Help with Nuclear translocation pipeline?



Hello everyone, I would like to say thanks up front!

I have a pipeline I have made and some images, but as a new user I can not upload it :frowning:

  1. I would like to setup a way in the pipeline to determine the average intensity of the uninfected samples, then use this + 1 standard deviation to set a threshold cutoff for what i consider p65 translocation. Then apply this to the cells and generate a % translocated above uninfected calculation as a separate output file.

  2. I would like to have a crop, background subtraction, illumination correction, and a merge for my channels as individual channel images and the merged. I would also like to save the outline of the nucleus and the cell on top of the gfp channel.

  3. I would like to calculate in addition to just median intensity the % translocation from the cytoplasm to nucleus. Then generate a image overlaid with this “localization” / “threshold” that reflects intensity?

  4. Help refining the de-clumping/ segmentation of the nucleus - or a way to discard/ or identify ones that are not individual cells via a label on the overlaid image that corresponds the intensity output file

At anyrate, I would love to have some help and feedback! This is not my first time using cell profiler, but is the first time i am trying to build an extensive pipeline.



Have you checked out our Translocation tutorial? It has many of the things you’re asking for.

As for 1), you can’t have CellProfiler run all images, make a calculation, then run all images AGAIN; you’ll need to run one pipeline to do the calculation you want, and another to apply it.

As for the others, I think if you search the issues individually you’ll find guidance on how to do them (search the program help, too, for words like “Overlay”); you can always upload your pipeline to GDrive/Dropbox/etc and link it here.

Good luck!


Hello, thank you for your response. I have been working on the pipline with your suggestions. The dropbox link for the pipeline and some test images is below. Any further suggestions would be great!

I am currently struggling with understanding why my intergrated intesnity levels for the positive control is so low. When I use FIJI to threshold/watershed for the nuclei and apply these as ROIs to the p65 image I get more believeable data (IE, the intergrated intensity for the positive control over all the nuclei is always 2-4 times higher than the negative control). Whereas, when i use the intergrated intesity values from cell profiler there is only 20-50 unit difference in the mean.

A6 and A11 are positive control for nuclear translocation
A7 and A12 are negative control



Without knowing how you do your FIJI analysis, I can’t say for sure why or how you’re getting the results that you are, but I’m suspicious that you see a 2x increase in translocation between the image on the left and the image on the right- neither looks particularly translocated to me, certainly not 2X.

Your images aren’t all of the same mean brightness either- just looking briefly at the outputs of MeasureImageIntensity, the ones in well A7 seem to be about 20-40% dimmer overall than the ones in well A6.

Finally, unless you expect nuclear size to be an interesting change to be measured by your perturbation, I would use mean or median rather than integrated intensity, as the latter contains a measure of size. Even better would be the ratio of mean intensity in the nucleus to mean in the cytoplasm.