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

Current intravital imaging techniques for the mouse central nervous system (CNS) do not simultaneously provide micrometer-scale spatial resolution, whole-brain coverage, and sub-minute temporal resolution, limiting organ-wide interrogation of CNS fluid dynamics in vivo. Here, we introduce intravital synchrotron radiation-based hard X-ray micro computed tomography (SRµCT), a modality that enables dynamic whole-brain imaging at micrometer-scale spatial resolution in living mice. We performed intravital SRµCT of mouse CNS fluid spaces at three synchrotron radiation facilities, imaging both anesthetized free-breathing and mechanically ventilated animals, with and without retrospective cardiac gating. This approach achieves complete brain coverage with temporal resolution of up to 23 s and voxel sizes down to 6.3 µm, at an effective spatial resolution better than 20 µm, enabling time-resolved visualization of cerebrospinal fluid (CSF) contrast distribution and quantitative analysis of tissue motion across the entire brain. By combining micrometer-scale resolution, whole-organ field of view, and dynamic intravital imaging, SRµCT closes a long-standing methodological gap between optical microscopy and magnetic resonance imaging. Intravital SRµCT provides access to spatiotemporal information that cannot be obtained with existing techniques and establishes a framework for testing and integrating mechanistic models of CSF dynamics and solute transport at the scale of the whole brain.