In the present study, the influence of topographic and mechanical cues on neuronal growth cones (NGCs) and network directionality in 3D-engineered cell culture models is explored. Two-photon polymerization (2PP) is employed to fabricate nanopillar arrays featuring tunable effective shear modulus. Large variations in mechanical properties are obtained by altering the aspect ratio of the nanostructures. The nanopillar arrays are seeded with different neuronal cell lines, including neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), I³Neurons, and primary hippocampal neurons. All cell types exhibit preferential orientations according to the nanopillar topology, as shown by neurites creating a high number of oriented orthogonal networks. Furthermore, the differentiation and maturation of NPCs are affected by the topographic and mechanical properties of the nanopillars, as shown by the expression of the mature neuronal marker Synapsin I. Lastly, NGCs are influenced by effective shear modulus in terms of spreading area, and stochastic optical reconstruction microscopy (STORM) is employed to assess the cytoskeleton organization at nanometric resolution. The developed approach, involving laser-assisted 3D microfabrication, neuro-mechanobiology, and super-resolution microscopy, paves the way for prospective comparative studies on the evolution of neuronal networks and NGCs in healthy and diseased (e.g., neurodegenerative) conditions.

To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present a method to improve the axial localization precision in SMLM by combining point-spread-function engineering with total internal reflection fluorescence (TIRF) fields with decay lengths that vary within the on-time of a fluorophore. Such time-varying illumination field intensity allows one to extract additional axial location information from the emitted photons. With this time varying illumination approach, we show that axial localization is improved two-fold over TIRF-based SMLM using astigmatic PSFs. We calculate theoretical resolution gains for various imaging conditions via the Cramér Rao Lower Bound (CRLB), a commonly used metric to compute the best attainable localization precision in SMLM.