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

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, Broca's 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.

Fig. 1.

Co-labeling of neurons and myelinated fibers stained with DiD in the human brain. (a) Images showing a portion of a brainstem slab (thickness: 300 μm) before (pre-SHORT) and after (post-SHORT) clearing and labeling with the SHORT method and after myelinated fiber labeling with the DiD probe (post-DiD). (b) Confocal spinning disk images of a SHORT-processed human brainstem slab (pons) labeled with DiD (magenta), an antibody against TH (yellow), and YOYO1 (cyan). Objective: 60X/1.4NA (voxel size: 0.11 μm × 0.11 μm × 1 μm). Single channel images are shown on the right. (c) Confocal spinning disk images of a SHORT-processed human brainstem slab (pons) labeled as in (b). Objective: 20X/0.75NA (voxel size: 0.32 μm × 0.32 μm × 2 μm). (d) Magnified inset images of (c). (e) Confocal spinning disk images of a SHORT-processed human brainstem slab (midbrain) labeled as in (b). Objective: 10X/0.5NA (voxel size: 0.65 μm × 0.65 μm × 2 μm). (f) Magnified inset images of (e). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Co-labeling and LSFM imaging of myelinated fibers in different human brain areas. (a) Maximum intensity projection (MIP) images (voxel size: 3.6 μm × 3.6 μm × 3.6 μm) of a SHORT-processed human brainstem slab (thickness: 300 μm) labeled for DiD (magenta), TH (yellow) and YOYO1 (cyan). (b) The multichannel MIP is shown from (a). (c) 3D rendering image of a 5 mm × 5 mm mm ROI is shown for (b). (d) Magnified inset images on the right highlight regions of interest in the white boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

LSFM images show the homogeneous staining of DiD along the thickness. MIP images of SHORT-processed human brain slab labeled with the DiD probe: (a) brainstem (300 μm-thick), (b) Broca’s area (500 μm-thick), (c) hippocampus (500 μm-thick) and (d) primary visual cortex (300 μm-thick). Orthogonal view images (xz and yz) and 3D rendering images are shown for a 5 × 5 mm ROI identified by the red boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Enhancement and orientation analysis of myelinated fibers in LSFM reconstructions of human brain tissue samples. (a–d) Average intensity projections of the 3D fiber orientation HSV colormaps generated by applying Foa3D to the mesoscopic reconstructions of the four anatomical brain regions imaged with LSFM (DiD fluorescence channel, isotropic pixel size: 3.6 μm); (e–h) ODFs of myelinated fibers (super-voxel size: 1 mm³); (i–l) orientation dispersion maps of myelinated fibers (isotropic pixel size: 250 μm); (m–p) 2mm × 2 mm × 0.25 mm volumetric ROIs (white boxes, ODF super-voxel size: 250 μm × 250 μm × 250 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Diagram illustrating the workflow for quantitative 3D myeloarchitectural analysis of human brain tissues. (a) Human brain slabs are processed with the SHORT method for tissue delipidation and antibody-mediated labeling of various neuronal populations. Next, in order to stain myelinated fibers, slabs are incubated in D-off solution, to slow the dye’s incorporation into the membrane, followed by incubation in D-on solution to allow DiD molecules to accumulate within the myelin sheath. A custom-built LSFM setup is used for volumetric imaging of the cleared and labeled brain slabs. (b) Post-processing of raw LSFM image stacks, which first undergo a composite affine transformation and, then, are automatically aligned using the ZetaStitcher tool for volumetric stitching of large, high-resolution microscopy data. (c) Main stages of the unsupervised analysis of 3D fiber orientations performed by the Foa3D Python tool based on Frangi filters. The figure is partially created in BioRender: https://BioRender.com/66xrdm3.