Uncovering Astrocyte Morphological Dynamics Using Optical Diffraction Tomography and Shape‐Based Trajectory Inference

Astrocytes, integral components of the central nervous system, are increasingly recognized for their multifaceted roles beyond support cells. Despite their acknowledged importance, understanding the intricacies of astrocyte morphological dynamics remains limited. Our study marks the first exploration of astrocytes using optical diffraction tomography (ODT), establishing a label-free, quantitative method to observe morphological changes in astrocytes over a 7-day in-vitro period. ODT offers quantitative insights into cell volume, dry mass, and area through label-free, real-time measurements-capabilities that are challenging to achieve with conventional imaging techniques. Through comprehensive analysis of 3D refractive index maps and shape characterization techniques, we capture the developmental trajectory and dynamic morphological transformations of astrocytes. Specifically, our observations reveal increased area and a transition to larger, flattened shapes, with alterations in cell volume and density, indicating shifts in cellular composition. By employing unsupervised clustering and pseudotime trajectory analysis, we introduce a novel morphological trajectory inference for neural cells, tracking the morphological evolution of astrocytes from elongated to evenly spread shapes. This analysis marks the first use of trajectory inference based solely on morphology for neural cell types, laying a foundation for future studies employing ODT to examine astrocyte dynamics and neural cell interactions across diverse substrates.