I have been trying to measure fluorescence intensity of cells stained for IL-10 (FITC_green) and TNF-alpha (Rhodamine_red) which are both in the cytoplasm. I use the nucleus (DAPI_blue), as primary objects to locate the secondary objects (Cells for TNF signal and Cells 2 for IL-10 signal). For the identification of the secondary object, I manually put in a thresh-hold value, 0.005. However after running my anaylsis, I found out the CP also picked up fluorescence intensities lower than the set threshhold value.
I expected all the recognised intensities to be above or equal to my threshhold, which is 0.005… Please I need to understand what is going on?
Here is a sheet containing the exported intensities
040717_m1gm_images.xlsx (88.1 KB)
This is the pipeline used for the analysis
trial pipeline 2_integrated.cppipe (23.8 KB)
Here is an image set
Thanks for your help.