Hi, I am just starting using the Cellprofiler, but unfortunately I am starting not with the easiest task… Previously I was doing this analysis manually by ImageJ, but now I want to perform a study with many time points, which brings me to the point when manual analysis takes too much time and effort.
What I have:
Nucleus labeled by DAPI
Cell membrane (in most cases) labeled by CD44
Golgi labeled by TGN46
And a fluorescent Shiga toxin (Stx)
What I need to find:
How the colocalisation between Stx and TGN46 changes during time (The toxin gets pretty fast into the cell and is transported to the Golgi and then further to ER). I have images taken each 5 minutes.
Main problem: Stx bind only to certain cells in the cell culture (around 20-50%). Therefore if I do the analysis on the whole image, I include also these Stx negative cells, which normally I would omit from analysis. Second problem: the amount of Stx which is bound to the cells can vary highly, so the best way calculate how much of the toxin is transported into the Golgi, is to find the proportion (%) between Stx colocalising with TGN46 and total Stx associated with the cell.
I am not sure if I explained it understandably…
I would like to know if such analysis in cell by cell manner for colocalisation of two signals would be possible by CellProfiler? I have been trying to make a pipeline for this, but I have not succeeded yet. Could you suggest what modules should I use and in which order?