The pial vasculature is the sole source of blood supply to the neocortex. The brain is contained within the skull, a vascularized bone marrow with a unique anatomical connection to the brain. Recent developments in tissue clearing have enabled unprecedented mapping of the entire pial and calvarial vasculature. However, what are the absolute flow rates values of those vascular networks? This information cannot accurately be retrieved with the commonly used bioimaging methods. Here, we introduce Pia-FLOW, a new approach based on large-scale fluorescence localization microscopy, to attain hemodynamic imaging of the whole murine pial and calvarial vasculature at frame rates up to 1000 Hz and spatial resolution reaching 5.4 μm. Using Pia-FLOW, we provide detailed maps of flow velocity, direction and vascular diameters which can serve as ground-truth data for further studies, advancing our understanding of brain fluid dynamics. Furthermore, Pia- FLOW revealed that the pial vascular network functions as one unit for robust allocation of blood after stroke.
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