Hierarchical X-ray microscopy and mesoscopic diffusion MRI in the same brain reveal the human connectome across scales

Matthieu Chourrout; Ting Gong; Richard Lee Schalek; Andrew Keenlyside; Yaël Balbastre; Neha Karlupia; Ricardo A. Gonzales; István N. Huszár; Eric Wanjau; Joseph Brunet; Theresa Urban; Hector Dejea; David Stansby; Kabilar Gunalan; Bess Glickman; Edward Gaibor; James J. Scherick; Kyriaki-Margarita Bintsi; Chiara Mauri; Cole Analoro; Satrajit Ghosh; Alexandre Bellier; Bruce R. Fischl; Jean C. Augustinack; Paul Tafforeau; Chiara Maffei; Peter Lee; Jeff William Lichtman; Anastasia Yendiki; Claire Walsh

doi:10.64898/2026.04.02.716198

NeurosciencebioRxivApr 6, 2026

Preprint

Hierarchical X-ray microscopy and mesoscopic diffusion MRI in the same brain reveal the human connectome across scales

Matthieu Chourrout iD, Ting Gong iD, Richard Lee Schalek iD, Andrew Keenlyside iD, Yaël Balbastre iD, Neha Karlupia iD, Ricardo A. Gonzales iD, István N. Huszár iD, Eric Wanjau iD, Joseph Brunet iD, Theresa Urban iD, Hector Dejea iD, David Stansby iD, Kabilar Gunalan iD, Bess Glickman iD, Edward Gaibor iD, James J. Scherick, Kyriaki-Margarita Bintsi iD, Chiara Mauri iD, Cole Analoro iD, Satrajit Ghosh iD, Alexandre Bellier iD, Bruce R. Fischl iD, Jean C. Augustinack iD, Paul Tafforeau iD, , Claire Walsh iD

Abstract

We present a multimodal pipeline for 3D imaging of cerebral white-matter archi-tecture across scales, from whole-brain axonal projections down to individual myelinated axons. After diffusion MRI, an adult ex vivo human hemisphere undergoes label-free imaging with Hierarchical Phase-Contrast Tomography (HiP-CT) from 20 {micro}m/voxel in the whole hemisphere to 2 {micro}m/voxel in areas of interest, with intrinsic cross-scale alignment. A 4 cm tissue block extracted from the hemisphere is reimaged with HiP-CT at 0.857 {micro}m/voxel, enabling direct visualisation of single myelinated axons. After osmium staining, micro-CT at 0.364 {micro}m/voxel and electron microscopy at 4 nm/voxel are acquired in biop-sies from the tissue block to validate the presence of myelinated axons in the label-free HiP-CT contrast. Spanning three orders of magnitude in resolution, these co-registered multimodal datasets bridge microscopic wiring and macro-scopic brain organisation, providing a foundation for anatomically grounded whole-brain connectomics.

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