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Journal of Biomedical Optics

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos
Author(s): Paolo Pozzi; Laura Sironi; Laura D’Alfonso; Margaux Bouzin; Maddalena Collini; Giuseppe Chirico; Piersandro Pallavicini; Franco Cotelli; Efrem A. Foglia
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Paper Abstract

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 <italic<μ</italic<m) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms–3 <italic<μ</italic<m time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos <italic<in vivo</italic<. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos’ vessels.

Paper Details

Date Published: 20 June 2014
PDF: 12 pages
J. Biomed. Opt. 19(6) 067007 doi: 10.1117/1.JBO.19.6.067007
Published in: Journal of Biomedical Optics Volume 19, Issue 6
Show Author Affiliations
Paolo Pozzi, Univ. degli Studi di Milano-Bicocca (Italy)
Laura Sironi, Univ. degli Studi di Milano-Bicocca (Italy)
Laura D’Alfonso, Univ. degli Studi di Milano-Bicocca (Italy)
Margaux Bouzin, Univ. degli Studi di Milano-Bicocca (Italy)
Maddalena Collini, Univ. degli Studi di Milano-Bicocca (Italy)
Giuseppe Chirico, Univ. degli Studi di Milano-Bicocca (Italy)
Piersandro Pallavicini, Univ. degli Studi di Pavia (Italy)
Franco Cotelli, Univ. degli Studi di Milano (Italy)
Efrem A. Foglia, Univ. degli Studi di Milano (Italy)

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