Cfi apo tirf 100 1.49 n a oil objective

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.

Fixed and stained INS-1E cells, EndoC-βH1, dispersed human pancreatic islets and live GFP–ELKS-expressing pancreatic islets were imaged using TIRFM performed on an inverted research microscope Nikon Eclipse Ti-E (Nikon) with a perfect focus system (PFS) (Nikon), equipped with a Nikon CFI Apo TIRF 100×1.49 NA oil objective (Nikon), Evolve 512 EMCCD (Photometrics) or Prime BSI camera (Photometrics) or CoolSNAP HQ2 CCD camera (Roper Scientific) and controlled with the MetaMorph 7.7.11.0 software (Molecular Devices). Images were projected onto the chip of an Evolve 512 camera with an intermediate lens 2.5× (Nikon C mount adapter 2.5×) or onto a CoolSNAP HQ2 or a Prime BSI chip without the lens. In all cases the final magnification was equal to 0.065 μm/pixel. To keep cells at 37°C a stage top incubator INUBG2E-ZILCS (Tokai Hit) was used. For excitation, 491 nm 100 mW Stradus (Vortran), 561 nm 100 mW Jive (Cobolt) and 642 nm 110 mW Stradus (Vortran) lasers were used. The microscope was equipped with an ET-GFP 49002 filter set (Chroma) for imaging of proteins tagged with GFP, an ET-mCherry 49008 (Chroma) and an ET-405/488/561/647 filter set. For presentation, images were adjusted for brightness using ImageJ 1.50b.