Tirf microscope

Transfected PC12 cells were bathed in Buffer A (145 mM NaCl, 5 mM KCl, 1.2 mM Na₂HPO₄, 10 mM D-glucose and 20 mM HEPES, pH 7.4). The cells were then visualized using an inverted Roper Scientific TIRF microscope equipped with a perfect focus system and an ILas² double laser illuminator (Roper Scientific). The microscope was fitted with a Nikon CFI Apo TIRF × 100 (1.49 numerical aperture) oil objective (Nikon Instruments) and an Evolve 512 delta EMCCD camera (Photometrics). Metamorph software was used for movie acquisition (Metamorph 7.7.8, Molecular Devices) at 50 Hz with 16,000 frames acquired for each cell kept at 37 °C. For PALM, a 405 nm laser was used to photoactivate the cells expressing Sx1A-mEos2, and a 561 nm laser was used for excitation of the resulting photo-converted single-molecule fluorescence signal. The sample was illuminated simultaneously with both the lasers. To isolate the mEos2 signal from auto-fluorescence and background signals, we used a double beam splitter (LF488/561-A-000, Semrock) and a double band emitter (FF01-523/610-25, Semrock, USA). To spatially distinguish and temporally separate the stochastically activated molecules during acquisition, the respective power of the lasers was adjusted (405 nm laser used 4–6% of initial laser power (200 mW) and 561 nm laser used 75–80% of initial laser power (200 mW)).

Transfected PC12 cells were bathed in Buffer A (145 mM NaCl, 5 mM KCl, 1.2 mM Na₂HPO₄, 10 mM D-glucose and 20 mM HEPES, pH 7.4). The cells were then visualized using an inverted Roper Scientific TIRF microscope equipped with a perfect focus system and an iLas2 double-laser illuminator (Roper Scientific). The microscope was fitted with a Nikon CFI Apo TIRF × 100 (1.49 NA) oil objective (Nikon Instruments) and an Evolve 512 delta EMCCD camera (Photometrics). Metamorph software was used for movie acquisition (Metamorph 7.7.8, Molecular Devices) at 50 Hz with 16,000 frames acquired for each cell kept at 37 °C. A 491 nm laser was used to photoactivate the cells expressing Dyn1aa-GFP, Dyn1ab-GFP, and Dyn1bb-GFP in both control (before) and 2 mM Ba²⁺ (during) stimulation conditions. TIRF angle was calibrated each imaging session and TIRF critical angle was ~70°. PC12 cells were selected based on cell morphology (proper attachment to the cover-glass and presence of filipodia).
The LFA refers to areas with comparatively less Dyn1-GFP fluorescence observed throughout the acquisition period, compared to that of HFA which exhibited high intensity Dyn1-GFP. Since the distribution of HFAs and LFAs varied dynamically, ROIs of equal size were meticulously chosen for each movie to ensure they were within either HFAs or LFAs throughout the duration of the acquisition.