Abstract

We developed a two-photon autofluorescence lifetime imaging system with excitations selected from the supercontinuum generated from a photonic crystal fiber. The system excites multiple endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and tryptophan, simultaneously and produces coregistered two-photon autofluorescence images of a biological sample. The technology provides a unique approach to investigate the cellular metabolic activity and protein expression in cells that are potentially important for noninvasive precancer diagnostics. We demonstrated that by taking the tryptophan fluorescence as a reference the ratio of NADH to the tryptophan signal serves as a sensitive indicator of cellular metabolism. The ratio can also clearly differentiate normal cells from cancer cells. The tryptophan fluorescence lifetime images of cells shows that the lifetime of tryptophan fluorescence, varying over a wide range, may be highly dependent on the expression and structure of the protein that tryptophan is packed in.

© 2009 Optical Society of America

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2008

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

D. Li, W. Zheng, and J. Y. Qu, Opt. Lett. 33, 2365 (2008).
[CrossRef] [PubMed]

2007

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

2006

2005

J. A. Palero, V. O. Boer, J. C. Vijverberg, H. C. Gerritsen, and H. J. C. M. Sterenborg, Opt. Express 13, 5363 (2005).
[CrossRef] [PubMed]

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

2004

2002

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

1999

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

1998

G. A. Wagnieres, W. M. Star, and B. C. Wilson, Photochem. Photobiol. 68, 603 (1998).
[PubMed]

1993

A. A. Rehms and P. R. Callis, Chem. Phys. Lett. 208, 276 (1993).
[CrossRef]

1962

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

Alberts, B.

B. Alberts, Essential Cell Biology (Garland Science, 2004).

Andersen, T. V.

Anvari, B.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Backman, V.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Baldwin, A.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Barni, S.

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

Boer, V. O.

Bottiroli, G.

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

Callis, P. R.

A. A. Rehms and P. R. Callis, Chem. Phys. Lett. 208, 276 (1993).
[CrossRef]

Chance, B.

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

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]

Denicke, S.

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Diagaradjane, P.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Ehlers, J. E.

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Eickhoff, J.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Feld, M.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Fitzpatrick, A. G.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Fukui, K.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Georgakoudi, I.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Gericke, K. H.

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Gerritsen, H. C.

Greiner, C.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Higashi, T.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Hilligsoe, K. M.

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Hunter, M.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Isobe, K.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Itoh, K.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Jobsis, F.

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

Kao, F. J.

F. J. Kao, Microsc. Res. Tech. 63, 175 (2004).
[CrossRef] [PubMed]

Kim, Y. L.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer Academic/Plenum, 1999).

Levitt, J. M.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Li, D.

Locatelli, D.

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

Matsunaga, S.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Molmer, K.

Mujat, C.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Munger, K.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Nielsen, C. K.

Niesner, R. A.

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Palero, J. A.

Paulsen, H. N.

Qu, J. Y.

Quentmeier, S.

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Ramanujam, N.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Rehms, A. A.

A. A. Rehms and P. R. Callis, Chem. Phys. Lett. 208, 276 (1993).
[CrossRef]

Riching, K. M.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

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]

Skala, M. C.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[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]

Star, W. M.

G. A. Wagnieres, W. M. Star, and B. C. Wilson, Photochem. Photobiol. 68, 603 (1998).
[PubMed]

Sterenborg, H. J. C. M.

Stucenski, L. A.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Tian, F.

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

Vijverberg, J. C.

Wagnieres, G. A.

G. A. Wagnieres, W. M. Star, and B. C. Wilson, Photochem. Photobiol. 68, 603 (1998).
[PubMed]

Watanabe, W.

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Webb, W. W.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

White, J. G.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Wilson, B. C.

G. A. Wagnieres, W. M. Star, and B. C. Wilson, Photochem. Photobiol. 68, 603 (1998).
[PubMed]

Wong, M. S.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Wu, Y.

Yaseen, M. A.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Yu, J.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Zheng, W.

Biophys. J.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Chem. Phys. Lett.

A. A. Rehms and P. R. Callis, Chem. Phys. Lett. 208, 276 (1993).
[CrossRef]

Int. J. Cancer

C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Munger, and I. Georgakoudi, Int. J. Cancer 122, 363 (2008).
[CrossRef]

J. Phys. Chem. B

S. Quentmeier, S. Denicke, J. E. Ehlers, R. A. Niesner, and K. H. Gericke, J. Phys. Chem. B 112, 5768 (2008).
[CrossRef] [PubMed]

Jpn. J. Appl. Phys. Part 2

K. Isobe, W. Watanabe, S. Matsunaga, T. Higashi, K. Fukui, and K. Itoh, Jpn. J. Appl. Phys. Part 2 44, L167 (2005).
[CrossRef]

Lasers Surg. Med.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Microsc. Res. Tech.

F. J. Kao, Microsc. Res. Tech. 63, 175 (2004).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Photochem. Photobiol.

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

G. A. Wagnieres, W. M. Star, and B. C. Wilson, Photochem. Photobiol. 68, 603 (1998).
[PubMed]

Proc. Natl. Acad. Sci. USA

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Science

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

Other

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Kluwer Academic/Plenum, 1999).

B. Alberts, Essential Cell Biology (Garland Science, 2004).

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Figures (2)

Fig. 1
Fig. 1

Schematic of the TPEF imaging system of two-color excitation. PCF, photonic crystal fiber; M, mirror; DM, dichroic mirror; F1 and F2, bandpass filters; FB: fiber bundle; SP, shortpass filter; L, lens.

Fig. 2
Fig. 2

Representative TPEF spectra and images. a, Spectra of tryptophan solution and SiHa cells at 600 nm excitation; b, spectra of free NADH solution and SiHa cells at 700 nm excitation; c, tryptophan fluorescence image of SiHa cells; d, NADH fluorescence image of SiHa cells; e, ratio image of NADH to tryptophan fluorescence for control sample of SiHa cells; f, ratio image of SiHa cells treated with NaCN; g, ratio image of SiHa cells treated with CCCP; h, tryptophan fluorescence average lifetime image of SiHa cells.

Tables (1)

Tables Icon

Table 1 Ratio of NADH to Tryptophan Fluorescence Signal a, b, c

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