TIRF microscopy was performed as previously described (Sochacki et al., 2012 (link); Larson et al., 2014 (link)). Briefly, an inverted fluorescence microscope (IX-81; Olympus, Center Valley, PA) with a 100×/1.45 numerical aperture objective (Olympus) was used for TIRF imaging. Fluorescence was excited alternatively with 488- or 561-nm laser lines that were combined and passed through a 488/561DM filter cube (Semrock, Rochester, NY). Emission was spectrally separated using a 565DCXR dichroic mirror and projected side by side on an electron-multiplying charge-coupled device camera (DU 897; Andor, Belfast, UK) with a DualView image splitter (Photometrics, Tucson, AZ) containing 525Q/50 and 605Q/55 filters. The green and red images were superimposed in postprocessing by acquiring an image of 100-nm yellow-green beads (Invitrogen) that were visible in both channels, mapping the position of six beads in both channels, and then superimposing the channels using projective image transform. This protocol was performed each day before experimental images were recorded. Bead imaging was also used to confirm the uniformity and quality of the TIRF illumination field before each experiment.
Images were acquired with IQ2 software (Andor) successively in the green and then the red channels with an exposure time of 500 ms and a 500-ms pause between pairs of images. Pixel size was 160 nm.
Images were acquired with IQ2 software (Andor) successively in the green and then the red channels with an exposure time of 500 ms and a 500-ms pause between pairs of images. Pixel size was 160 nm.
