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Microcirculation plays a key role in the maintenance and hemodynamics of tissues and organs also due to its extensive interaction with the immune system. A critical limitation of state-of-the-art clinical techniques to characterize the blood flow is their lack of the spatial resolution required to scale down to individual capillaries. On the other hand the study of the blood flow through auto- or cross-correlation methods fail to correlate the flow speed values with the morphological details required to describe an intricate network of capillaries. Here we propose to use a newly developed technique (FLICS, FLow Image Correlation Spectroscopy) that, by employing a single raster-scanned xy-image acquired in vivo by confocal or multi-photon excitation fluorescence microscopy, allows the quantitative measurement of the blood flow velocity in the whole vessel pattern within the field of view, while simultaneously maintaining the morphological information on the immobile structures of the explored circulatory system. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The whole analytical dependence of the CCFs on the flow speed amplitude and the flow direction has been reported recently. We report here the derivation of approximated analytical relations that allows to use the CCF peak lag time and the corresponding CCF value, to directly estimate the flow speed amplitude and the flow direction. The validation has been performed on Zebrafish embryos for which the flow direction was changed systematically by rotating the embryos on the microscope stage. The results indicate that also from the CCF peak lag time it is possible to recover the flow speed amplitude within 13% of uncertainty (overestimation) in a wide range of angles between the flow and the image scanning direction.
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