Abstract

We show that fluorescence lifetime is a powerful contrast mechanism that can enhance the whole-body imaging of fluorescent proteins (FPs), in the presence of background tissue autofluorescence (AF). The nonexponential AF decay is characterized from time-domain (TD) measurements on multiple nude mice and separated from the FP fluorescence using a linear fit to a priori basis functions. We illustrate this approach using an orthotopic mouse tumor model of breast adenocarcinoma. We also report that four commonly used FPs show distinct lifetimes, indicating their suitability for in vivo lifetime multiplexing. These results suggest the potential for exploiting fluorescence lifetime for imaging FPs for a variety of whole-body small-animal imaging applications.

© 2009 Optical Society of America

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N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

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[CrossRef] [PubMed]

A. T. N. Kumar, S. B. Raymond, B. J. Bacskai, and D. A. Boas, Opt. Lett. 33, 470 (2008).
[CrossRef] [PubMed]

2007 (2)

2006 (1)

R. M. Hoffman and M. Yang, Nat. Protocols 1, 1429 (2006).
[CrossRef]

2005 (2)

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

2003 (1)

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

2002 (1)

R. K. Jain, L. L. Munn, and D. Fukumura, Nat. Rev. Cancer 2, 266 (2002).
[CrossRef] [PubMed]

1999 (1)

P. I. H. Bastiaens and A. Squire, Trends Cell Biol. 9, 48 (1999).
[CrossRef] [PubMed]

Arridge, S. R.

Bacskai, B. J.

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

A. T. N. Kumar, S. B. Raymond, B. J. Bacskai, and D. A. Boas, Opt. Lett. 33, 470 (2008).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

P. I. H. Bastiaens and A. Squire, Trends Cell Biol. 9, 48 (1999).
[CrossRef] [PubMed]

Boas, D. A.

A. T. N. Kumar, S. B. Raymond, B. J. Bacskai, and D. A. Boas, Opt. Lett. 33, 470 (2008).
[CrossRef] [PubMed]

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

Deliolanis, N. C.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Dunn, A. K.

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

French, P. M. W.

Fukumura, D.

R. K. Jain, L. L. Munn, and D. Fukumura, Nat. Rev. Cancer 2, 266 (2002).
[CrossRef] [PubMed]

Grimm, J.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Hajnal, J. V.

Hoffman, R. M.

R. M. Hoffman and M. Yang, Nat. Protocols 1, 1429 (2006).
[CrossRef]

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Jain, R. K.

R. K. Jain, L. L. Munn, and D. Fukumura, Nat. Rev. Cancer 2, 266 (2002).
[CrossRef] [PubMed]

Jiang, P.

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Kambara, H.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Kasmieh, R.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Kumar, A. T. N.

A. T. N. Kumar, S. B. Raymond, B. J. Bacskai, and D. A. Boas, Opt. Lett. 33, 470 (2008).
[CrossRef] [PubMed]

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

Lingna, L.

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Mahmood, U.

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

McGinty, J.

Moossa, A. R.

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Munn, L. L.

R. K. Jain, L. L. Munn, and D. Fukumura, Nat. Rev. Cancer 2, 266 (2002).
[CrossRef] [PubMed]

Neil, M. A. A.

Ntziachristos, V.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Penman, S.

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Pittet, M. J.

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

Raymond, S. B.

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

A. T. N. Kumar, S. B. Raymond, B. J. Bacskai, and D. A. Boas, Opt. Lett. 33, 470 (2008).
[CrossRef] [PubMed]

Ripoll, J.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Saeki, Y.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Sardini, A.

Shah, K.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Shih, H.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Soloviev, V. Y.

Squire, A.

P. I. H. Bastiaens and A. Squire, Trends Cell Biol. 9, 48 (1999).
[CrossRef] [PubMed]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Tahir, K. B.

Tam, J. M.

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

Tannous, B. A.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Tsien, R. Y.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Upadhyay, R.

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

Weissleder, R.

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Wurdinger, T.

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Yang, M.

R. M. Hoffman and M. Yang, Nat. Protocols 1, 1429 (2006).
[CrossRef]

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

Zacharakis, G.

