Samples for in vivo imaging were prepared as follows: at room temperature, 10 μl of cells were placed inside of a ring of petroleum jelly onto a 24×60 mm no. 1.5 coverslip and allowed to settle for 1–3 min. Then, a 22×22 mm no. 1.5 coverslip with 5 μl of immobilization buffer (10 mM HEPES, 5 mM EGTA, pH 7.4) was inverted onto the larger cover glass to form a sealed observation chamber. For TIRF imaging, we used a Nikon Eclipse Ti-U inverted light microscope equipped with a 60×/1.49 NA objective lens and a 40 mW 488 nm diode laser (Spectraphysics) (Lechtreck, 2013 (link)). Images were recorded at 10 fps using the iXon X3 DU897 EMCCD camera (Andor) and the Elements software package (Nikon). ImageJ (National Institutes of Health) and the KymoResliceWide plug-in were used to analyze the recordings and generate kymograms. Kymograms, individual frames, and videos were cropped and adjusted for brightness and contrast in ImageJ and Photoshop CC 2018 (Adobe); Illustrator CC 2018 (Adobe) was used to assemble the figures. Still images mostly represent 10-frame walking averages.
For drug treatments, cells were resuspended in M medium with 20 mM mM LiCl or 20 mM NaPPi, pH 6.9; the experiments were repeated twice or more.
For drug treatments, cells were resuspended in M medium with 20 mM mM LiCl or 20 mM NaPPi, pH 6.9; the experiments were repeated twice or more.