Single-molecule microscopy for tracking

For single-molecule microscopy, a Nikon Eclipse Ti with a TIRF objective (× 100, Apo, NA: 1.49) was used. Image acquisition was accomplished using a back-illuminated EMCCD camera (Hamamatsu). The center of a 20-fold expanded beam from a 100-mW multiline argon laser (JDS Uniphase, laser head: 2219-G5MLS) was focused on the back focal plane and operated during image acquisition with 150 to 200 W/cm². A 514-nm laser diode was used as excitation source. For image acquisition, the program Andor Solis 4.21 was applied. Streams of 1500 frames of 20 ms were acquired. Cells continued to grow after imaging, showing that there is little to no photo damage during acquisition. Acquired streams were loaded into Fiji ImageJ [75 (link)], and pixel sizes (106 nm) and time increments were calibrated. Tracking of single molecules was achieved using u-track 2.0 [76 (link)]. Only trajectories with at least four frames were used for further analysis to calculate the diffusion coefficients. Trajectory x/y coordinates and diffusion coefficients were calculated using a custom-made MATLAB script which included localization error. At least 1000 tracks were used for each analysis so that the CDF curves had at least 10⁴ points.