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

IEEE Trans. Med. Imaging (1)

A. T. N. Kumar, S. B. Raymond, A. K. Dunn, B. J. Bacskai, and D. A. Boas, IEEE Trans. Med. Imaging 27, 1152 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

N. C. Deliolanis, R. Kasmieh, T. Wurdinger, B. A. Tannous, K. Shah, and V. Ntziachristos, J. Biomed. Opt. 13, 044008 (2008).
[CrossRef] [PubMed]

Mol. Imaging (1)

J. M. Tam, R. Upadhyay, M. J. Pittet, R. Weissleder, and U. Mahmood, Mol. Imaging 6, 269 (2007).
[PubMed]

Nat. Methods (1)

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, Nat. Methods 2, 905 (2005).
[CrossRef] [PubMed]

Nat. Protocols (1)

R. M. Hoffman and M. Yang, Nat. Protocols 1, 1429 (2006).
[CrossRef]

Nat. Rev. Cancer (1)

R. K. Jain, L. L. Munn, and D. Fukumura, Nat. Rev. Cancer 2, 266 (2002).
[CrossRef] [PubMed]

Opt. Lett. (2)

Proc. Natl. Acad. Sci. USA (2)

M. Yang, L. Lingna, P. Jiang, A. R. Moossa, S. Penman, and R. M. Hoffman, Proc. Natl. Acad. Sci. USA 100, 14259 (2003).
[CrossRef] [PubMed]

G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, Proc. Natl. Acad. Sci. USA 102, 18252 (2005).
[CrossRef] [PubMed]

Trends Cell Biol. (1)

P. I. H. Bastiaens and A. Squire, Trends Cell Biol. 9, 48 (1999).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1
Fig. 1

(a) Continuous-wave component (signal inside dashed circle, yellow online) of the reflectance TD fluorescence measurement (excitation, 488 nm ; emission, 515 nm longpass) of an anesthetized nude mouse placed in supine position. 2 × 10 6 EGFP/CFP expressing breast-tumor cell lines (MDA-MB-231) were implanted in the mammary fat pad. The images shown were obtained three weeks post implantation. (b) Amplitude components of the AF (red online, large area inside circle) and FP (green online, small part inside circle) decays, a AF and a FP , obtained by fitting Eq. (1) to the raw TD fluorescence data are shown as a single RGB image. The dashed lines indicate the illumination area ( 2.5 cm 2 ) . The background image of the mouse is shown in white.

Fig. 2
Fig. 2

(a) Representative AF decays obtained from two control nude mice and from a control region of a mouse with a localized tumor. (b) Global AF decay generated from the control mouse data (black) compared with the decays of FPs measured in vitro. The FP decays were exponential with lifetimes: GFP, 2.8 ns ; dsRed, 2.5 ns ; mCherry, 1.5 ns ; and tdTomato, 3.4 ns . tdTomato, mCherry, and dsRed were excited at 543 nm ( 10 nm width) and detected with a 580 nm long-pass filter. GFP was excited at 480 nm ( 10 nm width) and detected using a 515 nm long-pass filter. The insets in (b) show the CW reflectance image (left) of tubes containing equal concentrations of tdTomato, dsRed, and mCherry, and the amplitude components (right) of a three-exponential fit using the averaged decays of the three FPs. The amplitudes are shown as red (dsRed), green (mCherry), and blue (tdTomato).

Fig. 3
Fig. 3

Simulations to test the efficiency of AF separation. (a)–(c) Continuous-wave images of simulated exponential decays within a 1 cm diameter area added to an experimetal AF image from a control mouse with planar illumination. The ratios between the FP and AF images correspond to ratios of the maximum CW intensities of the FP and AF decays. (d)–(f) show the decoupled AF (red online, exterior color) and the FP (green online, interior color) decay amplitudes obtained using Eq. (1), as the red and green components of an RGB image matrix. Thus yellow (online) indicates the degree of overlap. The FP amplitude is scaled by a factor of 2 in. (e) and 4 in. (f) for clarity. The corresponding FP:AF ratios recovered by the fit are indicated in (d)–(f).

Equations (1)

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U ( r d , t ) = a AF ( r d ) B ( t ) + a FP ( r d ) exp ( t τ FP ) ,

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