Research Papers: Sensing

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos

[+] Author Affiliations
Paolo Pozzi

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Laura Sironi

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Laura D’Alfonso

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Margaux Bouzin

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Maddalena Collini

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Giuseppe Chirico

Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy

Piersandro Pallavicini

Università di Pavia, Dipartimento di Chimica, viale Taramelli, 12 – 27100 Pavia, Italy

Franco Cotelli

Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, I-20133, Milano, Italy

Efrem A. Foglia

Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, I-20133, Milano, Italy

J. Biomed. Opt. 19(6), 067007 (Jun 20, 2014). doi:10.1117/1.JBO.19.6.067007
History: Received March 20, 2014; Revised May 15, 2014; Accepted May 19, 2014
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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 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms–3 μm 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 in vivo. 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.

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© 2014 Society of Photo-Optical Instrumentation Engineers

Citation

Paolo Pozzi ; Laura Sironi ; Laura D’Alfonso ; Margaux Bouzin ; Maddalena Collini, et al.
"Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos", J. Biomed. Opt. 19(6), 067007 (Jun 20, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.6.067007


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