Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy alters the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here a nanobody is presented that binds the calcium sensor synaptotagmin‐1 (NbSyt1). This nanobody functions as an intrabody (iNbSyt1) in living neurons and is minimally invasive, leaving synaptic transmission almost unaffected, as suggested by the crystal structure of the NbSyt1 bound to Synaptotagmin‐1 and by the physiological data. Its single‐domain nature enables the generation of protein‐based fluorescent reporters, as showcased here by measuring spatially localized presynaptic Ca2+ with a NbSyt1‐ jGCaMP8 chimera. Moreover, the small size of NbSyt1 makes it ideal for various super‐resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience with unprecedented precision across multiple spatiotemporal scales.

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy changes the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here we introduce a nanobody that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions in living neurons as an intrabody (iNbSyt1) and is minimally invasive, leaving synaptic transmission almost unaffected, as demonstrated by the crystal structure of the NbSyt1 bound to synaptotagmin-1 and by our physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as we showcase here by measuring spatially-localized presynaptic Ca2+ with an NbSyt1- jGCaMP8 chimera. Moreover, its small size makes the NbSyt1 ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience at a higher precision than possible in the past, over multiple spatiotemporal scales.