Redox‐Activated Probes Enable High‐Contrast Live Imaging of Native Postsynaptic Scaffolds

Direct visualization of postsynaptic scaffolds in living neurons is essential for dissecting synaptic dynamics and plasticity. Existing methods for live synapse visualization have major constraints, relying on genetic engineering or multistep application of live-cell incompatible antibodies or nanobodies. Available affinity probes and delivery strategies lack the required contrast due to incomplete or excess delivery. Here, we introduce Sylives, a set of compact, synthetic fluorescent peptides that enable high-contrast live imaging of inhibitory (gephyrin) and excitatory (PSD-95) postsynaptic scaffolds in native neurons. Critically, by pre-purification of the redox-cleavable CPP-probe conjugate we overcome side-product formation of in-situ coupling strategies, achieving reliable cytosolic delivery and restored scaffold binding after intracellular reduction. The Sylive design addresses the need for nanomolar probe levels versus micromolar CPP for clean labelling and efficient delivery by decoupling targeting and uptake. Through quantitative evaluation of uptake and off-target binding, we defined a transferrable parameter space for effective intracellular delivery. Near traceless Sylive uptake and target specificity are validated by direct comparison to transiently expressed proteins and immunolabeling in fixed neurons. The reduction-sensitive Sylive conjugates enable high-contrast, specificity-restored labelling of endogenous postsynaptic sites without genetic modification and offer a modular platform for targeting alternative intracellular proteins in living primary neurons.