Significance: Digital Holographic Microscopy (DHM) provides label-free quantitative phase images (QPIs) of living cells and has become a powerful tool for studying cellular morphology and dynamics. While most DHM studies have focused on cell-level analysis, the quantitative characterization of neuronal network organization and maturation from DHM images remains largely unexplored, highlighting the need for dedicated computational approaches. Aim: We aimed to develop an automated framework combining deep-learning-based image analysis and graph theory to quantitatively characterize the organization, connectivity, and maturation of neuronal networks in primary rat cortical cultures imaged by DHM. Approach: Two U-Net convolutional neural networks were trained on manually annotated DHM phase images to segment neuronal cell bodies and neurites. The resulting segmentation maps were used to infer putative morphological connections between neurons and generate graph representations of neuronal networks, referred to as graph fingerprints. A panel of 18 connectomics-inspired graph features was then computed to characterize local and global properties of network organization across four stages of culture maturation. Results: The mean area under the receiver operating characteristic curves was 0.98 for cell-body and 0.91 for neurite segmentation, indicating near-perfect identification. Graph-theoretical analysis revealed reproducible topological changes during network maturation in vitro, including increased density, reduced modularity, and progressive network integration. Correlation analysis showed that the 18 graph features grouped into two highly correlated families. A Random Forest classifier identified density and modularity as the most informative descriptors, achieving an accuracy of 87% in classifying maturation stages of neuronal cultures. Conclusions: Our results demonstrate that combining DHM, deep-learning-based segmentation, and graph-theoretical analysis enables quantitative characterization of neuronal network organization and maturation from label-free phase images. This framework provides a foundation for future studies of pharmacological experiments, neuronal network phenotyping, and human induced pluripotent stem cell (hiPSC)-derived neuronal cultures, where quantitative assessment of network organization remains a major challenge.
Yazdani, Z., Belanger, E., Moreaud, M., Llinares, J., Allard, A., Marquet, P., Desrosiers, P.
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