Super-resolution imaging provides unprecedented visualization of sub-cellular structures, but the two main techniques used, single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED), are not easily reconciled. We present a protocol to super-impose nanoscale protein distribution reconstructed with SMLM to sub-cellular morphology obtained in STED. We describe steps for tracking cells on etched coverslips and registering images from two different microscopes with 30-nm accuracy. In this protocol, synaptic proteins are mapped in the dendritic spines of primary neurons. For complete details on the use and execution of this protocol, please refer to Inavalli et al. 1 .