To understand how the molecular machinery of synapsesworks, itis essential to determine an inventory of synaptic proteins at a subsynapticresolution. Nevertheless, synaptic proteins are difficult to localizebecause of the low expression levels and limited access to immunostainingepitopes. Here, we report on the exTEM (epitope-exposedby expansion-transmission electron microscopy) method that enables the imagingof synaptic proteins in situ. This method combinesTEM with nanoscale resolution and expandable tissue-hydrogel hybridsfor enhanced immunolabeling with better epitope accessibility viamolecular decrowding, allowing successful probing of the distributionof various synapse-organizing proteins. We propose that exTEM canbe employed for studying the mechanisms underlying the regulationof synaptic architecture and function by providing nanoscale moleculardistribution of synaptic proteins in situ. We alsoenvision that exTEM is widely applicable for investigating proteinnanostructures located in densely packed environments by immunostainingof commercially available antibodies at nanometer resolution.