Disordered glass nanowire substrates produce in vivo-like astrocyte morphology revealed by optical diffraction tomography

Astrocytes, integral components of the central nervous system (CNS), fulfill crucial roles such as maintaining ion homeostasis, providing neuroprotection, and contributing to the blood-brain barrier. Their distinctive, star-like morphology is essential to these functions, and abnormalities in astrocyte structure are linked to numerous neurological disorders. However, our understanding of astrocyte morphology, particularly in vivo, remains limited. Traditional imaging methods, such as fluorescence microscopy, introduce challenges like restricting continuous observation and comprehensive morphological analysis. In this study, we present a novel approach utilizing optical diffraction tomography (ODT), an advanced imaging technique that generates 3D refractive index profiles, to image and quantify detailed astrocyte morphology. We demonstrate, for the first time, the application of ODT to image samples through and on disordered glass nanowire (NW) substrates, overcoming the typical challenges posed by nanostructures, which can disrupt phase reconstruction. Crucially, we show that disordered glass nanowire (NW) substrates can induce in vivo-like astrocyte morphology in cultured rat cortical astrocytes. Compared to traditional glass substrates, astrocytes grown on disordered glass NWs substrates exhibited enhanced process branching and greater total arbor length--features typically observed in their natural, in vivo state, a state of advanced maturation. This finding underscores the significant influence of substrate topography on astrocyte structure and highlights the unique potential of nanostructured environments to mimic physiological conditions. By leveraging ODT, we were able to monitor astrocyte behavior on these substrates, providing unprecedented insights into their morphological dynamics. Our study pioneers the use of nanostructured substrates for reconstructing astrocyte morphology and sets the stage for further exploration of how microenvironmental cues shape astrocyte morphology and behavior.