Double-helix optical point spread function enables real-time mesoscopic 3D functional microangiography in the living mouse brain and skull.

Zhang, Baoyuan; Guo, Shiyao; Tang, Lin; Chen, Yi; Glandorf, Lukas; Jessen, Etienne; Chang, Xuyang; Jin, Tian et al. · Nat Commun · 2026

basic_science · Level V

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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<sup>3</sup> 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.