I’m quite confident it should be possible to quantify your phenotype using the Worm Toolbox.
There are a few alternative approaches:
A. If you can use a pharynx-specific marker, use IdentifyPrimaryObjects on this channel and measure intensity in GFP for pharynx object.
B. If you do not have a pharynx marker, you can still extract fluorescence from the approximate position of the pharyngeal bulb by using the Worm Toolbox to untangle clustered worms, ‘straighten’ the worms, and map them to a common low-resolution atlas.
First of all, you need a good foreground/background segmentation of the worms, if possible using a bright-field image of the worms.
Using non-touching worms (selected by size-filtering or EditObjectsManually), use the UntangleWorms module, in the ‘Training’ mode, and train a model of the variability in shape of your worms (the .xml file produced can be used for all your experiments, as long as you use the same microscope settings & worms). Thereafter, use the UntangleWorms module in the ‘Untangle’ mode, followed by ‘StraightenWorms’, where you specify the GFP channel as the image to straighten, and specify ~6 transverse segments (atlas sub-regions) from which GFP intensity is extracted. Without a common head-marker, you may not know in what end of the worm the pharyngeal bulb is located, but e.g. using the difference in GFP fluorescence between sub-segment 2of6 and 4of6 you should be able to separate the two phenotypes.
We will soon publish a number of sample pipelines on the website, and you’re welcome to send a sample image so that I can help you set up your pipeline.