TIRF Microscopy Imaging Protocol

Microscopy was performed using a Quorum Diskovery microscope that is comprised of a Leica DMi8 microscope equipped with a 63×/1.49 NA TIRF objective with a 1.8× camera relay (total magnification 108×). Imaging was done using 488-, 561, and 637-nm laser illumination and 527/30, 630/75, and 700/75 emission filters. The microscope was operated in TIRFM or widefield epifluorescence microscopy modes, as indicated. Images were acquired using a Zyla 4.2Plus sCMOS camera (Hamamatsu). Fixed-cell TIRFM imaging was done at room temperature with samples mounted in PBS. For live-cell imaging experiments (Figs. 1 and 2), cells were maintained at constant 37 °C during imaging, in phenol-free DMEM/F12 media (Gibco) supplemented with 20 mM Hepes.