Abstract

A fluorescence lifetime imaging microscopy (FLIM) method was developed and applied to investigate metabolic function in living human normal esophageal (HET-1) and Barrett’s adenocarcinoma (SEG-1) cells. In FLIM, image contrast is based on fluorophore excited state lifetimes, which reflect local biochemistry and molecular activity. Unique FLIM system attributes, including variable ultrafast time gating (≥200 ps), wide spectral tunability (337.1–960 nm), large temporal dynamic range (≥600 ps), and short data acquisition and processing times (15 s), enabled the study of two key molecules consumed at the termini of the oxidative phosphorylation pathway, NADH and oxygen, in living cells under controlled and calibrated environmental conditions. NADH is an endogenous cellular fluorophore detectable in living human tissues that has been shown to be a quantitative biomarker of dysplasia in the esophagus. Lifetime calibration of an oxygen-sensitive, ruthenium-based cellular stain enabled in vivo oxygen level measurements with a resolution of 8 µM over the entire physiological range (1–300 µM). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.

© 2006 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. “Cancer Facts and Figures 2005,” (American Cancer Society, Atlantic City, 2005).
  2. I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
    [PubMed]
  3. E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.
  4. H. Schneckenburger and K. König, “Fluorescence decay and imaging of NAD(P)H and flavins as metabolic indicators,” Opt Eng 31, 1447–1451 (1992).
    [Crossref]
  5. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).
  6. J. W. Dobrucki, “Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements,” J Photochem Photobiol B 65, 136–144 (2001).
    [Crossref]
  7. J. V. Houten and R. J. Watts, “Temperature dependence of the photophysical and photochemical properties of the Tris(2,2’-bipyridyl) ruthenium(II) ion in aqueous solution,” J Am Chem Soc 96, 4853–4858 (1976).
    [Crossref]
  8. W. Rudolph and M. Kempe, “Topical review: Trends in optical biomedical imaging,” J Mod Opt 44, 1617–1642 (1997).
    [Crossref]
  9. P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging,” Journal of Pathology 191, 229–234 (2000).
    [Crossref] [PubMed]
  10. Y. Chen and J. D. Mills, “Protein localization in living cells and tissues using FRET and FLIM,” Differentiation 71, 528–541 (2003).
    [Crossref] [PubMed]
  11. R. N. Day and D. W. Piston, “Spying on the hidden lives of proteins,” Nat Biotechnol 17, 425–426 (1999).
    [Crossref] [PubMed]
  12. P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
    [Crossref]
  13. W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
    [Crossref]
  14. D. Lubbers, “Optical sensors for clinical monitoring,” Acta Anaesthesiologica Scandinavica 39, 37–54 (1995).
    [Crossref]
  15. R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
    [Crossref] [PubMed]
  16. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
    [Crossref]
  17. H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
    [Crossref]
  18. G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
    [PubMed]
  19. O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
    [Crossref] [PubMed]
  20. V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
    [Crossref] [PubMed]
  21. N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
    [Crossref] [PubMed]
  22. I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
    [Crossref] [PubMed]
  23. R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
    [Crossref] [PubMed]
  24. B. W. Gibson, “The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation,” Int J Biochem Cell Biol 37, 927–934 (2005).
    [Crossref] [PubMed]
  25. L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).
  26. A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
    [Crossref] [PubMed]
  27. A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
    [Crossref] [PubMed]
  28. H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
    [Crossref] [PubMed]

2005 (2)

B. W. Gibson, “The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation,” Int J Biochem Cell Biol 37, 927–934 (2005).
[Crossref] [PubMed]

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

2004 (1)

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

2003 (6)

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
[Crossref]

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Y. Chen and J. D. Mills, “Protein localization in living cells and tissues using FRET and FLIM,” Differentiation 71, 528–541 (2003).
[Crossref] [PubMed]

A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
[Crossref] [PubMed]

2002 (2)

H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
[Crossref] [PubMed]

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

2001 (2)

