In vivo imaging of central nervous system fluid spaces using synchrotron radiation-based micro computed tomography

In this repository for our publication, we provide supplementary videos, code, hardware designs and tables with results data to enhance the reproducibility and facilitate broader adoption of in vivo synchrotron radiation-based hard X-ray micro-computed tomography (SRµCT). Documentation and data are published under Creative Commons Attribution 4.0 International, code under GNU General Public License v3.0 or later. Reconstructed 3D SRµCT datasets are available via individual dataset entries on Zenodo, due to the repository's 50 gigabyte limit for individual datasets. Links to these datasets are in the description below. For time series experiments, a single reconstructed 2D section is provided for each time point. Full 3D stacks are included for only one or two representative time points due to their cumulative size of 2.7 terabytes. The remaining datasets are available upon request. Abstract Current approaches to in vivo imaging of the mouse central nervous system (CNS) do not offer a combination of micrometer resolution and a whole-brain field of view. To address this limitation, we introduce an approach based on synchrotron radiation-based hard X-ray micro computed tomography (SRµCT). We performed intravital SRµCT acquisitions of mouse CNS fluid spaces at three synchrotron radiation facilities. Imaging was conducted on both anesthetized free-breathing and ventilated animals, with and without retrospective cardiac gating. We achieved whole-brain imaging at 6.3 μm voxel length, observed the distribution of cerebrospinal fluid (CSF) contrast agent over time and quantified choroid plexus movement. SRμCT bridges the gap between multiphoton microscopy and magnetic resonance imaging, offering dynamic imaging with micrometer-scale resolution and whole-organ field of view. Intravital SRμCT will play a crucial role in validating and integrating hypotheses on CSF dynamics and solute transport by providing unique data that cannot be acquired otherwise. Links to tomography datasets ID17 Biomedical Beamline, European Synchrotron Radiation Facility (ESRF) Mouse17, Intra-cerebroventricular injection, retrospective cardiac gating https://doi.org/10.5281/zenodo.15684790 Mouse19, Contrast agent-free imaging Absorptionhttps://doi.org/10.5281/zenodo.15674822 Paganinhttps://doi.org/10.5281/zenodo.15674076 Mouse50, Intracisterna magna infusion, time series https://doi.org/10.5281/zenodo.15676994 Mouse63, Intra-cerebroventricular infusion, time series https://doi.org/10.5281/zenodo.15682921 BL20B2 Medical and Imaging I, Super Photon ring - 8 GeV (SPring-8) JP28, Intra-cerebroventricular infusion, time serieshttps://doi.org/10.5281/zenodo.15671325 JP34, Contrast agent-free imaging, peri-mortem morphological changeshttps://doi.org/10.5281/zenodo.15671360 Contrast agent calibration phantomshttps://doi.org/10.5281/zenodo.15675683 BMIT Biomedical Imaging and Therapy Facility, Canadian Light Source (CLS) CA019, Intra-cerebroventricular infusion Peri-mortem morphological changes https://doi.org/10.5281/zenodo.15677930 Choroid plexus motionhttps://doi.org/10.5281/zenodo.15690358