Abstract

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.

© 2016 Optical Society of America

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    [Crossref] [PubMed]
  2. B. Chance, “Mitochondrial NADH redox state, monitoring discovery and deployment in tissue,” Methods Enzymol. 385, 361–370 (2004).
    [Crossref] [PubMed]
  3. M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
    [Crossref] [PubMed]
  4. A. Mayevsky and B. Chance, “A new long-term method for the measurement of NADH fluorescence in intact rat brain with chronically implanted cannula,” Adv. Exp. Med. Biol. 37, 239–244 (1973).
    [Crossref] [PubMed]
  5. N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
    [Crossref] [PubMed]
  6. C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
    [Crossref] [PubMed]
  7. C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential,” PLoS One 7(11), e48014 (2012).
    [Crossref] [PubMed]
  8. K. Torno, B. K. Wright, M. R. Jones, M. A. Digman, E. Gratton, and M. Phillips, “Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH,” Curr. Microbiol. 66(4), 365–367 (2013).
    [Crossref] [PubMed]
  9. B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
    [Crossref] [PubMed]
  10. B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
    [Crossref] [PubMed]
  11. J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
    [Crossref] [PubMed]
  12. D. M. Jameson, V. Thomas, and D. M. Zhou, “Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase,” Biochim. Biophys. Acta 994(2), 187–190 (1989).
    [Crossref] [PubMed]
  13. 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]
  14. B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (1996).
    [Crossref] [PubMed]
  15. S. A. Sánchez, T. L. Hazlett, J. E. Brunet, and D. M. Jameson, “Aggregation states of mitochondrial malate dehydrogenase,” Protein Sci. 7(10), 2184–2189 (1998).
    [Crossref] [PubMed]
  16. 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]
  17. 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]
  18. P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
    [Crossref] [PubMed]
  19. M. Skala and N. Ramanujam, “Multiphoton redox ratio imaging for metabolic monitoring in vivo,” Methods Mol. Biol. 594, 155–162 (2010).
    [Crossref] [PubMed]
  20. T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
    [Crossref] [PubMed]
  21. A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
    [Crossref] [PubMed]
  22. S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
    [Crossref] [PubMed]
  23. C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
    [Crossref] [PubMed]
  24. A. V. Meleshina, V. V. Dudenkova, M. V. Shirmanova, V. I. Shcheslavskiy, W. Becker, A. S. Bystrova, E. I. Cherkasova, and E. V. Zagaynova, “Probing metabolic states of differentiating stem cells using two-photon FLIM,” Sci. Rep. 6, 21853 (2016).
    [Crossref] [PubMed]
  25. 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]
  26. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
    [Crossref] [PubMed]
  27. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
    [Crossref] [PubMed]
  28. M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, “Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli,” J. Biol. Chem. 263(33), 17715–17723 (1988).
    [PubMed]
  29. R. D. Fugate and P. S. Song, “Spectroscopic characterization of beta-lactoglobulin-retinol complex,” Biochim. Biophys. Acta 625(1), 28–42 (1980).
    [Crossref] [PubMed]
  30. R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
    [Crossref] [PubMed]
  31. D. A. Kolb and G. Weber, “Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide oxalate,” Biochemistry 14(20), 4471–4476 (1975).
    [Crossref] [PubMed]
  32. Q. Zhang, D. W. Piston, and R. H. Goodman, “Regulation of corepressor function by nuclear NADH,” Science 295(5561), 1895–1897 (2002).
    [PubMed]
  33. A. A. Heikal, “Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies,” Biomarkers Med. 4(2), 241–263 (2010).
    [Crossref] [PubMed]
  34. R. A. Neher and E. Neher, “Applying spectral fingerprinting to the analysis of FRET images,” Microsc. Res. Tech. 64(2), 185–195 (2004).
    [Crossref] [PubMed]
  35. J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
    [Crossref] [PubMed]
  36. A. Woehler, J. Wlodarczyk, and E. Neher, “Signal/noise analysis of FRET-based sensors,” Biophys. J. 99(7), 2344–2354 (2010).
    [Crossref] [PubMed]
  37. A. Zeug, A. Woehler, E. Neher, and E. G. Ponimaskin, “Quantitative intensity-based FRET approaches--a comparative snapshot,” Biophys. J. 103(9), 1821–1827 (2012).
    [Crossref] [PubMed]
  38. A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
    [Crossref] [PubMed]
  39. B. L. Scott and A. D. Hoppe, “Three-dimensional reconstruction of three-way fret microscopy improves imaging of multiple protein-protein interactions,” PLoS One 11(3), e0152401 (2016).
    [Crossref] [PubMed]
  40. R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
    [Crossref] [PubMed]
  41. D. M. Jameson and G. Mocz, “Fluorescence polarization/anisotropy approaches to study protein-ligand interactions: effects of errors and uncertainties,” Methods Mol. Biol. 305, 301–322 (2005).
    [PubMed]

