Abstract

Fluorescence lifetime imaging microscopy (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. In recent years, there has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of robust, fast, and cost-effective FLIM instrumentation suitable for in vivo tissue imaging. Unfortunately, the predominant multispectral FLIM approaches suffer from limitations that impede the development of high-speed instruments for in vivo applications. We present a cost-effective scanning multispectral FLIM implementation capable of achieving pixel rates on the order of tens of kilohertz, which will facilitate the evaluation of FLIM for in vivo applications.

© 2010 Optical Society of America

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  1. I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
    [CrossRef] [PubMed]
  2. P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
    [CrossRef] [PubMed]
  3. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
    [CrossRef]
  4. Y. Sun, R. Liu, D. S. Elson, C. W. Hollars, J. A. Jo, J. Park, and L. Marcu, Opt. Lett. 33, 630 (2008).
    [CrossRef] [PubMed]
  5. A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
    [CrossRef] [PubMed]
  6. J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
    [CrossRef] [PubMed]
  7. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
    [CrossRef] [PubMed]
  8. American National Standard for Safe Use of Lasers, ANSI Z136.1-2007 (American National Standards Institute, 2007).

2010 (1)

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

2009 (2)

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

2008 (1)

2007 (1)

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

2005 (1)

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Adame, J.

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

Brandon, J.

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Clubb, F.

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

Conti, C.

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Dabir, A. S.

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

DiGiovanni, J.

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Dunsby, C.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Eickhoff, J.

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

Elson, D. S.

Y. Sun, R. Liu, D. S. Elson, C. W. Hollars, J. A. Jo, J. Park, and L. Marcu, Opt. Lett. 33, 630 (2008).
[CrossRef] [PubMed]

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

French, P. M.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Galletly, N.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Gendron-Fitzpatrick, A.

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

Gimenez-Conti, I.

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Goldstein, L.

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Hollars, C. W.

Jo, J.

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

Jo, J. A.

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

Y. Sun, R. Liu, D. S. Elson, C. W. Hollars, J. A. Jo, J. Park, and L. Marcu, Opt. Lett. 33, 630 (2008).
[CrossRef] [PubMed]

Lakowicz, J. R.

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
[CrossRef]

Lanigan, P. M.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Lever, M. J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Liu, R.

Marcu, L.

McGinty, J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Munro, I.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Neil, M. A.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Pande, P.

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

Park, J.

Ramanujam, N.

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

Requejo-Isidro, J.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Riching, K. M.

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

Ryu, Y.

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

Skala, M. C.

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

Stamp, G. W.

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

Sun, Y.

Thomas, P.

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

Trivedi, C. A.

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

White, J. G.

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

J. Biomed. Opt. (2)

I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P. M. Lanigan, D. S. Elson, C. Dunsby, M. A. Neil, M. J. Lever, G. W. Stamp, and P. M. French, J. Biomed. Opt. 10, 051403 (2005).
[CrossRef] [PubMed]

A. S. Dabir, C. A. Trivedi, Y. Ryu, P. Pande, and J. A. Jo, J. Biomed. Opt. 14, 024030 (2009).
[CrossRef] [PubMed]

Opt. Lett. (1)

Photochem. Photobiol. (1)

P. Thomas, P. Pande, F. Clubb, J. Adame, and J. Jo, Photochem. Photobiol. 86, 727 (2010).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA (1)

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

Toxicol. Pathol. (1)

J. Brandon, C. Conti, L. Goldstein, J. DiGiovanni, and I. Gimenez-Conti, Toxicol. Pathol. 37, 733 (2009).
[CrossRef] [PubMed]

Other (2)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).
[CrossRef]

American National Standard for Safe Use of Lasers, ANSI Z136.1-2007 (American National Standards Institute, 2007).

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Figures (3)

Fig. 1
Fig. 1

Schematic of the FLIM system. The same lens collects the emission. Dichroic mirrors guide the laser beam to the sample and the emission beam to the FLIM collection system: BP, bandpass filter; DM, dichroic mirror.

Fig. 2
Fig. 2

(a) Normalized fluorescence intensity maps of the three fluorophores: emissions of POPOP peaks at 420 nm ( 20 dB SNR at 390 nm ), NADH at 450 nm ( 20 dB SNR at 450 nm ), and FAD at 520 nm ( 19 dB SNR at 550 nm ). (b) Lifetime maps. Color scale in nanoseconds. (c) Time-resolved spectroscopic measurements of the same fluorophore solutions taken using a calibrated spectrometer.

Fig. 3
Fig. 3

Multispectral FLIM images of an oral lesion in a hamster cheek pouch. (a) Normalized intensity maps at various emission bands. (b) Corresponding average lifetime maps. Color scale in nanoseconds. The result shows clear contrast between the lesion (arrows) and surrounding healthy tissue.

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