The actin cytoskeleton is a key component for neuronal architecture and function. Along the axon, actin filaments assemble into distinct subcellular structures in the initial segment, along the distal shaft and within presynaptic boutons. Despite advances in the field of super-resolution microscopy, little is known about the presynaptic actin cytoskeleton, because of difficulties in visualizing it alongside the highly enriched postsynaptic compartment.Our objective is to map the presynaptic actin cytoskeleton scaffold and identify its nanostructures to further understand its functions. We have set up a new model using micropatterns of postsynaptic adhesion proteins printed on the glass to induce the formation of isolated presynapses along axons. We first validated this model of presynapses-on-glass by characterizing the induction of presynaptic protein clusters along isolated axons in contact with the micropatterned dots, and confirmed that these clusters have spontaneously cycling of synaptic vesicles, a hallmark of functional presynapses in culture. We then imaged the nanoscale organization of actin in the micropattern-induced presynapses by Single-Molecule Localization Microscopy (SMLM), to determine if the induced presynapses-on-glass exhibit the nanostructures recently described by the team using a presynapse-on-beads model and CRISPR tagging of endogenous actin (Bingham et al., J Cell Biol. 2023).We are currently mapping the different presynaptic components and sub-compartments (active zone, pools of vesicles, scaffolds) together with actin using multicolor SMLM on micro pattern induced presynapses. Our results will refine the understanding of actin organization at presynapses, and gain insight into the function of the distinct presynaptic actin nanostructures.