Myelinated fiber labeling and orientation mapping of the human brain with light-sheet fluorescence microscopy

Year: 2025

Authors: Sorelli M., Di Meo D., Bradley S., Cheli F., Ramazzotti J., Caria F.F., Perego L., Destrieux C., Hof P.R., Pavone F.S., Mazzamuto G., Costantini I.

Autors Affiliation: European Lab Nonlinear Spect LENS, Sesto Fiorentino, Italy; Univ Florence, Dept Informat Engn, Florence, Italy; Univ Florence, Dept Biol, Florence, Italy; Univ Florence, Dept Phys & Astron, Florence, Italy; Univ Tours, UMR 1253, iBrain, INSERM, Tours, France; Icahn Sch Med Mt Sinai, Friedman Brain Inst, Nash Family Dept Neurosci, New York, NY 10029 USA; Icahn Sch Med Mt Sinai, Ctr Discovery & Innovat, New York, NY 10019 USA; CNR, Natl Inst Opt CNR INO, Sesto Fiorentino, Italy.

Abstract: The convoluted network of myelinated fibers that supports behavior, cognition, and sensory processing in the human brain is the source of its extraordinary complexity. Advancements in tissue optical clearing, 3D fluorescence microscopy, and automated image analysis have enabled unprecedented insights into the architecture of these networks. Here, we investigate the multiscale organization of myelinated fibers in human brain tissue from the brainstem, Brocas area, hippocampus, and primary visual cortex by exploiting a specific fiber staining method, light-sheet fluorescence microscopy (LSFM), and an advanced spatial orientation analysis tool. Using an optimized protocol that integrates tissue clearing with the lipophilic DiD probe to achieve uniform and deep myelinated fiber labeling, we generate micrometer-resolution volumetric reconstructions of multiple brain regions through an inverted LSFM. Automated image processing, employing unsupervised 3D multiscale Frangi filters, provides orientation distribution functions and local orientation dispersion maps. This enables precise characterization of the directionality of white matter bundles, linking mesoscopic structural properties to orientation details computed at the native micrometric resolution of the LSFM apparatus. The presented workflow illustrates a robust platform for large-scale, high-resolution brain mapping, which may facilitate the investigation of pathological alterations with unparalleled spatial resolution and, furthermore, the validation of other neuroimaging modalities.

Journal/Review: NEUROIMAGE

Volume: 323      Pages from: 121581-1  to: 121581-13

More Information: We express our gratitude to the donor involved in the body donation program of the Association des dons du corps du Centre Ouest, Tours, France, and Massachusetts General Hospital Autopsy Service, USA, who made this study possible by generously donating their body to science. This project received funding from the General Hospital Corporation Center of the National Institutes of Health under award number U01 MH117023, as well as from BRAIN CONNECTS (award number U01 NS132181). The content of this work is solely the responsibility of the authors and does not necessarily represent the o fficial views of the National Institutes of Health-USA. Additional support was provided by the European Union’s Horizon 2020 research and innovation Framework Programme under grant agreement No. 654148 (Laserlab-Europe), and by HORIZON-INFRA-2022-SERV-B-01 EBRAINS 2.0: A Research Infrastructure to Advance Neuroscience and Brain Health Horizon Europe-Framework Programme for Research and Innovation (2021-2027). This research also benefited from funding by the Italian Ministry for University and Research, through the Advanced Light Microscopy Italian Node of Euro-Bioimaging ERIC, and by the European Union-Next Generation EU, Mission 4 Component 1, CUP: B53C22001810006, Project IR0000023 SeeLife Strengthening the Italian Infrastructure of Euro-Bioimaging. Further support came from Fondazione Cassa di Risparmio di Firenze (project Human Brain Optical Mapping), by RICTD2025_2026-CUP: B97G24000240005, and from LENS and CNR for technical and scientific assistance to the Italian National Node FOE 2022-CUP: B53C24004790001. The University of Florence, Italy also contributed funding (D.R. n. 464 del 02/04/2024) for the project Smart hydrogels with enhanced toughness to enable human brain tissue clearing (SMART-brain), CUP: B97G24000240005. Finally, this research was supported by the Italian National Recovery and Resilience Plan (NRRP), M4C2, funded by the European Union-NextGenerationEU, IR0000011, CUP: B51E22000150006, Project EBRAINS-Italy.
KeyWords: Optical Clearing Methods; In-vivo; System; Tissue; Histology; Density; Pathway; Stain; Tool
DOI: 10.1016/j.neuroimage.2025.121581