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9 5 I H I G H L I G H T S 2 0 2 1

X-ray holography reveals how the local brain tissue environment affects axon morphology

X-ray nanoholotomography reveals how axon diameters and trajectories are modulated by other white matter structures. This impacts the non- invasive investigation of axon diameter with diffusion MRI and challenges current knowledge of how axons conduct signals.

The structure of axons, the brain s communication cables, affects how fast signals are conducted in the brain network. Axon diameter (AD) determines the conduction velocity (CV) of signals along myelinated axons and acts as an indicator of brain performance. It is also a potential biomarker of some neurodegenerative diseases that selectively damage certain sizes of axons. Currently, diffusion Magnetic Resonance Imaging (MRI) is the only method capable of probing AD in the living brain, but studies show that diffusion MRI overestimates AD when compared to light microscopy. The diffusion MRI approach assumes that axons have very simple cylindrical shapes. To improve the diffusion MRI estimates of AD, therefore, knowledge of three-dimensional axon morphologies is needed [1].

To investigate axon morphology, X-ray nanoholotomography (XNH) was used at beamline ID16A to image the monkey corpus callosum an organised white matter (WM) region consisting of

many axons aligned in the same direction. The ADs in the corpus callosum of the same monkey brain had previously been studied with diffusion MRI [2,3]. By acquiring four consecutive and overlapping holograms, the cylindrical field-of-view (FOV) spanned a diameter of 153.6 µm and height of 584.5 µm, and had a 75-nm isotropic voxel size.

Cells, blood vessels and vacuoles could be segmented and quantified within this volume (Figure 75a), demonstrating the intricate architecture of WM in which the cell clusters aligned either with the main direction of the axons, or were anchored to the blood vessels. Axons were segmented from one of the XNH volumes and were shown to have inherently complex morphologies. A diameter analysis concluded that the diameters of axons vary considerably along their lengths (Figure 75b), with larger axons having less specific mean diameters than smaller ones. This implies that single axon diameters cannot be accurately estimated from two-dimensional images, such as those from light or electron microscopy. By also quantifying the thickness of the signal-boosting myelin sheath, it could be shown that the diameter changes of axons affect the conduction velocities along them, challenging current knowledge of how axons communicate signals.

Simulations of diffusion within the axons established how the variations in diameter and trajectory (Figure 75c) of the axons cause an overestimation of AD with diffusion MRI. Looking ahead, the geometrical characterisations

Fig. 75: a) Three-dimensional visualisation of cell cluster (blue), blood vessel (red), and vacuole (green) segmentations in the extended FOV produced by overlapping XNH volumes. b) Left: Segmented axons from the splenium. Right: The total axon diameter distribution (black dotted line). The diameter distributions along the thinnest (blue) and thickest (green) axons are also shown. c) The trajectory variations of the axons were quantified in terms of the angle of segments of length L from the main axon direction.