Abstract

We report a wide-field fluorescence lifetime imaging microscope based on a low-repetition-rate (3.7-MHz) passively mode-locked diode-pumped laser source. This inexpensive and compact laser source operating in the visible and UV range can excite a wide range of fluorophores of biological interest. We demonstrate that the power of this laser source is highly sufficient for studying biological systems with low quantum yields (autofluorescence of tissues and stained living cells). The maximum measurable lifetime is also strongly increased with this laser source, as fluorescence intensity measurement can occur 250 ns after the excitation pulse.

© 2005 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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2003 (3)

M. J. Booth and T. Wilson, J. Microsc. (Oxford) 214, 36 (2003).
[CrossRef]

D. N. Papadopoulos, S. Forget, M. Delaigue, F. Druon, F. Balembois, and P. Georges, Opt. Lett. 28, 1838 (2003).
[CrossRef] [PubMed]

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

2002 (1)

2001 (1)

2000 (1)

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

1995 (2)

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

1977 (1)

J. Olmsted and D. R. Kearns, Biochemistry 16, 3647 (1977).
[CrossRef] [PubMed]

Alivisatos, A. P.

Balembois, F.

Berland, K. M.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Booth, M. J.

M. J. Booth and T. Wilson, J. Microsc. (Oxford) 214, 36 (2003).
[CrossRef]

Chemla, D. S.

Coppey, J.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Coppey-Moisan, M.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Dahan, M.

Davis, D. M.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Delaigue, M.

Denjean, P.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Dong, C. Y.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Draaijer, A.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Druon, F.

Durieux, C.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Edmann, R.

Elson, D. S.

Forget, S.

French, P. M. W.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, and R. Edmann, Opt. Lett. 27, 1409 (2002).
[CrossRef]

French, T.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Georges, P.

Gerritsen, H. C.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Gratton, E.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Houpt, P. M.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Kearns, D. R.

J. Olmsted and D. R. Kearns, Biochemistry 16, 3647 (1977).
[CrossRef] [PubMed]

Kemnitz, K.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Laurence, T.

Lauritsen, K.

Lévêque-Fort, S.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, and R. Edmann, Opt. Lett. 27, 1409 (2002).
[CrossRef]

Lever, M. J.

Levine, Y. K.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Olmsted, J.

J. Olmsted and D. R. Kearns, Biochemistry 16, 3647 (1977).
[CrossRef] [PubMed]

Pansu, R. B.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Papadopoulos, D. N.

Philips, D.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Pinaud, F.

Sabharwal, Y.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Sanders, R.

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Sauer, M.

Siegel, J.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, and R. Edmann, Opt. Lett. 27, 1409 (2002).
[CrossRef]

So, P. T. C.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Suhling, K.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Tramier, M.

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

Wahl, M.

Webb, S. E. D.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, and R. Edmann, Opt. Lett. 27, 1409 (2002).
[CrossRef]

Weiss, S.

Wilson, T.

M. J. Booth and T. Wilson, J. Microsc. (Oxford) 214, 36 (2003).
[CrossRef]

Yu, W. M.

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Anal. Biochem. (1)

R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, Anal. Biochem. 227, 302 (1995).
[CrossRef] [PubMed]

Biochemistry (1)

J. Olmsted and D. R. Kearns, Biochemistry 16, 3647 (1977).
[CrossRef] [PubMed]

Bioimaging (1)

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, Bioimaging 3, 49 (1995).
[CrossRef]

Biophys. J. (1)

M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R. B. Pansu, and M. Coppey-Moisan, Biophys. J. 78, 2614 (2000).
[CrossRef] [PubMed]

J. Microsc. (Oxford) (1)

M. J. Booth and T. Wilson, J. Microsc. (Oxford) 214, 36 (2003).
[CrossRef]

Opt. Lett. (3)

Rev. Sci. Instrum. (1)

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Philips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Experimental setup for wide-field fluorescence microscopy.

Fig. 2
Fig. 2

(a)–(c) Fixed prostate tissue investigated under 355 nm; (d)–(f) urothelial cell with the laser at 532 nm and a filter at the detection point (centered at a 550-nm width of 50 nm). (a), (d) Transmission images; (b), (e) fluorescence intensity images; (c), (f) fluorescence lifetime images.

Fig. 3
Fig. 3

(a) Transmission image of three HeLa cells grown on a coverslip and incubated with ethidium bromide. (b), (c) Corresponding intensity and FLIM image, respectively. (d), (e), (f) Time-gated intensity images taken along the fluorescence decay 300 ps, 45 ns, and 80 ns after the excitation pulse, respectively.

Fig. 4
Fig. 4

Typical fluorescence decay data inside a cell fitted with a single and a double exponential model.

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