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 mm3 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.
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Data availability
The main data supporting the finding of this study are available within the main text or Supplementary Information. Representative raw imaging datasets supporting the findings of this study have been deposited in Zenodo and are publicly available at https://doi.org/10.5281/zenodo.18876905. Source data are provided with this paper.
Code availability
Localization of fluorescence emitters was performed with the open-source TrackNTrace toolbox63. RBC tracking was performed using the SimpleTracker algorithm36. ADMM reconstruction was performed with the open-source DiffuserCam code69. Pseudocode outlining the main DH-PSF image reconstruction workflow is included in the Supplementary Information (Supplementary Algorithm 1). Custom MATLAB codes for data analysis which are available for research purposes from the corresponding author upon request.
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Acknowledgements
D.R. acknowledges grant support from the Swiss National Science Foundation (10.006.824 and 10.003.762). Z.C. acknowledges support from the Fundamental Research Funds for the Central Universities in China (13702150142) and the National Natural Science Foundation of China (62575214). W.W. acknowledges support from the National Natural Science Foundation of China (52275527).
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B.Z., Q.Z. and Z.C. conceived the experimental design. B.Z. and Q.Z. carried out the experiments. B.Z. and Q.Z. conducted data analysis. L.G., E.J. and D.S. carried out the segmentation and visualization of vessels. M.R. and T.J. assisted with the animal experiments. L.T. performed RBC staining. C.G. and B.W. contributed to the interpretation of the results. X.C. contributed to the optimization of the ADMM algorithm. S.G., X.D., Q.F., H.A. and W.H. contributed to the design and optimization of the phase mask. Y.C., X.L.D-B. and S.L. established the tumor model and contributed to the interpretation of the results. W.W., X.D., Z.C., D.R. and Q.Z. supervised the work. D.R. acquired funding for the project. B.Z. and Q.Z. drafted the manuscript. All authors reviewed the manuscript.
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Nature Communications thanks Xiaochuan Zhang, Robert Prevedel and the other anonymous reviewer for their contribution to the peer review of this work. [A peer review file is available].
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Zhang, B., Guo, S., Tang, L. et al. Double-helix optical point spread function enables real-time mesoscopic 3D functional microangiography in the living mouse brain and skull. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71746-9
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DOI: https://doi.org/10.1038/s41467-026-71746-9
