Live imaging and cortical ablation were performed on a Nikon Eclipse Ti-E inverted microscope equipped with a Yokogawa CSU-X1 spinning disk head, 1.4 NA 60X oil objective, Andor DU-897 EMCCD and a dedicated 100 mW 405 diode ablation laser, generously provided by the Nikon Centre of Excellence at Vanderbilt University. The instrument was controlled using Nikon Elements AR software. For ablation, a 1.4 μm x 1.4 μm ROI was used for all experiments. A DIC and/or fluorescence image was acquired before ablation, followed by ablation using a miniscanner. A pixel dwell time of 500 ms, 50% laser power was used for a duration of 1 s, followed by acquiring DIC or fluorescence images at 2 s intervals. Samples were maintained at 37°C with 5% CO2 using Tokai Hit Stage Incubator.
To image MIIA and MIIB in fixed ESC colonies, large image stitching was performed using the 60X objective in Elements software. Z sections were acquired at 1 mm intervals for the entire stitch and maximum projections were displayed and used for generating line scans. To image endogenous MIIA and MIIB in fixed HeLa cells, single slices through the middle of the cell were acquired using the 60X objective.
To image MIIA and MIIB in fixed ESC colonies, large image stitching was performed using the 60X objective in Elements software. Z sections were acquired at 1 mm intervals for the entire stitch and maximum projections were displayed and used for generating line scans. To image endogenous MIIA and MIIB in fixed HeLa cells, single slices through the middle of the cell were acquired using the 60X objective.