Abstract

Zebrafish has rapidly evolved as a powerful vertebrate model organism for studying human diseases. Here we first demonstrate a new label-free approach for in vivo imaging of microvasculature, based on the recent discovery and detailed characterization of the two-photon excited endogenous fluorescence in the blood plasma of zebrafish. In particular, three-dimensional reconstruction of the microvascular networks was achieved with the depth-resolved two-photon excitation fluorescence (TPEF) imaging. Secondly, the blood flow images, obtained by perpendicularly scanning the focal point across the blood vessel, provided accurate information for characterizing the hemodynamics of the circulatory system. The endogenous fluorescent signals of reduced nicotinamide adenine dinucleotide (NADH) enabled visualization of the circulating granulocytes (neutrophils) in the blood vessel. The development of acute sterile inflammation could be detected by the quantitative counting of circulating neutrophils. Finally, we found that by utilizing a short wavelength excitation at 650 nm, the commonly used fluorescent proteins, such as GFP and DsRed, could be efficiently excited together with the endogenous fluorophores to achieve four-color TPEF imaging of the vascular structures and blood cells. The results demonstrated that the multi-color imaging could potentially yield multiple view angles of important processes in living biological systems.

© 2014 Optical Society of America

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2012 (3)

2011 (3)

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, “Two-photon excited hemoglobin fluorescence,” Biomed. Opt. Express 2(1), 71–79 (2011).
[Crossref] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A 79(10), 737–745 (2011).
[Crossref] [PubMed]

2010 (3)

O. J. Tamplin and L. I. Zon, “Fishing at the cellular level,” Nat. Methods 7(8), 600–601 (2010).
[Crossref] [PubMed]

D. A. Boas and A. K. Dunn, “Laser speckle contrast imaging in biomedical optics,” J. Biomed. Opt. 15(1), 011109 (2010).
[Crossref] [PubMed]

C. Li, R. K. Pastila, C. Pitsillides, J. M. Runnels, M. Puoris’haag, D. Côté, and C. P. Lin, “Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence,” Opt. Express 18(2), 988–999 (2010).
[Crossref] [PubMed]

2009 (3)

I. V. Larina, S. Ivers, S. Syed, M. E. Dickinson, and K. V. Larin, “Hemodynamic measurements from individual blood cells in early mammalian embryos with Doppler swept source OCT,” Opt. Lett. 34(7), 986–988 (2009).
[Crossref] [PubMed]

L. V. Wang, “Multiscale photoacoustic microscopy and computed tomography,” Nat. Photonics 3(9), 503–509 (2009).
[Crossref] [PubMed]

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

2008 (3)

2007 (1)

H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood 109(12), 5208–5214 (2007).
[Crossref] [PubMed]

2006 (2)

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

G. O. Clay, A. C. Millard, C. B. Schaffer, J. Aus-der-Au, P. S. Tsai, J. A. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: Evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B 23(5), 932–950 (2006).
[Crossref]

2003 (1)

D. M. McDonald and P. L. Choyke, “Imaging of angiogenesis: From microscope to clinic,” Nat. Med. 9(6), 713–725 (2003).
[Crossref] [PubMed]

2000 (1)

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

1998 (1)

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

1997 (1)

Z. Lele, S. Engel, and P. H. Krone, “hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos,” Dev. Genet. 21(2), 123–133 (1997).
[Crossref] [PubMed]

1996 (1)

1994 (1)

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Aus-der-Au, J.

Barnathan, E. S.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Boas, D. A.

D. A. Boas and A. K. Dunn, “Laser speckle contrast imaging in biomedical optics,” J. Biomed. Opt. 15(1), 011109 (2010).
[Crossref] [PubMed]

Briolat, V.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Buck, C. A.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Cairo, M. S.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Chen, Y. S.

Chia, J. S.

Chia, T. H.

Chong, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Choyke, P. L.

