A correlative workflow for synaptic imaging by cryo-electron tomography

Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein synaptotagmin-1. Cryo-ET imaging at sites of colocalization predominantly revealed excitatory synapses. Our method allows structural studies of synapse-resident protein complexes in situ, facilitating investigations of the molecular architecture of synapses.