Fig. 5.

mIA nuclear spot (NS) analysis pipeline. (A) Schematic of the steps included in the NS pipeline. (B) Maximum projection of 3D image stack results in increased blur in 2D not seen in (C) single best focus plane image. (D) Best plane for each cell selected by defining plane with maximal variance. (E) Best focus plane projection results in 2D image with focused spot in each nucleus. (F) Spot segmentation minimizes the effect of dark pixels by normalizing pixel intensity before (G) local threshold is applied to maximize the signal to background ratio. (H) Final spot mask. 2D, two dimensional; 3D, three dimensional.