Processing steps for identification of RIF. (A) A single slice (2D) grey-scale image showing a nucleus, blue dashed line, with a single RIF. The RIF has been highlighted to show its appearance in subsequent slices and are colour-coded to C-G. (B) The same image is shown as a 3D intensity mesh and is also shown (C) for a single axis with coloured lines for the most central focal plane (n = magenta) and the 4 focal planes at 0.5 μm intervals beneath. (D) The same intensity mesh after filtering shows reduced noise. (E) A laplacian operator is applied to identify boundaries and a normalisation value of 1500 is added to ensure RIF values do not fall below zero. (F) A final filtering step removes noise before finally (G) a maximum entropy threshold is applied to identify RIF.