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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)
http://dx.doi.org/10.1364/BOE.5.000653


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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

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:
Microscopy

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

Citation
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)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-5-3-653


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