2016 (4)

S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
[Crossref] [PubMed]

C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
[Crossref] [PubMed]

A. V. Meleshina, V. V. Dudenkova, M. V. Shirmanova, V. I. Shcheslavskiy, W. Becker, A. S. Bystrova, E. I. Cherkasova, and E. V. Zagaynova, “Probing metabolic states of differentiating stem cells using two-photon FLIM,” Sci. Rep. 6, 21853 (2016).
[Crossref] [PubMed]

B. L. Scott and A. D. Hoppe, “Three-dimensional reconstruction of three-way fret microscopy improves imaging of multiple protein-protein interactions,” PLoS One 11(3), e0152401 (2016).
[Crossref] [PubMed]

2015 (2)

R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
[Crossref] [PubMed]

N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
[Crossref] [PubMed]

2014 (2)

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
[Crossref] [PubMed]

2013 (2)

K. Torno, B. K. Wright, M. R. Jones, M. A. Digman, E. Gratton, and M. Phillips, “Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH,” Curr. Microbiol. 66(4), 365–367 (2013).
[Crossref] [PubMed]

A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013).
[Crossref] [PubMed]

2012 (5)

A. Zeug, A. Woehler, E. Neher, and E. G. Ponimaskin, “Quantitative intensity-based FRET approaches--a comparative snapshot,” Biophys. J. 103(9), 1821–1827 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
[Crossref] [PubMed]

C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
[Crossref] [PubMed]

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

2011 (2)

D. J. Bonda, H. G. Lee, A. Camins, M. Pallàs, G. Casadesus, M. A. Smith, and X. Zhu, “The sirtuin pathway in ageing and Alzheimer disease: Mechanistic and therapeutic considerations,” Lancet Neurol. 10(3), 275–279 (2011).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

2010 (3)

M. Skala and N. Ramanujam, “Multiphoton redox ratio imaging for metabolic monitoring in vivo,” Methods Mol. Biol. 594, 155–162 (2010).
[Crossref] [PubMed]

A. A. Heikal, “Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies,” Biomarkers Med. 4(2), 241–263 (2010).
[Crossref] [PubMed]

A. Woehler, J. Wlodarczyk, and E. Neher, “Signal/noise analysis of FRET-based sensors,” Biophys. J. 99(7), 2344–2354 (2010).
[Crossref] [PubMed]

2009 (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]

J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy,” J. Cell Biol. 185(7), 1135–1148 (2009).
[Crossref] [PubMed]

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[Crossref] [PubMed]

2008 (3)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

2007 (2)

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]

2006 (1)

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

2005 (1)

D. M. Jameson and G. Mocz, “Fluorescence polarization/anisotropy approaches to study protein-ligand interactions: effects of errors and uncertainties,” Methods Mol. Biol. 305, 301–322 (2005).
[PubMed]

2004 (2)

B. Chance, “Mitochondrial NADH redox state, monitoring discovery and deployment in tissue,” Methods Enzymol. 385, 361–370 (2004).
[Crossref] [PubMed]

R. A. Neher and E. Neher, “Applying spectral fingerprinting to the analysis of FRET images,” Microsc. Res. Tech. 64(2), 185–195 (2004).
[Crossref] [PubMed]

2002 (1)

Q. Zhang, D. W. Piston, and R. H. Goodman, “Regulation of corepressor function by nuclear NADH,” Science 295(5561), 1895–1897 (2002).
[PubMed]

1998 (1)

S. A. Sánchez, T. L. Hazlett, J. E. Brunet, and D. M. Jameson, “Aggregation states of mitochondrial malate dehydrogenase,” Protein Sci. 7(10), 2184–2189 (1998).
[Crossref] [PubMed]

1996 (1)

B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (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]

1989 (1)

