Prior proscan 3

Live-cell imaging was performed on a Nikon EclipseTE2000-E inverted microscope equipped with a Nikon 20X (0.45 NA) Plan Fluor objective or 60X (1.40 NA, oil immersion) and an Andor iXon3 high sensitivity EMCCD camera (Andor Technology, Belfast, UK). Multi-point x-y-z coordinates were set using a Prior Proscan III motorized stage (Prior Scientific, Rockland, MA). To account for Z-drift, images were captured in five Z-planes (10 micron below initial plane to 10 microns above) with the Elements AR software used to determine the in-focus plane for collection. For fluorescence quantitation (data presented in figures are from single representative experiments), individual cells were selected from multiple fields of view using ImageJ software. Background-subtracted fluorescence intensity was measured at each timepoint (to derive F_t). Relative fluorescence (F_t/F₀) was calculated as a ratio relative to fluorescence at timepoint zero. For experiments where X-Y drift occurred, the TurboReg and StackReg plugins (Philippe Thévenaz) were utilized to align images before analyses.

Cells are grown to approximately 50–60% confluent on 35 mm glass bottom dishes (MatTek or InVitro Scientific) before imaging. Live-cell time-lapse imaging is performed using an automated Olympus IX81 inverted widefield microscope equipped with a motorized stage (Prior Proscan III, Prior Scientific), Hamamatsu ORCA-R2 CCD camera, 200 watt LumenPro200 metal halide lamp (Prior Scientific) and customized stage-top incubation system (InVivo Scientific) at 37 °C and 90% humidity using Slidebook6 (3I). Olympus objectives used are 10 X 0.40PH2, 20X/0.7NA PH2 UPLAPO Plan Apo and 40X/0.75NA PH2 UPLFLN SemiApo. Time-lapse microscopy images are taken every 10 min using mCherry and EGFP excitation/emission and phase-contrast microscopy. Autofocusing is applied in the transmitted light channel, images are binned 2 × 2 and fluorescent exposures are 10–30 ms. Spinning disc confocal time-lapse microscopy is from an Olympus CV1000 equipped with a spinning disc confocal head and environmental control at 37 °C and 80% humidity; images are every 10 min using 488 nm and 568 nm laser illumination, a red LED for transmitted light, and an Olympus 20 X 0.75NA objective. Fixed-cell images are acquired using mCherry, EGFP, Cy5/Aleca647 and/or Hoechst/DAPI illumination; imaging conditions are identical between all imaging sessions.