Oh! I see…
In trying to figure out how the measuring intensity module works on the cell profiler, I identified my secondary object (a surface marker) using 4 different manually set thresholds, 0.005, 0.01, 0.1 and 1. I subsequently measured the secondary object size and intensity.
With 0.005 I got the following measurements:
I went further to increase the threshold to 0.01 and got the following measurements:
I noticed the mean cell area reduced which makes sense because object areas with intensities less than 0.01 was cut off. This resulted in the mean intensity being higher.
With 0.1, I got these:
I noticed the mean cell area became same as the nuclei area. This made me think the threshold was higher than the highest intensity on the image. However looking at the intensity measurements, a mean of 0.012 (lower than the mean calculated when 0.01 was used as the threshold) was generated. Given that the threshold is high, I didn’t expect CP to give me any values.
I went on to use 1 as my threshold value and I got these measurements:
I got exactly same measurements generated using 0.1 as threshold.
Questions: Please can you explain why I got similar values using thresholds 0.1 and 1? Also, does CP put the set object identification threshold into consideration while calculating object intensity?