Characterizing the nanoscale organization of chemical synapses by expansion-Airyscan microscopy

Abstract Transsynaptic nanocolumns are nanoscale alignments of pre- and postsynaptic proteins that ensure efficient synaptic transmission. Autoantibodies against the transsynaptic protein LGI1, implicated in LGI1 autoimmune encephalitis, are known to disrupt synaptic function, but their impact on nanocolumn architecture remains unclear. To investigate this, we employed a multi-modal super-resolution imaging strategy combining post-gelation immunolabeling, expansion microscopy with Airyscan super-resolution imaging and compared the results with another advanced super-resolution microscopy technique – d STORM. By physically expanding hippocampal neuron cultures 10.3-fold, our approach enabled decrowding of dense synaptic regions and improved epitope accessibility as well as labeling efficiency. Post-expansion immunolabeling followed by multicolor Airyscan imaging achieved 20–30 nm resolution, allowing detailed visualization of transsynaptic nanocolumns. With this approach we observed LGI1 autoantibody-induced sharpening of Munc13-1 – GluA1 alignment and a shift in AMPA receptor positioning. These findings highlight how advanced expansion-based imaging enables quantitative analysis of nanoscale synaptic alterations in disease contexts.