Neurons are highly polarized cells that display characteristic differences in the organization of their organelles in axons and dendrites. The kinases SadA and SadB (SadA/B) promote the formation of distinct axonal and dendritic extensions during the development of cortical and hippocampal neurons. Here, we show that SadA/B are required for the specific dynamics of axonal mitochondria. Ankyrin B (AnkB) stimulates the activity of SadA/B that function as regulators of mitochondrial dynamics through the phosphorylation of tau. Suppression of SadA/B or AnkB in cortical neurons induces the elongation of mitochondria by disrupting the balance of fission and fusion. SadA/B-deficient neurons show an accumulation of hyper-fused mitochondria and activation of the integrated stress response (ISR). The normal dynamics of axonal mitochondria could be restored by mild actin destabilization. Thus, the elongation after loss of SadA/B results from an excessive stabilization of actin filaments and reduction of Drp1 recruitment to mitochondria.

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.

Advanced 3D imaging techniques and image segmentation and classification methods can transform biomedical research by offering insights into the human brain cytoarchitecture under pathological conditions. We propose a comprehensive pipeline for 3D imaging and automated quantitative cellular phenotyping on Formalin-Fixed Paraffin-Embedded human brain specimens. We exploit the versatility of our method by applying it to different human specimens from both adult and pediatric, normal and abnormal brain regions. Quantitative data on neuronal morphology, local density, and spatial clustering level are obtained from a machine-learning-based analysis of the 3D cytoarchitectural organization of cells identified by different molecular markers in two subjects with malformations of cortical development. This approach grants access to a wide range of clinical specimens, allowing for volumetric imaging and quantitative analysis of human brain samples at cellular resolution. Possible genotype-phenotype correlations can be unveiled, providing insights into the pathogenesis of various brain diseases and enlarging treatment opportunities.

The intricate macroscopic and microscopic structure of the human brain poses a significant challenge for techniques that rely on diffuse near-infrared light for functional imaging and diagnostics.¹ The complex and heterogeneous composition of brain tissue influences light propagation, affecting the accuracy of these techniques. Current optical methods, such as functional near-infrared spectroscopy (fNIRS) and diffuse optical tomography (DOT), focus primarily on monitoring hemodynamic responses by mapping blood oxygenation changes in different brain regions.^{2 – 5} Among these, time-resolved techniques play a crucial role in extracting absolute tissue oxygen saturation values and estimating the reduced scattering coefficient,⁶ both of which are essential for functional and clinical assessments. A key advantage of these methods is their ability to separate extra-cerebral and cerebral contributions, as different photon propagation times correspond to different penetration depths. This feature enhances the accuracy of functional imaging and disease diagnostics by isolating signals from deeper brain structures, as long as light transport through brain tissue is accurately modeled.

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 micrometerresolution 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.