D. M. Jameson, V. Thomas, and D. M. Zhou, “Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase,” Biochim. Biophys. Acta 994(2), 187–190 (1989).
[Crossref] [PubMed]

1988 (1)

M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, “Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli,” J. Biol. Chem. 263(33), 17715–17723 (1988).
[PubMed]

1980 (1)

R. D. Fugate and P. S. Song, “Spectroscopic characterization of beta-lactoglobulin-retinol complex,” Biochim. Biophys. Acta 625(1), 28–42 (1980).
[Crossref] [PubMed]

1975 (1)

D. A. Kolb and G. Weber, “Quantitative demonstration of the reciprocity of ligand effects in the ternary complex of chicken heart lactate dehydrogenase with nicotinamide adenine dinucleotide oxalate,” Biochemistry 14(20), 4471–4476 (1975).
[Crossref] [PubMed]

1973 (1)

A. Mayevsky and B. Chance, “A new long-term method for the measurement of NADH fluorescence in intact rat brain with chronically implanted cannula,” Adv. Exp. Med. Biol. 37, 239–244 (1973).
[Crossref] [PubMed]

Alfonso-García, A.

R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
[Crossref] [PubMed]

Andersen, P. E.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Andrews, L. M.

B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
[Crossref] [PubMed]

Bain, A. J.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Becker, W.

S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
[Crossref] [PubMed]

A. V. Meleshina, V. V. Dudenkova, M. V. Shirmanova, V. I. Shcheslavskiy, W. Becker, A. S. Bystrova, E. I. Cherkasova, and E. V. Zagaynova, “Probing metabolic states of differentiating stem cells using two-photon FLIM,” Sci. Rep. 6, 21853 (2016).
[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]

Blacker, T. S.

T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
[Crossref] [PubMed]

Bonda, D. J.

D. J. Bonda, H. G. Lee, A. Camins, M. Pallàs, G. Casadesus, M. A. Smith, and X. Zhu, “The sirtuin pathway in ageing and Alzheimer disease: Mechanistic and therapeutic considerations,” Lancet Neurol. 10(3), 275–279 (2011).
[Crossref] [PubMed]

Breymayer, J.

S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
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S. A. Sánchez, T. L. Hazlett, J. E. Brunet, and D. M. Jameson, “Aggregation states of mitochondrial malate dehydrogenase,” Protein Sci. 7(10), 2184–2189 (1998).
[Crossref] [PubMed]

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N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
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A. V. Meleshina, V. V. Dudenkova, M. V. Shirmanova, V. I. Shcheslavskiy, W. Becker, A. S. Bystrova, E. I. Cherkasova, and E. V. Zagaynova, “Probing metabolic states of differentiating stem cells using two-photon FLIM,” Sci. Rep. 6, 21853 (2016).
[Crossref] [PubMed]

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M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

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B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (1996).
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S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
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R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
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C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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Cinquin, O.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
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A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
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R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
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K. Torno, B. K. Wright, M. R. Jones, M. A. Digman, E. Gratton, and M. Phillips, “Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH,” Curr. Microbiol. 66(4), 365–367 (2013).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
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Donovan, P. J.

C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
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T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, and M. R. Duchen, “Separating NADH and NADPH fluorescence in live cells and tissues using FLIM,” Nat. Commun. 5, 3936 (2014).
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A. V. Meleshina, V. V. Dudenkova, M. V. Shirmanova, V. I. Shcheslavskiy, W. Becker, A. S. Bystrova, E. I. Cherkasova, and E. V. Zagaynova, “Probing metabolic states of differentiating stem cells using two-photon FLIM,” Sci. Rep. 6, 21853 (2016).
[Crossref] [PubMed]

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C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
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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).
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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).
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P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
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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).
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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, “In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia,” J. Biomed. Opt. 12(2), 024014 (2007).
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Flanagan, L. A.

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential,” PLoS One 7(11), e48014 (2012).
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M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
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A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
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[Crossref] [PubMed]

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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).
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N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
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M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, “Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli,” J. Biol. Chem. 263(33), 17715–17723 (1988).
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R. Datta, A. Alfonso-García, R. Cinco, and E. Gratton, “Fluorescence lifetime imaging of endogenous biomarker of oxidative stress,” Sci. Rep. 5, 9848 (2015).
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N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
[Crossref] [PubMed]

K. Torno, B. K. Wright, M. R. Jones, M. A. Digman, E. Gratton, and M. Phillips, “Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH,” Curr. Microbiol. 66(4), 365–367 (2013).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
[Crossref] [PubMed]

C. Stringari, J. L. Nourse, L. A. Flanagan, and E. Gratton, “Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential,” PLoS One 7(11), e48014 (2012).
[Crossref] [PubMed]

C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
[Crossref] [PubMed]

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Grigiene, J.

