Fig. 4

Outline of the GFP image segmentation protocol for balanced mutants. a Representative images acquired in brightfield and green channels. First, the well is segmented based on intensity in the green channel, then the image is inverted (bottom). The brightfield image is subjected to pre-processing to enhance the contrast for ease of segmentation. The segmented worms are tagged in blue and excluded objects are tagged in yellow (top). Exclusion criteria are specified in Additional file 4. The user can modulate these morphological parameters per requirement. By subtracting the well mask from the segmented worms, we obtain the worm mask. b Identification of heterozygous animals (Green head ID). Once worm mask is defined, green heads are segmented and tagged in red, excluded objects are tagged in yellow. Exclusion is based on area and intensity levels. The user can modulate these morphological parameters per requirement. The next step subtracts the identified green heads from the worm bodies. As a result, segmented worms appear blue without the green heads (Worm minus green head). c Background subtraction. Segmented worms are dilated in order to facilitate measurement of the intensity of the immediate background. d Target linking in order to compose one final target. Worm mask, dilated worm and worm minus green head targets are linked together. The final target shows the three measurement regions: the worms in blue, the immediate background in cyan and the green head in red. e Identified targets with the corresponding measurements. Intensity of the worm (Int worm), intensity of the immediate background (Int Bkgd), background subtraction (Int worm - Int Bkgd), worm length and identification of green head (Green Head ID). Targets 8 and 9 correspond to heterozygous worms with a green head ID greater than 0. Additional measures like area, major axis length, X/Y position and form factor can be added per user requirement