D. M. McDonald and P. L. Choyke, “Imaging of angiogenesis: From microscope to clinic,” Nat. Med. 9(6), 713–725 (2003).
[Crossref] [PubMed]

Christensen, R. D.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Cines, D. B.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Clay, G. O.

Côté, D.

Dickinson, M. E.

Drobizhev, M.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Dunn, A. K.

D. A. Boas and A. K. Dunn, “Laser speckle contrast imaging in biomedical optics,” J. Biomed. Opt. 15(1), 011109 (2010).
[Crossref] [PubMed]

Ellis, R.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Engel, S.

Z. Lele, S. Engel, and P. H. Krone, “hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos,” Dev. Genet. 21(2), 123–133 (1997).
[Crossref] [PubMed]

Gillan, E. R.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Greenbaum, L.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Handin, R. I.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Herbomel, P.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Holtom, G. R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Hsieh, T. Y.

Hug, B. A.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Hughes, T. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Ivers, S.

Jin, H.

H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood 109(12), 5208–5214 (2007).
[Crossref] [PubMed]

Kissa, K.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Kleinfeld, D.

Konkle, B. A.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Krone, P. H.

Z. Lele, S. Engel, and P. H. Krone, “hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos,” Dev. Genet. 21(2), 123–133 (1997).
[Crossref] [PubMed]

Larin, K. V.

Larina, I. V.

Lavie, R.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Lele, Z.

Z. Lele, S. Engel, and P. H. Krone, “hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos,” Dev. Genet. 21(2), 123–133 (1997).
[Crossref] [PubMed]

Levene, M. J.

Li, C.

Li, D.

Li, L.

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

Y. Zeng, J. Xu, D. Li, L. Li, Z. Wen, and J. Y. Qu, “Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging,” Opt. Lett. 37(13), 2490–2492 (2012).
[Crossref] [PubMed]

Lin, C. P.

Lin, H. F.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Lin, W. L.

Liu, H. W.

Liu, T. M.

Loscalzo, J.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Lu, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Luo, Y.

Makarov, N. S.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Malik, Z.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

McCrae, K. R.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

McDonald, D. M.

D. M. McDonald and P. L. Choyke, “Imaging of angiogenesis: From microscope to clinic,” Nat. Med. 9(6), 713–725 (2003).
[Crossref] [PubMed]

McEver, R. P.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Millard, A. C.

Min, W.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Mordelet, E.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Murayama, E.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Pastila, R. K.

Pitsillides, C.

Pober, J. S.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Pollak, E. S.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Puoris’haag, M.

Qu, J. Y.

Rebane, A.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Rothmann, C.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Roy, R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Runnels, J. M.

Schaffer, C. B.

Schmidt, A. M.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Schwartz, B. S.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Shi, Y. Q.

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

Spencer, D. D.

Squier, J. A.

Stern, D. M.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Suen, Y.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Syed, S.

Tamplin, O. J.

O. J. Tamplin and L. I. Zon, “Fishing at the cellular level,” Nat. Methods 7(8), 600–601 (2010).
[Crossref] [PubMed]

Tárnok, A.

V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A 79(10), 737–745 (2011).
[Crossref] [PubMed]

Tillo, S. E.

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

Tsai, C. K.

Tsai, P. S.

Tuchin, V. V.

V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A 79(10), 737–745 (2011).
[Crossref] [PubMed]

van de Ven, C.

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

Wang, L. V.

L. V. Wang, “Multiscale photoacoustic microscopy and computed tomography,” Nat. Photonics 3(9), 503–509 (2009).
[Crossref] [PubMed]

Webb, W. W.

Wen, Z.

Y. Zeng, J. Xu, D. Li, L. Li, Z. Wen, and J. Y. Qu, “Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging,” Opt. Lett. 37(13), 2490–2492 (2012).
[Crossref] [PubMed]

H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood 109(12), 5208–5214 (2007).
[Crossref] [PubMed]

Wen, Z. L.