M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
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B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (1996).
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M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

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S. A. Sánchez, T. L. Hazlett, J. E. Brunet, and D. M. Jameson, “Aggregation states of mitochondrial malate dehydrogenase,” Protein Sci. 7(10), 2184–2189 (1998).
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B. L. Scott and A. D. Hoppe, “Three-dimensional reconstruction of three-way fret microscopy improves imaging of multiple protein-protein interactions,” PLoS One 11(3), e0152401 (2016).
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N. Plotegher, C. Stringari, S. Jahid, M. Veronesi, S. Girotto, E. Gratton, and L. Bubacco, “NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells,” FASEB J. 29(6), 2484–2494 (2015).
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D. M. Jameson, V. Thomas, and D. M. Zhou, “Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase,” Biochim. Biophys. Acta 994(2), 187–190 (1989).
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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]

Jones, M. R.

K. Torno, B. K. Wright, M. R. Jones, M. A. Digman, E. Gratton, and M. Phillips, “Real-time analysis of metabolic activity within Lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH,” Curr. Microbiol. 66(4), 365–367 (2013).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, J. Markham, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy,” Biophys. J. 103(1), L7–L9 (2012).
[Crossref] [PubMed]

B. K. Wright, L. M. Andrews, M. R. Jones, C. Stringari, M. A. Digman, and E. Gratton, “Phasor-FLIM analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells,” Microsc. Res. Tech. 75(12), 1717–1722 (2012).
[Crossref] [PubMed]

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S. Kalinina, J. Breymayer, P. Schäfer, E. Calzia, V. Shcheslavskiy, W. Becker, and A. Rück, “Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping,” J. Biophotonics 2016, 297 (2016).
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C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
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M. R. Kasimova, J. Grigiene, K. Krab, P. H. Hagedorn, H. Flyvbjerg, P. E. Andersen, and I. M. Møller, “The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions,” Plant Cell 18(3), 688–698 (2006).
[Crossref] [PubMed]

Keely, P. J.

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[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]

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B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (1996).
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A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
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[Crossref] [PubMed]

Lakowicz, J. R.

B. Kierdaszuk, H. Malak, I. Gryczynski, P. Callis, and J. R. Lakowicz, “Fluorescence of reduced nicotinamides using one- and two-photon excitation,” Biophys. Chem. 62(1-3), 1–13 (1996).
[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]

Lee, C.

R. A. Colyer, C. Lee, and E. Gratton, “A novel fluorescence lifetime imaging system that optimizes photon efficiency,” Microsc. Res. Tech. 71(3), 201–213 (2008).
[Crossref] [PubMed]

Lee, H. G.

D. J. Bonda, H. G. Lee, A. Camins, M. Pallàs, G. Casadesus, M. A. Smith, and X. Zhu, “The sirtuin pathway in ageing and Alzheimer disease: Mechanistic and therapeutic considerations,” Lancet Neurol. 10(3), 275–279 (2011).
[Crossref] [PubMed]

Levic, D. S.

A. Venkateswaran, K. R. Sekhar, D. S. Levic, D. B. Melville, T. A. Clark, W. M. Rybski, A. J. Walsh, M. C. Skala, P. A. Crooks, E. W. Knapik, and M. L. Freeman, “The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development,” Cancer Res. 74(1), 38–43 (2014).
[Crossref] [PubMed]

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M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, “Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli,” J. Biol. Chem. 263(33), 17715–17723 (1988).
[PubMed]

Li, E.

