Multimodal imaging links molecular identity with nanoscale-resolved 3D cell morphology in opossum cortical cultures

In the mammalian central nervous system (CNS), the ability to regenerate after injury drops sharply in the first weeks after birth, but how this change is reflected in cell morphology and organization is still not fully understood. Here we cultivated cortical cell cultures from the gray short-tailed opossum (Monodelphis domestica) and compared cells derived from early postnatal pups (postnatal day 3-6, "Pyoung") with those from older pups (postnatal day 16-18, "Pold"), which differ in their capacity to regenerate after injury. For each culture, we assessed marker-defined cellular composition using fluorescently labeled markers for neurons, glia, and immature/progenitor cells, and characterized their nanoscale-resolved three-dimensional morphology using label-free scanning ion conductance microscopy (SICM). Pyoung cultures exhibited active change in shape over time, contained a higher proportion of immature SOX2⁺ cells, and grew more neurites (TUJ1⁺) into the lesion site after injury. Moreover, Pold cultures showed more stable soma size (e.g., thickness, area, volume), a larger astroglial (SOX9⁺) population early in culture, and a glia-dominated lesion response. These early astroglia included more immature or reactive (vimentin⁺) astrocytes. SICM further revealed cellular bridges across the lesion, ridges left in the substrate by the injury, and aligned neurite outgrowth. Across all measurements, changes in molecular markers were associated with corresponding changes in cell morphology. This multimodal combination of fluorescence labeling and SICM therefore relates marker-defined cellular composition to physical organization across micro- and nanoscale features during and after postnatal in vitro development.