J. W. Dobrucki, “Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements,” J Photochem Photobiol B 65, 136–144 (2001).
[Crossref]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

2000 (1)

P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging,” Journal of Pathology 191, 229–234 (2000).
[Crossref] [PubMed]

1999 (2)

R. N. Day and D. W. Piston, “Spying on the hidden lives of proteins,” Nat Biotechnol 17, 425–426 (1999).
[Crossref] [PubMed]

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

1997 (2)

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

W. Rudolph and M. Kempe, “Topical review: Trends in optical biomedical imaging,” J Mod Opt 44, 1617–1642 (1997).
[Crossref]

1995 (1)

D. Lubbers, “Optical sensors for clinical monitoring,” Acta Anaesthesiologica Scandinavica 39, 37–54 (1995).
[Crossref]

1994 (1)

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

1992 (2)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

H. Schneckenburger and K. König, “Fluorescence decay and imaging of NAD(P)H and flavins as metabolic indicators,” Opt Eng 31, 1447–1451 (1992).
[Crossref]

1991 (1)

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

1985 (1)

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

1976 (1)

J. V. Houten and R. J. Watts, “Temperature dependence of the photophysical and photochemical properties of the Tris(2,2’-bipyridyl) ruthenium(II) ion in aqueous solution,” J Am Chem Soc 96, 4853–4858 (1976).
[Crossref]

2nd,

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Alfano, R.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Aylott, J. W.

H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
[Crossref] [PubMed]

Backman, V.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Badizadegan, K.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Beamish, J. A.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Beer, D. G.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Berg, K.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Bleday, R. S.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Boone, C. W.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Bowman, D.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Brogan, A. P.

A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
[Crossref] [PubMed]

Carr-Locke, D. L.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Chang, C. C.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Chang, T. Y.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Chen, L. B.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Chen, Y.

Y. Chen and J. D. Mills, “Protein localization in living cells and tissues using FRET and FLIM,” Differentiation 71, 528–541 (2003).
[Crossref] [PubMed]

Crum, C. P.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Cummins, D. L.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Dasari, R. R.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Davis, S.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Day, R. N.

R. N. Day and D. W. Piston, “Spying on the hidden lives of proteins,” Nat Biotechnol 17, 425–426 (1999).
[Crossref] [PubMed]

Demidenko, E.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Dmitrovsky, E.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Dobrucki, J. W.

J. W. Dobrucki, “Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements,” J Photochem Photobiol B 65, 136–144 (2001).
[Crossref]

Draaijer, A.

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

Dragnev, K. H.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Eigenbrodt, E.

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

Feld, M. S.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Friis, R.

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

Fung, P. C.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Galati, A. J.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Georgakoudi, I.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Gerbracht, U.

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

Gerritsen, H. C.

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

Gibson, B. W.

B. W. Gibson, “The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation,” Int J Biochem Cell Biol 37, 927–934 (2005).
[Crossref] [PubMed]

Glasgold, M.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Glasgold, R.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Grinberg, O.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Hanash, S. M.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Harris, C. C.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Houten, J. V.

J. V. Houten and R. J. Watts, “Temperature dependence of the photophysical and photochemical properties of the Tris(2,2’-bipyridyl) ruthenium(II) ion in aqueous solution,” J Am Chem Soc 96, 4853–4858 (1976).
[Crossref]

Howell, B. G.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Hughes, S. J.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Iannettoni, M. D.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Jacobson, B. C.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Johnson, M. L.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

Kaighn, M. E.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Kempe, M.

W. Rudolph and M. Kempe, “Topical review: Trends in optical biomedical imaging,” J Mod Opt 44, 1617–1642 (1997).
[Crossref]

Khan, N.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Kohn, H.

A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
[Crossref] [PubMed]

König, K.

H. Schneckenburger and K. König, “Fluorescence decay and imaging of NAD(P)H and flavins as metabolic indicators,” Opt Eng 31, 1447–1451 (1992).
[Crossref]

Kopelman, R.

