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of the cortex and hippocampal brain areas. Synchrotron X-rays with energy in the range 17- 40 keV were used. The applied label-free virtual-histological approach allowed the 3D visualisation of neuronal and glial cell populations within extended dissected brain samples, as well as the identification of pathological neurons by detecting intra- cellular hyper-density (ICHD) in hippocampal and cortical brain layers involved in AD. This density-based signal was identified as amyloid-β and hyper-phosphorylated tau protein lesions with calcium- and iron-metal involvement by extensive comparison of the X-PCI-CT data to immunohistochemistry for amyloid and tau proteins, to intra-cellular elemental distributions measured via X-ray fluorescence nanoscopy (carried out at ID16A on thin sections), to high-field 9.4 T magnetic resonance imaging and to transmission electron microscopy.

Figure 68 shows the results of the correlation of multimodal imaging of neurons affected by amyloid and tau pathology (hyper-dense cells) in the model of AD acquired by XNH (maximum intensity projection image (Figure 68a) and 3D rendering (Figure 68b)) and X-ray fluorescence nanoscopy (normal neurons (Figure 68c) and diseased neurons (Figure 68d)). Figure 69 reports multiscale hierarchical visualisations of hippocampal neurons affected by AD at the organ ((Figure 69a) 33 µm3 voxel X-PCI-CT), tissue-layer ((Figure 69b) 0.33 µm3 voxel X-PCI-CT) and intra-cellular level ((Figure 69c) 1003 nm3 voxel XNH).

As a proof-of-concept, X-PCI-CT was also used to analyse hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyper-density particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy.

The multiscale and multimodal imaging approach used in this study, encompassing highly sensitive and complementary techniques, gave rise to neuroimaging datasets of unprecedented quality and enabled the depiction of cells affected by AD in 3D and in their almost natural environment. With these findings, a previously

Fig. 68: Multimodal imaging of neurons affected by amyloid and tau pathology (hyper- dense cells) in a mouse model of Alzheimer s disease.

Fig. 69: Multiscale visualisations of hippocampal neurons affected by Alzheimer s disease (hyper-dense cells).

unattainable quality of visualisation of anatomical and pathological 3D brain layers from the organ level to the intracellular level was achieved. The ability to volumetrically measure extensive populations of degenerating hippocampal cells in deep brain layers