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

Wick, T. M.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Williamson, A.

Wu, P. C.

Wu, Y. C.

W. Zheng, Y. C. Wu, D. Li, and J. Y. Qu, “Autofluorescence of epithelial tissue: Single-photon versus two-photon excitation,” J. Biomed. Opt. 13(5), 054010 (2008).
[Crossref] [PubMed]

Xie, X. S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Xu, C.

Xu, J.

Y. Zeng, J. Xu, D. Li, L. Li, Z. Wen, and J. Y. Qu, “Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging,” Opt. Lett. 37(13), 2490–2492 (2012).
[Crossref] [PubMed]

H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood 109(12), 5208–5214 (2007).
[Crossref] [PubMed]

Yan, B.

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

Yeh, C. Y.

Zapata, A.

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

Zeng, Y.

Zhang, W. Q.

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

Zharov, V. P.

V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A 79(10), 737–745 (2011).
[Crossref] [PubMed]

Zheng, W.

Zimmerman, G. A.

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

Zon, L. I.

O. J. Tamplin and L. I. Zon, “Fishing at the cellular level,” Nat. Methods 7(8), 600–601 (2010).
[Crossref] [PubMed]

Biol. Chem. (1)

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: A model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Biomed. Opt. Express (2)

Blood (3)

E. R. Gillan, R. D. Christensen, Y. Suen, R. Ellis, C. van de Ven, and M. S. Cairo, “A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: Significant induction of peripheral and bone marrow neutrophilia,” Blood 84(5), 1427–1433 (1994).
[PubMed]

D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, E. S. Barnathan, K. R. McCrae, B. A. Hug, A. M. Schmidt, and D. M. Stern, “Endothelial cells in physiology and in the pathophysiology of vascular disorders,” Blood 91(10), 3527–3561 (1998).
[PubMed]

H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood 109(12), 5208–5214 (2007).
[Crossref] [PubMed]

Cytometry A (1)

V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A 79(10), 737–745 (2011).
[Crossref] [PubMed]

Dev. Genet. (1)

Z. Lele, S. Engel, and P. H. Krone, “hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos,” Dev. Genet. 21(2), 123–133 (1997).
[Crossref] [PubMed]

Immunity (1)

E. Murayama, K. Kissa, A. Zapata, E. Mordelet, V. Briolat, H. F. Lin, R. I. Handin, and P. Herbomel, “Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development,” Immunity 25(6), 963–975 (2006).
[Crossref] [PubMed]

J. Biol. Chem. (1)

L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang, and Z. L. Wen, “Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration,” J. Biol. Chem. 287(30), 25353–25360 (2012).
[Crossref] [PubMed]

J. Biomed. Opt. (2)

D. A. Boas and A. K. Dunn, “Laser speckle contrast imaging in biomedical optics,” J. Biomed. Opt. 15(1), 011109 (2010).
[Crossref] [PubMed]

W. Zheng, Y. C. Wu, D. Li, and J. Y. Qu, “Autofluorescence of epithelial tissue: Single-photon versus two-photon excitation,” J. Biomed. Opt. 13(5), 054010 (2008).
[Crossref] [PubMed]

J. Opt. Soc. Am. B (2)

Nat. Med. (1)

D. M. McDonald and P. L. Choyke, “Imaging of angiogenesis: From microscope to clinic,” Nat. Med. 9(6), 713–725 (2003).
[Crossref] [PubMed]

Nat. Methods (2)

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods 8(5), 393–399 (2011).
[Crossref] [PubMed]

O. J. Tamplin and L. I. Zon, “Fishing at the cellular level,” Nat. Methods 7(8), 600–601 (2010).
[Crossref] [PubMed]

Nat. Photonics (1)

L. V. Wang, “Multiscale photoacoustic microscopy and computed tomography,” Nat. Photonics 3(9), 503–509 (2009).
[Crossref] [PubMed]

Nature (1)

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Lett. (3)

Other (7)

V. Kumar, A. K. Abbas, N. Fausto, and J. Aster, Robbins and cotran: pathologic basis of disease (Elsevier, 2009).

R. F. Tuma, W. N. Durán, and K. Ley, Handbook of Physiology: Microcirculation (Elsevier, 2008).

J. K. Li, Dynamics of the vascular system (World scientific, 2004).

M. Westerfield, The zebrafish book: a guide for the laboratory use of zebrafish (Daniorerio) (University of Oregon Press, 2000).