H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
[Crossref] [PubMed]

Kouba, D. J.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Kowalski, J.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Kuick, R. D.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Kuppusamy, P.

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Kutala, V. K.

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[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,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

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

Levine, Y. K.

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

Liegeois, N. J.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Lubbers, D.

D. Lubbers, “Optical sensors for clinical monitoring,” Acta Anaesthesiologica Scandinavica 39, 37–54 (1995).
[Crossref]

Mamelak, A. J.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Matsukura, N.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Mazurek, S.

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

Mills, J. D.

Y. Chen and J. D. Mills, “Protein localization in living cells and tissues using FRET and FLIM,” Differentiation 71, 528–541 (2003).
[Crossref] [PubMed]

Minn, F. K.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Muller, M. G.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Murphy, K.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Mycek, M.-A.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
[Crossref]

Naito, Z.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Nowaczyk, K.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

Orringer, M. B.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Pandian, R. P.

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Parinandi, N. L.

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Perelman, L. T.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Pinto, J.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Piston, D. W.

R. N. Day and D. W. Piston, “Spying on the hidden lives of proteins,” Nat Biotechnol 17, 425–426 (1999).
[Crossref] [PubMed]

Presek, P.

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

Reddel, R. R.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Resau, J. H.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Rudolph, W.

W. Rudolph and M. Kempe, “Topical review: Trends in optical biomedical imaging,” J Mod Opt 44, 1617–1642 (1997).
[Crossref]

Sanders, R.

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

Sauder, D. N.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Savage, H.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Schantz, S.

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Schneckenburger, H.

H. Schneckenburger and K. König, “Fluorescence decay and imaging of NAD(P)H and flavins as metabolic indicators,” Opt Eng 31, 1447–1451 (1992).
[Crossref]

Sheets, E. E.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Shen, J.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Shepherd, E. L.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Sloboda, R. D.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Soldes, O. S.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Song, J.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Steele, G. D.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Stoner, G. D.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Sun, D.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Swartz, H.

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Szmacinski, H.

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

Tadrous, P. J.

P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging,” Journal of Pathology 191, 229–234 (2000).
[Crossref] [PubMed]

Terasaki, M.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Thomas, G. A.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Thompson, I. A.

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Tzu, J.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Urayama, P.

W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
[Crossref]

Urayama, P. K.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Van Dam, J.

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Walker, E. S.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Wallace, M. B.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Watts, R. J.

J. V. Houten and R. J. Watts, “Temperature dependence of the photophysical and photochemical properties of the Tris(2,2’-bipyridyl) ruthenium(II) ion in aqueous solution,” J Am Chem Soc 96, 4853–4858 (1976).
[Crossref]

Welss, M. J.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Widger, W. R.

A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
[Crossref] [PubMed]

Wong, J. R.

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Xu, H.

H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
[Crossref] [PubMed]

Yadava, N.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

You, M.

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

Zahurak, M.

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Zhang, Q.

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Zhong, W.

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
[Crossref]

Zweier, J. L.

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Acta Anaesthesiologica Scandinavica (1)

D. Lubbers, “Optical sensors for clinical monitoring,” Acta Anaesthesiologica Scandinavica 39, 37–54 (1995).
[Crossref]

Analyst (1)

H. Xu, J. W. Aylott, and R. Kopelman, “Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging,” Analyst 127, 1471–1477 (2002).
[Crossref] [PubMed]

Antioxid Redox Signal (1)

V. K. Kutala, N. L. Parinandi, R. P. Pandian, and P. Kuppusamy, “Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells,” Antioxid Redox Signal 6, 597–603 (2004).
[Crossref] [PubMed]

Appl Phys B: Lasers and Optics (1)

P. K. Urayama, W. Zhong, J. A. Beamish, F. K. Minn, R. D. Sloboda, K. H. Dragnev, E. Dmitrovsky, and M.-A. Mycek, “A UV-visible fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution,” Appl Phys B: Lasers and Optics 76, 483–496 (2003).
[Crossref]

