. 1
Fig. 1

Left, diagram of the nonlinear spectral imaging system. Right, diagram of the spectral data recording on the EMCCD chip.

Fig. 2
Fig. 2

Effect of simultaneous background recording on the quality of the spectral image of in vivo Caucasian human skin. (Left) Individual intensity and real color RGB images of the three spectra that are recorded for each pixel (row1 = background, row2 = emission spectrum, row3 = background). (Top right) Spectra averaged over the ROI (see white rectangle). (Right bottom) Background corrected intensity and real color RGB image. XY images are 224 × 224 pixels, corresponding to 70 × 70 µm; image depth is 40 µm. Excitation power is 15 mW, acquisition time is 128 µs per pixel (6,5 s per image), and excitation wavelength is 760 nm.

Fig. 3
Fig. 3

3D nonlinear spectral imaging of in vivo human skin (Caucasian skin type). Background corrected real color RGB images are shown. XY images are 224 × 224 pixels, corresponding to 70 × 70 µm2; imaging depth is 40 µm. Excitation power is 15 mW, acquisition time is 128 µs per pixel (6,5 s per image), and excitation wavelength is 760 nm.

Fig. 4
Fig. 4

Nonlinear spectral imaging of the epidermal / dermal junction of Caucasian (red lines) and Asian (blue lines) skin. In the background corrected real color RGB images, ROIs are indicated by white squares; EC = epidermal cells, mEC = melanized epidermal cells, C = collagen fibers and E = elastic fibers. For these ROIs, the average spectra are shown on the right, where the red line indicates Caucasian skin and the blue line Asian skin (gray line is the spectrum of nonmelanized EC, added for comparison). The images are 224 × 224 pixels, corresponding to 70 × 70 µm2. Excitation power is 15 mW, acquisition time is 128 µs per pixel (6,5 s per image) and excitation wavelength is 760 nm.

Fig. 5
Fig. 5

Nonlinear spectral imaging of the epidermal / dermal junction of three volunteers with Caucasian skin. The images are 224 × 224 pixels, corresponding to 70 × 70 µm2. Excitation power is 15-20 mW, acquisition time is 128 µs per pixel (6.5 s per image) and excitation wavelength is 760 nm.

Fig. 6
Fig. 6

(Top) Effect of spectral averaging on the quality of real color RGB images. For each pixel, the spectrum is averaged over N = 1 (blue area and spectrum), N = 9 (red) or 25 pixels (green); the intensity is not averaged, see text. (Center) RGB images of Asian human skin (same as Fig. 4) for the three levels of averaging. The RGB colors on the vertical white lines are horizontally extended in the bars left of the images. (Bottom) Ternary plots displaying the relative R, G, and B contributions in the pixels of five ROIs (indicated by white boxes in the RGB image): EC = epidermal cells (light blue), pmEC = partly melanized epidermal cells (light green), mEC = melanized epidermal cells (dark green), E = elastic fibers (orange) and C = collagen fibers (violet).

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