TIRF imaging data were collected using an inverted microscope (IX71; Olympus) equipped with a 150×/1.45 NA oil-immersion, TIRF objective (U-APO; Olympus). A 637-nm laser diode (HL63133DG; Thorlabs) was used for AF647 excitation, and a 488-nm laser (Cyan Scientific; Spectra-Physics) was used for fluorescence excitation of mNG. A quad-band dichroic and emission filter set (LF405/488/561/635-A; Semrock) was used for sample illumination and emission. Emission light was separated onto different quadrants of an EMCCD camera (iXon 897; Andor Technologies), using a custom-built two-channel splitter with a 655-nm dichroic (Semrock) and 584/20-nm and 690/20-nm additional emission filters. Images had a pixel size of 0.106 μm and were acquired at 10 frames/second (100 ms exposure time).
The analysis algorithms and parameters used to track individual mNG molecules were previously described in Schwartz et al. (2017) (link). Briefly, tracks were analyzed using home-built MATLAB code that localized single particles per frame and determined connections between frames. The collected tracks were fitted to a two-component distribution to determine the off-rates of Syk at the membrane (Schwartz et al., 2017 (link)).
The analysis algorithms and parameters used to track individual mNG molecules were previously described in Schwartz et al. (2017) (link). Briefly, tracks were analyzed using home-built MATLAB code that localized single particles per frame and determined connections between frames. The collected tracks were fitted to a two-component distribution to determine the off-rates of Syk at the membrane (Schwartz et al., 2017 (link)).
