Expansion microscopy reveals nano-scale insights into the human neuromuscular junction

The neuromuscular junction (NMJ) is a specialized synapse that relays signals from the lower motor neuron to the skeletal muscle. Here, we detail the development and application of expansion microscopy (ExM) as a highly accessible, relatively cheap, powerful, and reproducible tool with which to obtain high-resolution insights into the subcellular structure and function of NMJs from whole-mount preparations, previously only achievable using super-resolution microscopy. ExM is equally applicable to both mouse and human tissue samples, facilitating high-resolution comparative analyses. Qualitative and quantitative analysis of ExM images reveals significant differences in the distribution of acetylcholine receptors, synaptic vesicles, and voltage-gated Na⁺ 1.4 (NaV1.4) channels between human and mouse NMJs that are not readily observable using conventional confocal microscopy. We conclude that ExM offers a cost-effective and adaptable approach to facilitate nano-scale imaging of the NMJ.