Abstract The brain is the most complex organ in the human body. Understanding its neural connections and molecular anatomy requires imaging technology that is capable of mapping the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. The currently best available option is correlative light and electron microscopy (CLEM), which combines the resolving power and global contrast of EM with the high molecular specificity of fluorescence microscopy. While very powerful, CLEM is laborious, low throughput, expensive and often inadequate in spatial correlation resolution. To date, there is no imaging technology that can resolve specific proteins within the ultrastructure of a synapse. We recently developed an all-optical method called pan-Expansion Microscopy (pan-ExM) that has the potential to allow standard confocal microscopy users to do just that: by combining ~20-fold linear expansion of biological samples with novel bulk (pan) staining of total proteins, hallmark ultrastructural features such as post- and pre-synaptic densities and mitochondria cristae can now be resolved by their characteristic pan-staining pattern, analogous to heavy-metal stains in EM. Panluminate Inc. is a new company built around this core technology. Our proposed Phase I project is to validate pan-ExM protocols that will establish pan-ExM as a brain imaging technology. Our overall goal is to enable every neuroscientist to locate specific protein labels within their 3D contextual compartments as well as perform EM-like neuronal tracing in brain tissue. We specifically propose to (1) validate and optimize antibody-labeling compatible protocols for synapse identification, and (2) optimize and validate a novel lipid pan-stain we developed to delineate cellular boundaries to complement our proteome pan-stain. Ultimately, the developments proposed here will enable Panluminate to streamline and deploy pan-ExM sample preparation kits to every neuroscientist interested in simultaneous 3D ultrastructural analysis, antibody labeling, and neuronal tracing in brain tissue and to offer pan-ExM sample preparation and imaging as a service.