Molecular Decrowding by Tissue Expansion Allows Precise Determination of the Spatial Distribution of Synaptic Proteins at a Nanometer Scale by exTEM.

Abstract

To understand how the molecular machinery of synapses works, it is essential to determine an inventory of synaptic proteins at a subsynaptic resolution. Nevertheless, synaptic proteins are difficult to localize because of the low expression levels and limited access to immunostaining epitopes. Here, we report on the exTEM (<b>e</b>pitope-exposed by e<b>x</b>pansion-<b>t</b>ransmission <b>e</b>lectron <b>m</b>icroscopy) method that enables the imaging of synaptic proteins <i>in situ</i>. This method combines TEM with nanoscale resolution and expandable tissue-hydrogel hybrids for enhanced immunolabeling with better epitope accessibility via molecular decrowding, allowing successful probing of the distribution of various synapse-organizing proteins. We propose that exTEM can be employed for studying the mechanisms underlying the regulation of synaptic architecture and function by providing nanoscale molecular distribution of synaptic proteins <i>in situ</i>. We also envision that exTEM is widely applicable for investigating protein nanostructures located in densely packed environments by immunostaining of commercially available antibodies at nanometer resolution.