Our recent collaborative work has delivered whole brain and nerve cord connectomes (synaptic resolution wiring diagrams) for adult Drosophila – the first for an animal that can walk (or fly) with complex visual, motor and cognitive behaviour. We have also identified over 11,000 cell types within these electron microscopy (EM) connectomes, defining their conserved neural circuit architecture. However, only a few hundred of these cell types have been molecularly characterised as far as the genes they express. Moreover, the precise positioning along neurites of key signalling molecules such as gap junctions, fast acting neurotransmitters and neuropeptides and their receptors is a crucial determinant of circuit function, but this information is largely unknown. We propose to optimise new expansion microscopy methods that use total protein staining to label neuronal ultrastructure with EM-like contrast in samples physically expanded up 20x. This super-resolution approach will allow key signalling proteins to be localised within individual neurons and to obtain the complete morphology of all neurons within a brain. In Drosophila neuronal morphologies can be directly matched across brains so this will allow molecular annotation of the connectome. We will focus on Drosophila but the pipeline will be applicable in other species.