Arch Biochem Biophys (1)

R. P. Pandian, V. K. Kutala, N. L. Parinandi, J. L. Zweier, and P. Kuppusamy, “Measurement of oxygen consumption in mouse aortic endothelial cells using a microparticulate oximetry probe,” Arch Biochem Biophys 420, 169–175 (2003).
[Crossref] [PubMed]

Biochemistry (1)

N. Khan, J. Shen, T. Y. Chang, C. C. Chang, P. C. Fung, O. Grinberg, E. Demidenko, and H. Swartz, “Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism,” Biochemistry 42, 23–29 (2003).
[Crossref] [PubMed]

Br J Cancer (1)

O. S. Soldes, R. D. Kuick, I. A. Thompson, 2nd, S. J. Hughes, M. B. Orringer, M. D. Iannettoni, S. M. Hanash, and D. G. Beer, “Differential expression of Hsp27 in normal oesophagus, Barrett’s metaplasia and oesophageal adenocarcinomas,” Br J Cancer 79, 595–603 (1999).
[Crossref] [PubMed]

Cancer Cells (1)

L. B. Chen, M. J. Welss, S. Davis, R. S. Bleday, J. R. Wong, J. Song, M. Terasaki, E. L. Shepherd, E. S. Walker, and G. D. Steele, “Mitochondria in living cells: effects of growth factors and tumor promoters, alterations in carcinoma cells, and targets for therapy,” Cancer Cells 3, 433–443 (1985).

Cancer Lett. (1)

R. Glasgold, M. Glasgold, H. Savage, J. Pinto, R. Alfano, and S. Schantz, “Tissue Autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis,” Cancer Lett. 82, 33–41 (1994).
[Crossref] [PubMed]

Cancer Research (2)

G. D. Stoner, M. E. Kaighn, R. R. Reddel, J. H. Resau, D. Bowman, Z. Naito, N. Matsukura, M. You, A. J. Galati, and C. C. Harris, “Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells,” Cancer Research 51, 365–371 (1991).
[PubMed]

I. Georgakoudi, B. C. Jacobson, M. G. Muller, E. E. Sheets, K. Badizadegan, D. L. Carr-Locke, C. P. Crum, C. W. Boone, R. R. Dasari, J. Van Dam, and M. S. Feld, “NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes,” Cancer Research 62, 682–687 (2002).
[PubMed]

Differentiation (1)

Y. Chen and J. D. Mills, “Protein localization in living cells and tissues using FRET and FLIM,” Differentiation 71, 528–541 (2003).
[Crossref] [PubMed]

Exp Dermatol (1)

A. J. Mamelak, J. Kowalski, K. Murphy, N. Yadava, M. Zahurak, D. J. Kouba, B. G. Howell, J. Tzu, D. L. Cummins, N. J. Liegeois, K. Berg, and D. N. Sauder, “Downregulation of NDUFA1 and other oxidative phosphorylation-related genes is a consistent feature of basal cell carcinoma,” Exp Dermatol 14, 336–348 (2005).
[Crossref] [PubMed]

Gastroenterology (1)

I. Georgakoudi, B. C. Jacobson, J. Van Dam, V. Backman, M. B. Wallace, M. G. Muller, Q. Zhang, K. Badizadegan, D. Sun, G. A. Thomas, L. T. Perelman, and M. S. Feld, “Fluorescence, Reflectance, and Light-Scattering Spectroscopy for Evaluating Dysplasia in patients With Barrett’e Esopghagus,” Gastroenterology 120, 1620–1629 (2001).
[Crossref] [PubMed]

Int J Biochem Cell Biol (1)

B. W. Gibson, “The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation,” Int J Biochem Cell Biol 37, 927–934 (2005).
[Crossref] [PubMed]

J Am Chem Soc (1)

J. V. Houten and R. J. Watts, “Temperature dependence of the photophysical and photochemical properties of the Tris(2,2’-bipyridyl) ruthenium(II) ion in aqueous solution,” J Am Chem Soc 96, 4853–4858 (1976).
[Crossref]