M. S. Levin, B. Locke, N. C. Yang, E. Li, and J. I. Gordon, “Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli,” J. Biol. Chem. 263(33), 17715–17723 (1988).
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C. Stringari, R. A. Edwards, K. T. Pate, M. L. Waterman, P. J. Donovan, and E. Gratton, “Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH,” Sci. Rep. 2, 568 (2012).
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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).
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A. Woehler, J. Wlodarczyk, and E. Neher, “Signal/noise analysis of FRET-based sensors,” Biophys. J. 99(7), 2344–2354 (2010).
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A. Zeug, A. Woehler, E. Neher, and E. G. Ponimaskin, “Quantitative intensity-based FRET approaches--a comparative snapshot,” Biophys. J. 103(9), 1821–1827 (2012).
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J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
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[Crossref] [PubMed]

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C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
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[Crossref] [PubMed]

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008).
[Crossref] [PubMed]

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Q. Zhang, D. W. Piston, and R. H. Goodman, “Regulation of corepressor function by nuclear NADH,” Science 295(5561), 1895–1897 (2002).
[PubMed]

Zhang, X. H.

C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
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C. Kang, H. L. Wu, C. Zhou, S. X. Xiang, X. H. Zhang, Y. J. Yu, and R. Q. Yu, “Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage,” Anal. Chim. Acta 910, 36–44 (2016).
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Zhou, D. M.

D. M. Jameson, V. Thomas, and D. M. Zhou, “Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase,” Biochim. Biophys. Acta 994(2), 187–190 (1989).
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Figures (7)

Fig. 1
Fig. 1 a) Phasor representation of a single exponential component on the universal circle (blue circle) and a component of unmodulated light at the origin (black circle). b) Experimental value of the free-NADH lifetime (blue circle). When different amounts of unmodulated light are added to the sample, the phasor position moves towards the origin according to phasor addition. In this figure, we use a physical source of unmodulated light to shift the position of the phasor of the free-NADH. A mathematical addition of a constant to each pixel of the decay gives the same plot.
Fig. 2
Fig. 2 Relationship between the experimental value of the g coordinate of the phasor plot and the value of g calculated using Eq. (2), for different amounts of external light L
Fig. 3
Fig. 3 Linear combination of free and bound NADH. a) The blue and red circles indicate the phasor position for free and bound NADH, respectively. Underlying the circles is the experimentally determined phasors of the free and bound form of NADH. Every possible combination in a pixel of free and bound NADH will be on the green line joining the phasor of the free and bound NADH. b) Upon addition of the external light, the phasor of the free and bound NADH moves toward the origin, but in different amounts as indicated by the red line. However, all points of the shifted red line have the same concentration equal to the concentration of the calibration solution since the solution phasor has moved by a given amount along the line of linear combination with the phasor at the origin.
Fig. 4
Fig. 4 a) Intensity image of a CHO-K1 cell in a gray scale. b) The phasor plot shows two characteristic clusters shown by the red and green cursors. c) All pixels in the red cursor are painted in red and all pixels in the green cursor are painted in green.
Fig. 5
Fig. 5 a) Same CHO-K1 cell as in Fig. 4 with external light added. Note that the contrast of the image is quite different from Fig. 4(a). b) Comparing panels b in Figs. 4 and 5 we can determine that some pixels of the image have moved more than others toward the origin, indicating a different concentration of NADH in those pixels. c) Same image colored according the cursors selection in panel b.
Fig. 6
Fig. 6 The cell phasor plots are shown in the background of each of the panels in this figure together with the phasor plot obtained with a certain amount of external light added. a) The amount of light added moves the red line to the position shown in panel a). All pixels with phasors along the red line have equal concentration (0.29 mM). These pixels correspond to the nucleus and part of the cytoplasm of the cell as shown in Fig. 5(b). b) Using less external light, the red line moves away from the origin. All points along this line have a concentration of 0.89mM. These points are selected by the red cursor in Fig. 5(b) and they map to the brighter pixels in the cytoplasm. c) Increasing the amount of added light, the red line moves toward the origin. The pixels selected in this region of the phasor plot map to certain regions of the cytoplasm where the concentration is about 0.12mM. d) Zoomed part of panel c near the origin.
Fig. 7
Fig. 7 a) Intensity image of NADH in a CHO-K1 cell. The color scale is in counts/pixel/frame. b) NADH concentration image according to the method described in this paper. The concentration scale was calibrated using a solution 1mM free-NADH.

Equations (5)

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

M= L L+F
g= g 0 L L+F
K D  =  [E] [L] [EL]
[ EL ]=  ( K D + E 0 + L 0 ) [ ( K D + E 0 + L o ) 2 4 E 0 L 0 ] 2
EL%= ( [EL] [E] )*100

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