title: In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence.
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.
authors.affiliation: Biophotonics Research Laboratory, Department of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ; These authors contributed equally to this work.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Molecular Neuroscience, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ; These authors contributed equally to this work.
authors.forename: Bo
authors.initials: B
authors.lastname: Yan
authors.affiliation: Biophotonics Research Laboratory, Department of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Qiqi
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Biophotonics Research Laboratory, Department of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Sicong
authors.initials: S
authors.lastname: He
authors.affiliation: Biophotonics Research Laboratory, Department of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Molecular Neuroscience, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ; Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ; zilong@ust.hk.
authors.forename: Zilong
authors.initials: Z
authors.lastname: Wen
authors.affiliation: Biophotonics Research Laboratory, Department of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ; Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Jianan Y
authors.initials: JY
authors.lastname: Qu
keywords: (170.1470) Blood or tissue constituent monitoring; (180.4315) Nonlinear microscopy; (180.5810) Scanning microscopy