Abstract

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.

© 2011 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
    [CrossRef] [PubMed]
  2. 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]
  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]
  4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express 14(10), 4395–4402 (2006).
    [CrossRef] [PubMed]
  5. E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
    [CrossRef] [PubMed]
  6. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
    [CrossRef] [PubMed]
  7. 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]
  8. V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
    [CrossRef] [PubMed]
  9. V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009).
    [CrossRef]
  10. 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]
  11. Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009).
    [CrossRef] [PubMed]
  12. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008).
    [CrossRef] [PubMed]
  13. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
    [CrossRef] [PubMed]
  14. 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]
  15. J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009).
    [CrossRef]
  16. P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. Van Sark, D. J. Van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, “High sensitivity spectrograph for use in fluorescence microscopy,” Appl. Spectrosc. 55(8), 1005–1012 (2001).
    [CrossRef]
  17. A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. K. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
    [CrossRef] [PubMed]
  18. J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
    [CrossRef] [PubMed]
  19. A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011).
    [CrossRef] [PubMed]
  20. G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
    [CrossRef] [PubMed]
  21. A. Amelink and H. J. Sterenborg, “Measurement of the local optical properties of turbid media by differential path-length spectroscopy,” Appl. Opt. 43(15), 3048–3054 (2004).
    [CrossRef] [PubMed]
  22. J. A. Gardecki and M. Maroncelli, “Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy,” Appl. Spectrosc. 52(9), 1179–1189 (1998).
    [CrossRef]
  23. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
    [CrossRef] [PubMed]
  24. R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996).
    [CrossRef] [PubMed]
  25. K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
    [CrossRef] [PubMed]
  26. N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
    [CrossRef] [PubMed]
  27. G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
    [CrossRef] [PubMed]
  28. M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
    [CrossRef] [PubMed]
  29. T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
    [CrossRef] [PubMed]
  30. Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
    [CrossRef] [PubMed]

2011 (2)

2009 (3)

J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009).
[CrossRef]

V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009).
[CrossRef]

Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009).
[CrossRef] [PubMed]

2008 (5)

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (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]

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[CrossRef] [PubMed]

D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett. 33(20), 2365–2367 (2008).
[CrossRef] [PubMed]

2007 (4)

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[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]

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (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]

2006 (1)

2005 (4)

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]

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

2004 (1)

2003 (2)

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (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)

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

2001 (1)

1998 (1)

1996 (1)

R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996).
[CrossRef] [PubMed]

1992 (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

1988 (1)

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [PubMed]

1970 (1)

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [PubMed]

1962 (1)

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

Abramovic, Z.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Altmeyer, P.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Amelink, A.

Andersson, A.

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
[CrossRef] [PubMed]

Asselbergs, M. A. H.

Bader, A. N.

Balaban, R. S.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

Baumgärtl, H.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Bendsoe, N.

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
[CrossRef] [PubMed]

Birch, D. J.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [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]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Blinova, K.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Boja, E. S.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Cantu, G.

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
[CrossRef] [PubMed]

Carroll, S.

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

Chance, B.

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [PubMed]

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

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]

Cohen, P.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

Colonna, A.

Combs, C. A.

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

de Beer, E. L.

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. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[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]

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

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (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]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Esposito, A.

Evans, N. D.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

Falck, B.

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
[CrossRef] [PubMed]

Frederix, P. L. T. M.

Galeotti, T.

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [PubMed]

Gardecki, J. A.

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]

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Gerritsen, H. C.

A. Esposito, A. N. Bader, S. C. Schlachter, D. J. van den Heuvel, G. S. K. Schierle, A. R. Venkitaraman, C. F. Kaminski, and H. C. Gerritsen, “Design and application of a confocal microscope for spectrally resolved anisotropy imaging,” Opt. Express 19(3), 2546–2555 (2011).
[CrossRef] [PubMed]

A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011).
[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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[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]

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

P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. Van Sark, D. J. Van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, “High sensitivity spectrograph for use in fluorescence microscopy,” Appl. Spectrosc. 55(8), 1005–1012 (2001).
[CrossRef]

Ghukasyan, V. V.

V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009).
[CrossRef]

Gnudi, L.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

Griffiths, G. L.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Hamelink, W.

Heikal, A. A.

Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009).
[CrossRef] [PubMed]

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]

Herde, M.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Herman, B. A.

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
[CrossRef] [PubMed]

Hou, H.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[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]

Jo, J. A.

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
[CrossRef] [PubMed]

Jobsis, F.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

Jobsis, P. D.

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

Johan van Voskuilen, C.

Johnson, M. L.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

Kaminski, C. F.

