Plan apo tirf 1.49na objective

Primary neuron cultures were plated on poly-L-lysine-coated coverglass bottom dishes as described under Cell Culture. Neurons were imaged using TIRF microscopy to minimize phototoxicity, improve resolution, and maximize acquisition speed. Images were acquired using a 100× Plan Apo TIRF 1.49NA objective (Nikon). Images were acquired on a 5 s interval for 5 min, except for pharmacological treatments in which three positions were acquired on a 15 s interval for 45 min, and optogenetic activation experiments which were acquired on a 5 s interval for 15 min. Images were acquired with 1 × 1 binning, or 2 × 2 binning for optogenetic activation experiments, and wherever more than two fluorescent channels were acquired (MIM + EVL + LifeAct experiments, MIM + EVL + Arp3/MRLC experiments) in order to decrease the light dose. Exposure times ranged between 70 and 200 ms at 100% laser power at TIR critical angle for each channel. For optogenetic activation experiments, whole cells were illuminated with vertical incident 488 nm laser light (non-TIRF) at 10% power for 400 ms exposure every 5 s during the stimulation phase.

Cells expressing Lifeact-iRFP670 and MLC-mRuby2 were used in this experiment to analyze actin dynamics during protrusion at the leading edge. Cells were plated as described above and ROCK inhibitor was added at indicated concentrations to dissolve SCABs. Cells were imaged using TIRF illumination with a ×100 Plan Apo TIRF 1.49NA objective on a Nikon Ti-E inverted microscope equipped with a Hamamatsu ORCA-Flash 4.0 V2 cMOS camera, Photometrics Evolve 512 EMCCD camera. Images were acquired on a 20-s interval for indicated duration. For EVL overexpression time-lapse experiments, cells expressing Lifeact-mEGFP and MLC-mRuby2, with or without iRFP670-EVL, were co-cultured and imaged as described above.
Kymography analysis of actin dynamics: Similar approach as above was used to register the kymographs, with the exception that maximum intensity projection of the fluorescent images from the entire time series was used to guide the positioning of the kymograph line.