Cells were imaged in epifluorescence after fixation with 4% paraformaldehyde (Electron Microscopy Sciences) solution in PBS and subsequent washes with PBS. Images were acquired on a Deltavision system (Applied Precision) equipped with a 60X 1.42 numerical aperture oil objective and SoftWoRx software (Applied Precision). Deconvolution of fluorescence images using measured point-spread function was done in the SoftWoRx software, and final images were processed using FiJi32 (link) or Metamorph software.
All total internal reflection fluorescence microscopy experiments were performed on a custom-built microscope based on an Olympus IX-70 microscope, equipped with a UApoN 100× 1.49 NA oil objective, a 488-nm, 594-nm, and a 647-nm laser. The cells were imaged under azimuthal TIR-FM33 (link) using a Cairn Opto-Split III with a cube and appropriate filters and a Hamamatsu Flash 4.0 camera. Multiple colors were aligned by using calibration data obtained with 500 nm fluorescent beads (TetraSpeck Fluorescent Microspheres Size Kit, T14792, Invitrogen) mounted on a slide34 (link). The microscope was pre-warmed to 37ºC prior to imaging and temperature was maintained during imaging.
All total internal reflection fluorescence microscopy experiments were performed on a custom-built microscope based on an Olympus IX-70 microscope, equipped with a UApoN 100× 1.49 NA oil objective, a 488-nm, 594-nm, and a 647-nm laser. The cells were imaged under azimuthal TIR-FM33 (link) using a Cairn Opto-Split III with a cube and appropriate filters and a Hamamatsu Flash 4.0 camera. Multiple colors were aligned by using calibration data obtained with 500 nm fluorescent beads (TetraSpeck Fluorescent Microspheres Size Kit, T14792, Invitrogen) mounted on a slide34 (link). The microscope was pre-warmed to 37ºC prior to imaging and temperature was maintained during imaging.
