The Nanoimager platform enables single-molecule neuroimaging of cultured neuronal cells and brain tissue sections, with a resolution reaching 20 nm. Combining automation features with super-resolution imaging allows the quick and efficient identification of region of interests to then gather localizations from thousands of synaptic proteins. It also facilitates acquisition of data on single molecule distribution, co-localization, or postsynaptic density profiles. Single-molecule imaging has the potential to help establish and characterize new disease models to enable robust testing of novel therapeutics and facilitate drug development. With its compact design, lack of need for a dark room or additional infrastructure, the Nanoimager is the most accessible super-resolution tool ever designed.