Ilas2 double laser illuminator

For live-cell TIRF microscopy, transfected cells were visualized on a TIRF microscope (Roper Technologies) equipped with an ILas² double laser illuminator (Roper Technologies), a CFI Apo TIRF 100× (1.49-NA) objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Image acquisition was performed using Metamorph software (version 7.7.8; Molecular Devices). Cells were bathed in buffer A.

Single-particle tracking photoactivated localization microscopy of MCF and MDCK cells transfected with Cav1-mEos2 was performed on the Roper Scientific total internal reflection fluorescence (TIRF) microscope equipped with an iLas² double-laser illuminator (Roper Technologies), a CFI Apo TIRF 100× (1.49 NA) objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Images were acquired using Metamorph software (version 7.78; Molecular Devices) at 50 Hz, and 16,000 frames were acquired per cell. A 405-nm laser was used to photoconvert mEos2, with simultaneous 561-nm exposure to excite the photoconverted mEos2. For stochastic photoconversion of mEos2 molecules, a low amount (3–5%) of 405-nm laser and 75–80% of 561-nm laser was used. Data analysis was performed as previously described (Bademosi et al., 2017 (link); Kasula et al., 2016 (link)) using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).

For live-cell oblique illumination, neurons were visualized at 50-Hz (10,000–20,000 frames by image streaming) and 20-ms exposure, at 37°C on a microscope equipped with an ILas² double-laser illuminator (Roper Technologies), a CFI Apo TIRF 100× 1.49 NA objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Image acquisition was performed using Metamorph software (MetaMorph Microscopy Automation and Image Analysis Software, version 7.7.8; Molecular Devices). A quadruple beam splitter (LF 405/488/561/635-A-000-ZHE; Semrock) and a QUAD band emitter (FF01-446/510/581/703-25; Semrock) were used.