Fig. 2: Simultaneous tracking of the reserve and recycling pool of SVs in live hippocampal neurons using DsdTIM.

a Graphical representation of the Dual-pulse sub-diffractional Tracking of Internalized Molecules (DsdTIM) protocol. Hippocampal neurons expressing Synaptotagmin1-pHluorin (Syt1pH) were stimulated for one min with high K⁺ medium containing anti-green fluorescent protein (GFP) Atto565-tagged nanobodies (At565Nb; red). After stimulation, the excess nanobodies were washed off, and the neurons were chased for 48 h in conditioning medium. After the 48 h chase, the labelled SVs were imaged to assess their mobility at 50 Hz. Immediately afterwards, the same neurons were stimulated for a second time, pulsing for five min with high K⁺ imaging buffer containing anti-GFP Atto647N-tagged nanobodies (At647Nb; green). After this re-stimulation, the excess At647Nb were washed off, and the neurons were chased for 10 min in a low K⁺ imaging buffer. To detect nanobodies inside individual SVs, the neurons were imaged using dual-colour single-molecule tracking at 50 Hz. Graphic created with BioRender.com. b Representative epifluorescence image of an axonal segment expressing Syt1pH acquired before incubation with At647Nb. The dashed boxes in (b) highlight (i) a presynaptic compartment and (ii) a peri-synaptic axonal segment. c–e Maximum intensity projection of (c) Syt1pH/At565Nb (reserve pool; 48 h chase), (d) Syt1pH/At647Nb (recycling pool; 10 min chase), and (e) merged maximum intensity projection. f Trajectory map of tracked reserve SVs containing Syt1pH bound by At565Nb in the (i) presynapse and (ii) axonal segment. g Trajectory map of tracked recycling SVs containing Syt1pH bound by At647Nb in the (i) presynapse and (ii) axonal segment. h Merged trajectory maps. Scale bar 1 µm (b–e) and 200 nm (f–h). Data were obtained from ≥3 independent neuronal cultures.