Thank you mplace, thank you Anne.
I was more or less going towards the suggested pipeline. I’m still struggling with the illumination correction, I have to do it for the green channel (alexa 488) but not the red one (celltrace red).
I’m correct to think that I can use “IdentifyPrimaryObjects” on the red channel and use the “red” images created by the “ColorToGray” module, and then ask for the green fluorescence intensity of each “red” object? how I do that? When I use the “MeasureOjectIntensity” module, should I select the corrected green image and select the objects defined by the “identifyPrimaryOjects” module?
So, my pipeline could be something like (I’m using CellProfiler 2.2.0) (please, I will greatly appreciate any correction):
create 2 grayscales images for both fluo channels: green and red
background, block size 20, no rescale, no smoothing
apply on green image, substract
create objects called “cell” from the red image; here I’m still playing with the threshold strategy and method to distinguish clumpled objects.
on corrected green image and selecting objects “cell”
showing integrated intensity on original color image with outlines of cell objects.
What would be the closest measurement I could plot for size (equivalent of FSC in flow cytometry)? AreaShape_Area?
Can I plot Object diameter against IntegratedIntensity?