Abstract

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

© 2012 Optical Society of America

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2011 (3)

E. I. Galanzha and V. P. Zharov, Cytom. A 79, 746 (2011).
[CrossRef]

V. V. Tuchin, A. Tárnok, and V. P. Zharov, Cytom. A 79, 737 (2011).
[CrossRef]

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, Biomed. Opt. Express 2, 71 (2011).
[CrossRef]

2009 (2)

2008 (2)

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef]

2007 (2)

G. J. Lieschke, and P. D. Currie, Nat. Rev. Genet. 8, 353 (2007).
[CrossRef]

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

2004 (1)

2003 (1)

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

1998 (1)

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

Baker, J. R.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Bielinska, A. U.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Cao, Z.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Christiet, R.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Currie, P. D.

G. J. Lieschke, and P. D. Currie, Nat. Rev. Genet. 8, 353 (2007).
[CrossRef]

Cybulsky, M. I.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

Edwards, D. F.

D. F. Edwards, The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd ed.(University of Oregon, 1995).

Galanzha, E. I.

E. I. Galanzha and V. P. Zharov, Cytom. A 79, 746 (2011).
[CrossRef]

Georgakoudi, I.

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Keszler, B.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Laudanna, C.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

Ley, K.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

Li, D.

Lieschke, G. J.

G. J. Lieschke, and P. D. Currie, Nat. Rev. Genet. 8, 353 (2007).
[CrossRef]

Lin, C. P.

Luo, Y.

Majoros, I.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Myc, A.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Norris, T. B.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Nourshargh, S.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

Novak, J.

Prossin, A.

Qu, J. Y.

Star, W. M.

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

Tárnok, A.

V. V. Tuchin, A. Tárnok, and V. P. Zharov, Cytom. A 79, 737 (2011).
[CrossRef]

Thomas, T.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Tkaczyk, E. R.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Tuchin, V. V.

V. V. Tuchin, A. Tárnok, and V. P. Zharov, Cytom. A 79, 737 (2011).
[CrossRef]

Wagnières, G. A.

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

Wang, L. V.

L. V. Wang, Nat. Photon. 3, 503 (2009).
[CrossRef]

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Wei, X.

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Wilson, B. C.

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

Wu, Y.

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef]

Ye, J. Y.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Zeng, Y.

Zharov, V. P.

E. I. Galanzha and V. P. Zharov, Cytom. A 79, 746 (2011).
[CrossRef]

V. V. Tuchin, A. Tárnok, and V. P. Zharov, Cytom. A 79, 737 (2011).
[CrossRef]

Zheng, W.

Zhong, C. F.

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Biomed. Opt. Express (1)

Cytom. A (2)

E. I. Galanzha and V. P. Zharov, Cytom. A 79, 746 (2011).
[CrossRef]

V. V. Tuchin, A. Tárnok, and V. P. Zharov, Cytom. A 79, 737 (2011).
[CrossRef]

J. Biomed. Opt. (2)

C. F. Zhong, E. R. Tkaczyk, T. Thomas, J. Y. Ye, A. Myc, A. U. Bielinska, Z. Cao, I. Majoros, B. Keszler, J. R. Baker, and T. B. Norris, J. Biomed. Opt. 13, 034008 (2008).
[CrossRef]

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef]

Nat. Photon. (1)

L. V. Wang, Nat. Photon. 3, 503 (2009).
[CrossRef]

Nat. Rev. Genet. (1)

G. J. Lieschke, and P. D. Currie, Nat. Rev. Genet. 8, 353 (2007).
[CrossRef]

Nat. Rev. Immunol. (1)

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, Nat. Rev. Immunol. 7, 678 (2007).
[CrossRef]

Opt. Lett. (2)

Photochem. Photobiol. (1)

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

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

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef]

Other (1)

D. F. Edwards, The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd ed.(University of Oregon, 1995).

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Figures (2)

Fig. 1.
Fig. 1.

Fluorescence characteristics of plasma in zebrafish. (a) Wide-field images of zebrafish and zebrafish blood vessels; (b) and (c) TPEF images formed with the signals in 400–540 nm and 550–600 nm bands (λex=720nm), respectively; (d) TPEF spectral signals extracted from the regions of dorsal aorta and axial vein marked in (b) and (c); (e) spectra of the plasma TPEF signals at 600, 650, 720, 760, and 800 nm excitation and the NADH fluorescence extracted from surrounding tissue. Scale bars in (b) and (c): 20 µm.

Fig. 2.
Fig. 2.

Blood flow images of Tg (lyz: DsRed) transgenic zebrafish. (a) Blood flow image of axial vein in zebrafish at 650 nm excitation; (b) and (c) blood flow images formed with the signals in 550–600 nm and 400–540 nm bands at 650 nm excitation; (d) fluorescence spectral signals extracted from neutrophil, plasma and tissue; (e) and (f) blood flow images formed with DsRed signals and the signals below 550 nm at 720 nm excitation; (g) blood flow image of dorsal aorta in zebrafish at 720 nm excitation. Scale bars for all flow images are shown in (b): vertical (spatial scale), 25 µm; horizontal (temporal scale), 1 s.

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