Abstract Quantitative, volumetric imaging of cerebrovascular networks and microcirculation is essential for understanding brain function. However, rapid mesoscopic 3D imaging remains challenging because of fundamental trade-offs between spatiotemporal resolution, field of view, and sensitivity to functional parameters. Here we present a mesoscopic fluorescence imaging platform featuring a double-helix phase mask for real-time, depth-resolved measurements through the intact mouse skull. The compact phase-mask design is compatible with both laser-scanning and widefield microscopy. Using multifocal laser scanning, we demonstrate real-time volumetric in vivo imaging while discriminating calvarial from cerebral vasculature across 6.6×6.6×0.8 mm 3 volume. Beyond high-resolution structural imaging, perfusion time-to-peak values are extracted from the laser-scanning configuration while accurate flow velocity/direction information is provided via widefield tracking of fluorescently labeled cells. We demonstrate the platform’s capabilities by analyzing brain-layer-specific perfusion dynamics and vascular topology in glioma-bearing mouse brains, offering unprecedented views for probing cerebrovascular alterations in both physiological and pathological contexts.
Zhang et al. (Mon,) studied this question.