A. V. Gore, K. Monzo, Y. R. Cha, W. Pan, and B. M. Weinstein, “Vascular development in the zebrafish,” Cold Spring Harbor Perspectives in Medicine 2, (2012).

F. H. Martini, M. J. Timmons, and B. Tallitsch, Human Anatomy (Pearson Education, 2003)

G. E. Davis, A. N. Stratman, A. Sacharidou, and W. Koh, “Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting,” International Review of Cell and Molecular Biology208 (2011) Chap. 3.

Supplementary Material (2)

» Media 1: MP4 (8994 KB)     
» Media 2: MP4 (2967 KB)     

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Figures (5)

Fig. 1
Fig. 1 Schematic diagram of the time-resolved spectroscopic imaging system. PCF: Photonic crystal fiber; BS: beam splitter; M: mirror; DM: dichroic mirror; MM: movable mirror; BF: band-pass filter; SF: short-pass filter; FB: fiber bundle; M-TCSPC: time-correlated single photon counting (TCSPC) module equipped with a multichannel PMT array

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Fig. 2
Fig. 2 Two-photon excitation fluorescence characteristics of zebrafish plasma. (a) TPEF intensity as a function of excitation power at the wavelength of 720 nm; (b) TPEF spectra excited at different wavelengths; (c) TPEF decays excited at different wavelengths; (d) two-photon action cross-sections of plasma fluorescence at different excitation wavelengths.

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Fig. 3
Fig. 3 Microvascular imaging of zebrafish brain. (a) The location of the TPEF measurement in the brain; (b) TPEF spectra of the blood plasma and the brain tissue excited at 650nm; (c)-(f) Ratio imaging for microvascular vessel extraction; (c)-(e) TPEF images formed with the signals in the wavelength band of 364-481 nm, 364-416 nm, and 429-481 nm, respectively; (f) Ratio image by dividing (d) over (e). (g)-(j) TPEF images at different depths (detailed depth-resolved TPEF images are shown in Media 1); (k) three-dimensionally reconstructed microvascular network in the brain (see detail in Media 2); (l) lifetime decays of blood plasma and brain tissue TPEF signals; Image size: 90 µm × 90 µm.

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Fig. 4
Fig. 4 In vivo TPEF imaging cytometric detection of acute sterile inflammation (a) blood flow TPEF image of control zebrafish (Tg(lyz:DsRed2)); (b) blood flow TPEF image of control zebrafish (strain AB) with acute sterile inflammation; (c) absolute counting number of circulating neutrophils in control zebrafish embryo (Tg(lyz:DsRed2)) and zebrafish embryo (Tg(lyz:DsRed2) and strain AB with induced acute sterile inflammation; (d) the counting number of circulating neutrophils normalized to circulating erythrocytes. Scale bars for all the flow images are shown in (a): vertical (spatial scale), 25 µm; horizontal (temporal scale), 1s; * indicates t-test p value <<0.05.

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Fig. 5
Fig. 5 Simultaneous four-color imaging with single excitation wavelength. (a) color coded TPEF image taken in the blood island (red arrow indicates the direction of blood flow; the image size: 90 µm × 90 µm); (b) The measured fluorescence spectra of muscle cells, endothelial cells, blood plasma and mesenchyme under 650 nm two-photon excitation.

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Equations (1)

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η( λ ex )= ε I f τ P λ ex 2 P ave 2

Metrics