ILLUMINATING SYNAPSES: FROM GENETIC TAGGING TO NANOBODY-BASED IMAGING AS NEW TOOLS FOR VISUALIZING SYNAPTIC ARCHITECTURE

Elucidating synaptic architecture requires molecular probes that remain highly specific across imaging scales, from super-resolution light microscopy to electron microscopy. However, most current approaches for visualizing endogenous synaptic proteins rely on conventional antibodies, which are limited by large probe size, multivalency, poor penetration into the synaptic cleft, and variable specificity. Here, we present a complementary set of genetic and nanobody-based tools that enable nanoscale and ultrastructural visualization of synaptic proteins in their native context. First, we generated a knock-in mouse line in which endogenous Neuroligin-1 (Nlgn-1) is N-terminally tagged with a biotin acceptor peptide (bAP), enabling cell-selective biotinylation and high-affinity labeling with streptavidin conjugates without affecting synaptic function or behavior. Using super-resolution fluorescence microscopy and electron microscopy, we show that endogenous Nlgn-1 localizes to both excitatory and inhibitory synapses. Within the synaptic cleft, Nlgn-1 organizes into discrete nanodomains whose number scales with postsynaptic size, revealing a previously inaccessible level of synaptic organization. Second, to enable quantitative protein detection in spatially restricted compartments such as synapses, we developed ultra-small nanobody–gold probes consisting of a defined 1:1 conjugate between a GFP-specific single-domain antibody (~3 nm) and a 1.4 nm gold nanoparticle, with optional fluorescent labeling. These probes efficiently penetrate synapses and enable correlative light and electron microscopy of GFP-tagged proteins. Applied to presynaptic neurexins and postsynaptic AMPA receptors, this approach allowed direct molecular counting and nanoscale mapping, revealing their organization into discrete nanodomains. Together, these tools establish a versatile framework for quantitative, multi-scale analysis of synaptic architecture.