Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24 Hz, 40 ms after local calcium activity; endocytosis and exocytosis events at up to 11 Hz, 40 ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3 Hz, 70 ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time.

Arc (or Arg3.1), Activity Regulated-Cytoskeleton associated-protein is pivotal to mediate plastic responses in neuronal cells. In vitro and in vivo studies suggest its ability to form high- and low-order oligomers which are potentially involved in neuronal trafficking. Despite its important function, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we take advantage of several complementary advanced fluorescence microscopy and spectroscopy techniques to observe and quantify Arc oligomeric states in cellular environment especially in the synapses. In cells, we uncovered Arc-Arc intermolecular interactions, Arc tendency to form liquid condensates and to interact with lipid bilayers. High-order oligomers are found to localize at the excitatory synaptic compartment and to directly affects AMPA receptor surface levels. Together, our observations support the model by which Arc oligomerization mediates plasma- membrane negative inward curvature favoring AMPA receptors endocytosis.