Once a cell is picked the region occupied by the cell is identified by generating a binary mask and the center of mass of this mask corresponds to the center of the cell.
Scrambling was quantified by measuring the increase in Annexin-V fluorescence in EGFP-expressing cells by creating a binary mask from the EGFP channel for each Annexin-V-Alexa-568 frame in the time series using Fiji Image-J 1.49.
The RC was calculated by convolving a binary mask corresponding to the MV with a 3-D gaussian function of FWHM equal to 7 mm.
3D masks for Barr bodies, defined as volumes with an enrichment of Xist RNA, were generated by Otsu thresholding of the Xist RNA signals followed by transformation into a binary mask file and dilation by several pixels.
To assess the volume of the cell occupied by contractile apparatus, a binary mask of f-actin labelling was created and expressed as percentage of total area.
Fluorescence background between cells was removed by multiplication with a binary mask that was created from a copy of the original image using an intensity threshold so that only signals of cells were included as valid pixels.
The resultant image was thresholded by 0.15 to create a binary mask image containing 'ones' for voxels with a probability of greater than 15%% to belong to grey ore white matter and 'zeros' for voxels outside the brain.
