Cell cmr las 488

All experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Fluorophores were excited with a 488 laser (Cell CMR-LAS-488, Olympus), and monitored using an inverted TIRF-equipped microscope (IX83, Olympus) under a 150x/1.45NA objective (UapoN). The Z-drift compensation system (IX3-ZDC) was used to ensure a constant position of the focal plane during imaging. Near-TIRF was achieved by adjusting the incident angle to 63.7°, which is near the critical angle of 63.63°. Images were acquired every 50 ms using a cooled EMCCD camera (iXon life 888, ANDOR). Field simulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.).

All near-total internal reflection fluorescence (near-TIRF) experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Individual release events were evoked by 1Hz field stimulation for 200s, unless noted otherwise. Fluorophores were excited with a 488 laser (Cell CMR-LAS-488, Olympus), and monitored using an inverted TIRF-equipped microscope (IX83, Olympus) under a 150x/1.45NA objective (UApo N). The Z-drift compensation system (IX3-ZDC) was used to ensure constant position of the focal plane during imaging. Near-TIRF with a penetration depth of <1 μm was achieved by adjusting the incident angle to 63.7°, which is near the critical angle of 63.6°. Images were acquired every 50 ms (with an exposure time of 49.38 ms) using a cooled EMCCD camera (iXon life 888, ANDOR). Field simulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Samples were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2mM CaCl₂, 1mM MgCl₂, 10 mM HEPES, 15mM Glucose, 50 μM APV, 10 μM CNQX adjusted to pH 7.4).