Final image thresholding.

<p>(<b>A,B</b>) First, we use the seed image to calcuate the euclidean distance from each non-cell pixel to the nearest cell-pixel and subtract it from the composite image (cf <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057970#pone-0057970-g003" target="_blank">Figure 3e</a>). This penalizes cell movements between frames. (<b>C</b>) The phase image multiplied by a constant is subtracted from the distance transformed image shown in B. (<b>D</b>) The intensity of each pixel from (C) is normalized and compared to a pair of thresholds. There are two conditions under which a pixel is assigned as part of the cell: (i) If the value is higher than the <i>low threshold</i> AND present in the seed or (ii) If the value is higher than the <i>high threshold</i> regardless of wheather the pixel is present in the seed. (<b>E</b>) All pixels fulfilling the two conditions are included in the final segmentation. Note that by allowing for ‘new’ pixels to be included in the cell, albeit at a higher threshold, we fulfill the need to balance changes in cell volume and slight cell movements with robust segmentation.