Kao, F.-J.

V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009).
[CrossRef]

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]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Kennedy, J.

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [PubMed]

Khan, N.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Kristl, J.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Lakowicz, J. R.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[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]

Leroy, F.

Levine, R. L.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Li, D.

Lübbers, D. W.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Maroncelli, M.

Mayer, D. H.

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [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]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

Palero, J.

J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009).
[CrossRef]

Palero, J. A.

A. N. Bader, A.-M. Pena, C. Johan van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, “Fast nonlinear spectral microscopy of in vivo human skin,” Biomed. Opt. Express 2(2), 365–373 (2011).
[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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[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]

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

Paterson, P. E.

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [PubMed]

Pena, A.-M.

Pickup, J. C.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

Pottier, R.

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [PubMed]

Qu, J. Y.

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]

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Ramanujan, V. K.

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
[CrossRef] [PubMed]

Richards-Kortum, R.

R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996).
[CrossRef] [PubMed]

Riching, K. M.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (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]

Rolinski, O. J.

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

Ronquist, G.

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
[CrossRef] [PubMed]

Rothstein, E. C.

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[CrossRef] [PubMed]

Ruddy, B.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Schierle, G. S. K.

Schlachter, S. C.

Schoener, B.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

Sentjurc, M.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Sevick-Muraca, E.

R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996).
[CrossRef] [PubMed]

Shi, Z. D.

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Skala, M. C.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (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]

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]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[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]

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

A. Amelink and H. J. Sterenborg, “Measurement of the local optical properties of turbid media by differential path-length spectroscopy,” Appl. Opt. 43(15), 3048–3054 (2004).
[CrossRef] [PubMed]

Struk, A.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Stücker, M.

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Swartz, H. M.

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

van den Heuvel, D. J.

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. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[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]

van der Ploeg-van den Heuvel, A.

van Rossum, G. D.

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [PubMed]

Van Sark, W. G. J. H. M.

van Weelden, H.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[CrossRef] [PubMed]

Vaughan, E. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Venkitaraman, A. R.

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]

Vrotsos, K. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Weagle, G.

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [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]

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]

White, J. G.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[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]

Yan, L.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Yu, Q.

Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009).
[CrossRef] [PubMed]

Zheng, W.

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]

Annu. Rev. Phys. Chem. (1)

R. Richards-Kortum and E. Sevick-Muraca, “Quantitative optical spectroscopy for tissue diagnosis,” Annu. Rev. Phys. Chem. 47(1), 555–606 (1996).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Spectrosc. (2)

Biochemistry (1)

K. Blinova, R. L. Levine, E. S. Boja, G. L. Griffiths, Z. D. Shi, B. Ruddy, and R. S. Balaban, “Mitochondrial NADH fluorescence is enhanced by complex I binding,” Biochemistry 47(36), 9636–9645 (2008).
[CrossRef] [PubMed]

Biomed. Opt. Express (1)

Biophys. J. (2)

E. C. Rothstein, S. Carroll, C. A. Combs, P. D. Jobsis, and R. S. Balaban, “Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters,” Biophys. J. 88(3), 2165–2176 (2005).
[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]

Cancer Res. (1)

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005).
[CrossRef] [PubMed]

Eur. J. Biochem. (1)

T. Galeotti, G. D. van Rossum, D. H. Mayer, and B. Chance, “On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues,” Eur. J. Biochem. 17(3), 485–496 (1970).
[CrossRef] [PubMed]

Exp. Dermatol. (1)

G. Ronquist, A. Andersson, N. Bendsoe, and B. Falck, “Human epidermal energy metabolism is functionally anaerobic,” Exp. Dermatol. 12(5), 572–579 (2003).
[CrossRef] [PubMed]

Imaging Microsc. (1)

J. Palero, “Nonlinear spectral imaging microscopy,” Imaging Microsc. 11(1), 22–25 (2009).
[CrossRef]

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. (2)

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]

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]

J. Microsc. (1)

V. K. Ramanujan, J. A. Jo, G. Cantu, and B. A. Herman, “Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells,” J. Microsc. 230(3), 329–338 (2008).
[CrossRef] [PubMed]

J. Photochem. Photobiol. B (3)

Q. Yu and A. A. Heikal, “Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level,” J. Photochem. Photobiol. B 95(1), 46–57 (2009).
[CrossRef] [PubMed]

N. D. Evans, L. Gnudi, O. J. Rolinski, D. J. Birch, and J. C. Pickup, “Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus,” J. Photochem. Photobiol. B 80(2), 122–129 (2005).
[CrossRef] [PubMed]

