title: Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos.
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.
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pozzi
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Sironi
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: D'Alfonso
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Margaux
authors.initials: M
authors.lastname: Bouzin
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Collini
authors.affiliation: Università degli Studi di Milano-Bicocca, Dipartimento di Fisica, Piazza della Scienza 3, I-20126, Milano, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Chirico
authors.affiliation: Università di Pavia, Dipartimento di Chimica, viale Taramelli, 12 - 27100 Pavia, Italy.
authors.forename: Piersandro
authors.initials: P
authors.lastname: Pallavicini
authors.affiliation: Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, I-20133, Milano, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Cotelli
authors.affiliation: Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, I-20133, Milano, Italy.
authors.forename: Efrem A
authors.initials: EA
authors.lastname: Foglia
mesh_terms: D000818:Animals; D001783:Blood Flow Velocity; D003198:Computer Simulation; D004583:Electrons; D004625:Embryo, Nonmammalian / Q000502:physiology*; D004912:Erythrocytes / Q000166:cytology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006439:Hemodynamics; D007368:Interferometry / Q000379:methods; D008962:Models, Theoretical; D055095:Optics and Photonics; D017785:Photons; D012212:Rheology; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D015027:Zebrafish
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; C026188:rhodamine 6G