Hi, I have 3 channel fluorescent images of cells (ch1 = DAPI, ch2=Marker1 ch3=Marker2) and would like to classify cells according to the intensity of the nuclear marker 1 and 2.
What I do is to detect primary objects in the DAPI channel and then measure the brightness per object in channel 2 und 3.
At the moment, I classify then by absolute intensity thresholds eg.: celltype 1 = intensity > 0.04 in ch2 and intensity < 0.03 in ch3.
What I would like to include now, the consideration of the background. Instead of a hard threshold for the classification, I would like to have something like celltype 1 = intensity object/intensity background > 100.
Is it possible to classify according to results of calculations ?
If so, is it possible to define the background for the measurement of background intensity as all pixels, which are not included in the primary objects?
I would really appreciate any help or ideas on that.
Thanks and best regards