J Fluoresc (1)

H. C. Gerritsen, R. Sanders, A. Draaijer, and Y. K. Levine, “Fluorescence lifetime imaging of oxygen in living cells,” J Fluoresc 7, 11–16 (1997).
[Crossref]

J Mod Opt (1)

W. Rudolph and M. Kempe, “Topical review: Trends in optical biomedical imaging,” J Mod Opt 44, 1617–1642 (1997).
[Crossref]

J Org Chem (1)

A. P. Brogan, W. R. Widger, and H. Kohn, “Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties,” J Org Chem 68, 5575–5587 (2003).
[Crossref] [PubMed]

J Photochem Photobiol B (1)

J. W. Dobrucki, “Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements,” J Photochem Photobiol B 65, 136–144 (2001).
[Crossref]

J Phys D: Applied Physics (1)

W. Zhong, P. Urayama, and M.-A. Mycek, “Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing,” J Phys D: Applied Physics 36, 1689–1695 (2003).
[Crossref]

Journal of Pathology (1)

P. J. Tadrous, “Methods for imaging the structure and function of living tissues and cells: 2. fluorescence lifetime imaging,” Journal of Pathology 191, 229–234 (2000).
[Crossref] [PubMed]

Nat Biotechnol (1)

R. N. Day and D. W. Piston, “Spying on the hidden lives of proteins,” Nat Biotechnol 17, 425–426 (1999).
[Crossref] [PubMed]

Opt Eng (1)

H. Schneckenburger and K. König, “Fluorescence decay and imaging of NAD(P)H and flavins as metabolic indicators,” Opt Eng 31, 1447–1451 (1992).
[Crossref]

Proceedings of the National Academy of Sciences (1)

J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proceedings of the National Academy of Sciences 89, 1271–1275 (1992).
[Crossref]

Other (3)

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

E. Eigenbrodt, U. Gerbracht, S. Mazurek, P. Presek, and R. FriisT. G. Pretlow II and T. P. Pretlow, “Carbohydrate metabolism and neoplasia: new perspectives for diagnosis and therapy,” in Biochemical and Molecular Aspects of Selected Cancers, eds. (Academic Press, Inc., San Diego, 1994), 311–385.

“Cancer Facts and Figures 2005,” (American Cancer Society, Atlantic City, 2005).

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 (4)

Fig. 1.
Fig. 1.

(a) Fluorescence Lifetime Imaging Microscopy (FLIM) setup. Abbreviations: CCD–charge coupled device; HRI–high rate imager; INT–intensifier; TTL I/O–TTL input/output card; OD–optical discriminator. Abbreviations for optical components: BS–beam splitter; DC–dichroic mirror; FM–mirror on retractable ‘flip’ mount; L1, L2, L3, L4, L5–quartz lenses; M–mirror. Thick solid lines–light path; thin solid line–electronic path. The FLIM system can excite in the UV-NIR range, from 337–960 nm depending on the laser dye used. The nitrogen laser is a pulsed source with peak energy of approximately 1.3 mJ with reproducibility within ±2%. FLIM has a spatial resolution of 1.4 µm and (with structured illumination) can achieve an optical section down to 10 µm. A key advantage of this FLIM implementation is its ability to measure lifetimes of long-lived fluorophores, ranging from 750 ps to almost 1µs with a resolution of 50 ps. Two different culture dish systems provide temperature controlled units for cellular study under more physiologically-apt conditions. (b) Illustration of data collection for RTDP fluorescence at delays of 40, 140, 240 and 340 ns. The gate width was set to 50 ns in order to obtain sufficient fluorescence signal. Note that the excitation is very short compared (<1ns) to the long lifetimes observed for RTDP (hundreds of nanoseconds) and is hence depicted as a pulse. (c) RTDP Calibration Curve: Plot of relative lifetime (τ0/τ) vs. oxygen levels (µM). RTDP calibration indicated a linear relationship between oxygen levels and relative lifetime, which was in good agreement with the Stern-Volmer equation. Over multiple runs Kq=4.5±0.4×10-3 µM-1. The intercept ≠1, indicating some degree of experimental variance. The calibration could differentiate between oxygen levels differing by as little as 8µM.