G. Weagle, P. E. Paterson, J. Kennedy, and R. Pottier, “The nature of the chromophore responsible for naturally occurring fluorescence in mouse skin,” J. Photochem. Photobiol. B 2(3), 313–320 (1988).
[CrossRef] [PubMed]

J. Phys. Chem. C (1)

V. V. Ghukasyan and F.-J. Kao, “Monitoring cellular metabolism with fluorescence lifetime of reduced nicotinamide adenine dinucleotide,” J. Phys. Chem. C 113(27), 11532–11540 (2009).
[CrossRef]

J. Physiol. (1)

M. Stücker, A. Struk, P. Altmeyer, M. Herde, H. Baumgärtl, and D. W. Lübbers, “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis,” J. Physiol. 538(3), 985–994 (2002).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Photochem. Photobiol. Sci. (1)

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. Sterenborg, H. van Weelden, and H. C. Gerritsen, “In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues,” Photochem. Photobiol. Sci. 7(11), 1422–1425 (2008).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (3)

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]

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A. 89(4), 1271–1275 (1992).
[CrossRef] [PubMed]

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Science (1)

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, “Intracellular oxidation-reduction states in vivo,” Science 137(3529), 499–508 (1962).
[CrossRef] [PubMed]

Skin Pharmacol. Physiol. (1)

Z. Abramovic, M. Sentjurc, J. Kristl, N. Khan, H. Hou, and H. M. Swartz, “Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo,” Skin Pharmacol. Physiol. 20(2), 77–84 (2007).
[CrossRef] [PubMed]

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1
Fig. 1

Schematic diagram of the spectral imaging setup. The intrinsic emission of a sample is epi-detected without a pinhole. The signal is spectrally dispersed by two prisms, focused with an achromat, and detected with a back-illuminated CCD array.

Fig. 2
Fig. 2

Representative time-lapse RGB spectral images of mouse keratinocytes in vivo before (t < 0 min) and during (t > 0 min) ischemia showing increase and decrease in autofluorescence (AF). Red arrows: hair follicles. Colors represent emission wavelength as indicated in color bar. All images are 224 × 224 pixels, 100 µm × 100 µm.

Fig. 3
Fig. 3

A: Color map of the temporal behavior of the autofluorescence spectra obtained from the pixel-averaged spectral data from a region of interest (ROI; yellow box in Fig. 2). White line indicates the average peak wavelength (λ = 456 nm) of the autofluorescence spectra before ischemia. Black dotted line indicates the autofluorescence peak wavelength depicting spectral blue-shift after ischemia (t > 0 min) and a red-shift at t > 50 min. B: Normalized integrated autofluorescence intensity (integrated between 400 nm and 600 nm). C: ROI pixel-averaged autofluorescence spectra at representative time points showing spectral variation during ischemia. D: Difference spectra (relative to AF spectrum at t = 0 min) at representative time points.

Fig. 4
Fig. 4

A: Three spectral components used for spectral unmixing with peak/ full-width-at-half-maximum (FWHM) values of: (1) 448 nm/ 91 nm; (2) 459 nm/ 91 nm; and (3) 528 nm /77 nm, attributed to protein-bound and free NADH and flavins, respectively. Curves are normalized to the peaks’ amplitude. B: Spectral unmixing analysis showing the spectral components (1 to 3) attributed to: (1) protein-bound NADH; (2) free NADH; and (3) flavins, and the sum of the fitted components. Also shown is the residual of the fit. Fitting r2 coefficient of determination is 0.996. C: Color map of the χ2 error obtained by global fitting ten arbitrary difference spectra as a function of the peak wavelengths of the first two components. The intersection point of the two dotted lines indicates the local minimum (λ1 = 448 nm, λ2 = 459 nm). D and E: Dependence of the global fitting error to the peak wavelength of spectral component 1 (at λ2 = 459 nm) and peak wavelength of spectral component 2 (at λ1 = 448 nm), respectively.

Fig. 5
Fig. 5

A and B: Time-dependence of the relative amplitudes relative to the average component amplitudes at t < 0 min (normoxic) of each of the fitted components from two independent in vivo experiment sets. A minimum of 25 epidermal cells (within the region of interest as depicted in Fig. 2, yellow box), was measured for each time point, for each experiment. Reference (normoxic) condition (t ≤ 0 min), ischemia (t > 0 min).

Fig. 6
Fig. 6

Left axis: Shown are the amplitudes of the spectral components A1 and A2 attributed to protein-bound and free NADH, respectively, normalized to the average pre-ischemic values. Right axis: Relative ratio between the amplitudes of the two spectral components A2 and A1, attributed to free and protein-bound NADH, respectively.

Metrics