I’m trying to modify a pipeline downloaded from cellprofiler ‘Example Speckles’ for my assay. My goal is to find speckles (stress granules) in two separate channels and then use the relate objects module to find out how many of the speckles overlap. This is to classify the speckles as stress granules or not (since by our definition, both proteins would need to be present for the speckle to be designated as a stress granule). I am using two of the three channels imaged to assess this, and there is no cellular marker stain. I am however unsure about a couple of things:
- What does the EnhanceFeature module do?
- Using the IdentifyPrimaryObjects after EnhanceFeatures I’ve chosen Otsu (and three classes). If I do not modify the lower and maximum values for the threshold I get speckles both in my positive and negative control images . In fact, I get thousands more in my negative control due to the diffuse staining. If I set the lower threshold to 0,08, all of the speckles in both positive and negative control images dissappear. This seems very low already, but when I modify this to 0,001 I get quite a few speckles in my positive control, and a very small number in the negative control. Of those in the negative control, none are related in the RelateObjects module. This seems like the sweet spot, but arbitrarily set, so but do you have any guidance on how to determine this lower threshold value?
- In a different pipeline in CellProfiler, ‘Example Colocalization’ there are some CorrectIllumination modules added. When is that helpful? Can it hurt to add it and align the images in a speckle finding pipeline?
- Feel free to comment on other things in the pipeline if you find that I have done added something unneccessary, or have mis-used a function.
See the pipeline I’m working on and a positive and negative control in attached files
Thank you for your help.
img_000000000_568 Ex_000.tif (8.0 MB) img_000000000_633 to combine with 568_000.tif (8.0 MB)img_000000000_568 Ex_000.tif (8.0 MB) img_000000000_633 to combine with 568_000.tif (8.0 MB) Stress granules_20200318_revised pH2AX pipeline.cpproj (961.1 KB)