Fig. 2.
Fig. 2.

(a) Oxidative phosphorylation in mitochondria. Complex I (NADH dehydrogenase) converts NADH to NAD+ and passes on electrons to carrier CoenzymeQ (CoQ) while pumping hydrogen ions into the intermembrane space. Complex II (succinate dehydrogenase) can also generate and pass on electrons to CoQ via a complex internal mechanism which is initiated by conversion of succinate to fumarate, a step in the Krebs cycle. CoQ transfers the electrons to an intermediary complex III (CoQ – cytochrome C oxidoreductase), which in turns enhances the proton gradient by pumping hydrogen ions against the gradient. The electrons are transferred to complex IV (cytochrome oxidase) via cytochrome C (CytC) which in turn hydrolyses oxygen to water and also pumps protons against the gradient. The ATP synthase complex (complex V) moves protons down the gradient, converting osmotic energy to chemical energy via ATP synthesis from ADP in a process known as chemiosmotic coupling. A more detailed explanation of the process and the unique structure of ATP synthase can be found in any standard biochemistry text. (b) NADH fluorescence and (c) Mitotracker-stained SEG images. For the Mitotracker, excitation=543 nm and emission=636 nm. Mitotracker Red is a commercially available stain used for tracking mitochondria within living cells. Fluorescent signals from both markers were found to co-localize. Confocal intensity images of NADH fluorescence in HET (d) and SEG (e): Illustration of differences observed with the Zeiss 5 LSM. The SEG consistently presented a brighter signature than the HET by approximately 2.5-fold. (f) Plot of differences in NADH fluorescence intensity and lifetime observed between the HET and SEG over multiple measurements with the FLIM system. No significant differences in NADH lifetime were observed.

Fig. 3.
Fig. 3.

Clockwise from top left - (a) confocal fluorescence and (b) Differential Interference Contrast, or DIC images of SEG. SEG were incubated with the RTDP dye prior to imaging (see Methods section). FLIM images of SEG incubated with RTDP: (c) DIC, (d) fluorescence intensity in counts, (e) lifetime in ns and (f) oxygen in µM. Note that one cell in the bottom of (c) shifted position in (d) in the time lapse between these two images. (g) The results of depletion experiments on SEG (see Methods section). Cellular viability is compromised with the passage of time and this results in the lifetime/oxygen content leveling off towards the end.

Fig. 4.
Fig. 4.

RTDP fluorescence intensity (a,d), lifetime in ns (b,e) and oxygen in µM (c,f) maps of HET (a,b,c) and SEG (d,e,f). The intensity images (a,d) could not be reliably used to discriminate between the two different cell lines. The binary lifetime maps (b,e), on the other hand, plainly indicate different lifetimes for these two cellular species, with the SEG recording lower lifetimes than the HET. For the given case, τHET=225 ns and τSEG=170 ns. The mean lifetime difference was found to be Δτ=44±7.48 ns. Logically, this translated into higher oxygen levels in the SEG vs. the HET using the calibration derived earlier, as can be seen in the oxygen distribution maps (c,f). (g) illustrates the differences in oxygen levels within the HET and SEG as measured over multiple runs and assessed using the RTDP calibration. The mean difference between the two cell lines was hence evaluated as Δ[O]2=78±13 µM and this value was statistically significant (p<0.001).

Equations (4)

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

ln I i , p = t i t p + C
τ p = N ( t i 2 ) ( t i ) 2 N t i ln I i , p ( t i ) ( ln I i , p )
τ = 4.4975 * T + K , r 2 = 0.9867
τ 0 τ x = 1 + K q [ O 2 ] x

Metrics