Figure 3.

sptPALM of P2X7-Dendra2 receptors. Live cell sptPALM was performed at 50 Hz on dendrites expressing P2X7-Dendra2. (Aa) Epi-fluorescence of a P2X7-Dendra2 (green) expressing dendrite and FM4-64 (red) labeled synapses. FM4-64 was used for identification of “synaptic” and “extra-synaptic” trajectories. (Ab) Representative threshold image obtained from a super-resolved rendered image shows nanoclusters used for localization of trajectories. Trajectories were localized as “trapped” or “free” within nanoclusters. (Ac,Ad) Example of trajectories obtained using the sptPALM approach. Minimum 10-point long trajectories were used for diffusion measurements in order to reduce calculation error. (B) “Synaptic” and “Trapped” trajectories show slower diffusion compared to “Extra-synaptic” and “Free” trajectories, respectively (Kolmogorov-Smirnov statistical test ^***p < 0.001). Inset: Proportion of trajectories observed for each fraction (Synaptic + Extra-synaptic = 100% and Trapped + Free = 100%). (C) Mean square displacement (MSD) plot shows more confined P2X7Rs at synapses and within nanoclusters (trapped).