Abstract

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular auto fluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365nm the time-resolved autofluorescence decays, dominated by free–bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free–bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.

© 2006 Optical Society of America

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2006

2005

Y. Wu, P. Xi, J. Y. Qu, T. Cheung, and M. Yu, Opt. Express 13, 382 (2005).
[CrossRef] [PubMed]

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

2004

1999

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

1997

R. N. Fichorova, J. G. Rheinwald, and D. J. Anderson, Biol. Reprod. 57, 847 (1997).
[CrossRef] [PubMed]

1995

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

1992

H. Schneckenburger and K. Konig, Opt. Eng. 31, 1447 (1992).
[CrossRef]

1982

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

1976

A. Gafni and L. Brand, Biochemistry 15, 3165 (1976).
[CrossRef] [PubMed]

1970

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

1962

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

Anderson, D. J.

R. N. Fichorova, J. G. Rheinwald, and D. J. Anderson, Biol. Reprod. 57, 847 (1997).
[CrossRef] [PubMed]

Babai, F.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Balaban, R. S.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Balassy, A.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Barni, S.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Bird, D. K.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Blanchard, L.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Blinova, K.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Bose, S.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Bottiroli, G.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Brand, L.

A. Gafni and L. Brand, Biochemistry 15, 3165 (1976).
[CrossRef] [PubMed]

Brands, W. R.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

Brewer, M. A.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

Carroll, S.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Chance, B.

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

Cheung, T.

Cohen, P.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

Croce, A. C.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Drezek, R. A.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

Durocher, G.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Eliceiri, K. W.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Fichorova, R. N.

R. N. Fichorova, J. G. Rheinwald, and D. J. Anderson, Biol. Reprod. 57, 847 (1997).
[CrossRef] [PubMed]

Gaboury, L.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Gafni, A.

A. Gafni and L. Brand, Biochemistry 15, 3165 (1976).
[CrossRef] [PubMed]

Galeotti, T.

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

Harvey, J. I.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Heikal, A. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

Hirschberg, J. G.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Jobsis, F.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

Kasischke, K. A.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

Keely, P. J.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Kirkpatrick, N. D.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

Knutson, J. R.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Kohen, C.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Kohen, E.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Konig, K.

H. Schneckenburger and K. Konig, Opt. Eng. 31, 1447 (1992).
[CrossRef]

Locatelli, D.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Mayer, D. H.

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

Pal, P.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Pradhan, A.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Qu, J. Y.

Ramanujam, N.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Rheinwald, J. G.

R. N. Fichorova, J. G. Rheinwald, and D. J. Anderson, Biol. Reprod. 57, 847 (1997).
[CrossRef] [PubMed]

Salmon, J. M.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Schneckenburger, H.

H. Schneckenburger and K. Konig, Opt. Eng. 31, 1447 (1992).
[CrossRef]

Schoener, B.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

Sciola, L.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Smironov, A. V.

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

Souter, A. W.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Spano, A.

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Thorell, B.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Utzinger, U.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

VanRossum, G. D. V.

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

Vaughan, E. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Viallet, P.

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

Villeneuve, L.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Vishwasrao, H. D.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

Vrotsos, K. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Webb, W. W.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

White, J. G.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Wu, Y.

Xi, P.

Yan, L.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Yu, M.

Zou, C.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

Biochemistry

K. Blinova, S. Carroll, S. Bose, A. V. Smironov, J. I. Harvey, J. R. Knutson, and R. S. Balaban, Biochemistry 44, 2585 (2005).
[CrossRef] [PubMed]

A. Gafni and L. Brand, Biochemistry 15, 3165 (1976).
[CrossRef] [PubMed]

Biol. Reprod.

R. N. Fichorova, J. G. Rheinwald, and D. J. Anderson, Biol. Reprod. 57, 847 (1997).
[CrossRef] [PubMed]

Cancer Res.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Eur. J. Biochem.

T. Galeotti, G. D. V. VanRossum, D. H. Mayer, and B. Chance, Eur. J. Biochem. 17, 485 (1970).
[CrossRef] [PubMed]

J. Biol. Chem.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, J. Biol. Chem. 280, 25119 (2005).
[CrossRef] [PubMed]

J. Photochem. Photobiol., B

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, J. Photochem. Photobiol., B 31, 101 (1995).
[CrossRef]

Opt. Eng.

H. Schneckenburger and K. Konig, Opt. Eng. 31, 1447 (1992).
[CrossRef]

Opt. Express

Opt. Lett.

Photochem. Photobiol.

N. D. Kirkpatrick, C. Zou, M. A. Brewer, W. R. Brands, R. A. Drezek, and U. Utzinger, Photochem. Photobiol. 81, 125 (2005).
[CrossRef]

J. M. Salmon, E. Kohen, P. Viallet, J. G. Hirschberg, A. W. Souter, C. Kohen, and B. Thorell, Photochem. Photobiol. 36, 585 (1982).
[CrossRef] [PubMed]

A. C. Croce, A. Spano, D. Locatelli, S. Barni, L. Sciola, and G. Bottiroli, Photochem. Photobiol. 69, 364 (1999).
[CrossRef] [PubMed]

Science

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, Science 137, 499 (1962).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

Schematic of the time-resolved confocal fluorescence spectroscopy system.

Fig. 2
Fig. 2

(a) Normalized short lifetime DAS of SiHa and Ect 1 cells and the fluorescence spectrum of pure NADH. (b) Normalized long lifetime DAS of SiHa and Ect 1 cells and the fluorescence spectrum of LDH-bound NADH.

Fig. 3
Fig. 3

Variation of the A 1 A 2 ratio as a function of wavelength. (a) SiHa cells treated before and after with NaCN and CCCP. (b) Ect 1 cells treated before and after with NaCN and CCCP. (c) Comparison between SiHa and Ect 1 cells.

Tables (2)

Tables Icon

Table 1 Fitting Analysis of Cellular Fluorescence Excited at 365 nm

Tables Icon

Table 2 Analysis of Time-Resolved Fluorescence Measured from SiHa Cells with an Excitation Range from 365 to 405 nm

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