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Image Correlation Microscopy for flow mapping in vivo.
Chirico G., Ceffa N.G., Sironi L., Collini M. D'Alfonso L., Borzenkov M.
Microcirculation is a complex network of small vessels with a typical diameter ranging from 5 to 50 micrometers, delivers nutrient-rich oxygenated blood to tissues and organs. It plays therefore a crucial role in their maintenance and hemodynamics. Moreover, it interacts extensively with the immune system and its impairments and dysfunctions lead to several pathological conditions. Therefore, mapping the blood flow velocity in microvessels is crucial for the characterization of healthy and diseased organs. We describe a novel method (FLICS, FLow Image Correlation Spectroscopy) to extract flow speeds in complex vessel networks from a single raster-scanned optical $xy$-image, acquired $in vivo$ by confocal or two-photon excitation microscopy. 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 analytical expression of the CCF has been derived by applying scanning fluorescence correlation concepts to drifting optically resolved objects and the theoretical framework has been validated in systems of increasing complexity. The power of the technique is revealed by its application to the intricate murine hepatic microcirculatory system where blood flow speed has been mapped simultaneously in several capillaries from a single $xy$-image and followed in time at high spatial and temporal resolution.