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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 3 — Mar. 1, 2014
  • pp: 653–663

In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence

Yan Zeng, Bo Yan, Qiqi Sun, Sicong He, Jun Jiang, Zilong Wen, and Jianan Y. Qu  »View Author Affiliations

Biomedical Optics Express, Vol. 5, Issue 3, pp. 653-663 (2014)

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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

OCIS Codes
(170.1470) Medical optics and biotechnology : Blood or tissue constituent monitoring
(180.5810) Microscopy : Scanning microscopy
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: December 20, 2013
Revised Manuscript: January 29, 2014
Manuscript Accepted: January 30, 2014
Published: February 4, 2014

Yan Zeng, Bo Yan, Qiqi Sun, Sicong He, Jun Jiang, Zilong Wen, and Jianan Y. Qu, "In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence," Biomed. Opt. Express 5, 653-663 (2014)

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  1. R. F. Tuma, W. N. Durán, and K. Ley, Handbook of Physiology: Microcirculation (Elsevier, 2008).
  2. J. K. Li, Dynamics of the vascular system (World scientific, 2004).
  3. D. M. McDonald and P. L. Choyke, “Imaging of angiogenesis: From microscope to clinic,” Nat. Med.9(6), 713–725 (2003). [CrossRef] [PubMed]
  4. D. A. Boas and A. K. Dunn, “Laser speckle contrast imaging in biomedical optics,” J. Biomed. Opt.15(1), 011109 (2010). [CrossRef] [PubMed]
  5. 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]
  6. L. V. Wang, “Multiscale photoacoustic microscopy and computed tomography,” Nat. Photonics3(9), 503–509 (2009). [CrossRef] [PubMed]
  7. W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature461(7267), 1105–1109 (2009). [CrossRef] [PubMed]
  8. 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. B23(5), 932–950 (2006). [CrossRef]
  9. C. K. Tsai, Y. S. Chen, P. C. Wu, T. Y. Hsieh, H. W. Liu, C. Y. Yeh, W. L. Lin, J. S. Chia, and T. M. Liu, “Imaging granularity of leukocytes with third harmonic generation microscopy,” Biomed. Opt. Express3(9), 2234–2243 (2012). [CrossRef] [PubMed]
  10. V. V. Tuchin, A. Tárnok, and V. P. Zharov, “In vivo flow cytometry: A horizon of opportunities,” Cytometry A79(10), 737–745 (2011). [CrossRef] [PubMed]
  11. W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, “Two-photon excited hemoglobin fluorescence,” Biomed. Opt. Express2(1), 71–79 (2011). [CrossRef] [PubMed]
  12. 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. Express18(2), 988–999 (2010). [CrossRef] [PubMed]
  13. 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]
  14. O. J. Tamplin and L. I. Zon, “Fishing at the cellular level,” Nat. Methods7(8), 600–601 (2010). [CrossRef] [PubMed]
  15. D. Li, W. Zheng, and J. Y. Qu, “Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence,” Opt. Lett.33(20), 2365–2367 (2008). [CrossRef] [PubMed]
  16. C. Xu and W. W. Webb, “Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm,” J. Opt. Soc. Am. B13(3), 481–491 (1996). [CrossRef]
  17. M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011). [CrossRef] [PubMed]
  18. M. Westerfield, The zebrafish book: a guide for the laboratory use of zebrafish (Daniorerio) (University of Oregon Press, 2000).
  19. 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]
  20. 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,” Blood84(5), 1427–1433 (1994). [PubMed]
  21. 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]
  22. 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]
  23. 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).
  24. 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]
  25. T. H. Chia, A. Williamson, D. D. Spencer, and M. J. Levene, “Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding,” Opt. Express16(6), 4237–4249 (2008). [CrossRef] [PubMed]
  26. V. Kumar, A. K. Abbas, N. Fausto, and J. Aster, Robbins and cotran: pathologic basis of disease (Elsevier, 2009).
  27. 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,” Blood91(10), 3527–3561 (1998). [PubMed]
  28. H. Jin, J. Xu, and Z. Wen, “Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development,” Blood109(12), 5208–5214 (2007). [CrossRef] [PubMed]
  29. 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,” Immunity25(6), 963–975 (2006). [CrossRef] [PubMed]
  30. F. H. Martini, M. J. Timmons, and B. Tallitsch, Human Anatomy (Pearson Education, 2003)
  31. 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.

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