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.