I’m trying to measure (in CP) the localization of a protein to the Golgi. The protein undergoes a very large distribution change (from ER to Golgi) upon treatment, and I’m finding it difficult to get a Pipeline that can deal with this large morphological change.
In the two attached files, the top one shows ER localization of the GFP protein of interest. The red/orange dots are a dsRed Golgi marker. The second image shows localization of the GFP protein to the Golgi.
I can easily segment the red Golgi objects. I can also segment the GFP localized to the Golgi, as well as the disperse GFP, but only in two separate modules (otherwise I get a lot of false objects).
So my first thought was to segment the Golgi, and then measure the GFP intensity inside the Golgi objects. Across various test treatments, this gives me a measureable difference, but with a very intra-well range:
Is there some way to improve the localization calculation? Or perhaps a change in the logical approach to measure the difference between the two conditions? At this point we don’t have a cell body dye, but could add one if necessary to help improve the segmentation (esp when the GFP signal is disperse).