Measuring Object Intensity



I am quite new to cell profiler. I work with monocyte derived macrophages and I’m trying to compare the expression of pro-inflammatory and anti-inflammatory markers produced by the cells. I stained the cells for the nucleus (DAPI_blue), IL-10 (FITC_green) and TNF-alpha (Rhodamine_red) which are both in the cytoplasm. I created a pipeline to measure object intensity. I use the nucleus as my primary object to located the cells; I have two secondary objects, Cells for TNF signal and Cells 2 for IL-10 signal. However after running my anaylsis, I realized both Cells and Cells 2 measured the green and red object intensities.
I expected Cells to measure the red intensity only while Cells 2 measures green intensity only… Did I do something wrong?
This is my piplepline
trial pipeline 2_integrated.cppipe (24.0 KB)

This is an image set

Thanks for your help.


Hello there,

In your current module MeasureObjectIntensity you let dual objects pair with dual channels, it will result in 4 total measurements.

If you expect “Cells to measure the red intensity only while Cells 2 measures green intensity only”, then it should be set in 2 separated modules:

Otherwise, your pipeline looks great.



I get it now!

Thanks so much Minh.

Best wishes,


Hi there,
I’m new with CellProfiler. I have 2 channel image: Dapi and a nuclear marker in 488. I need to measure the intensity of the 488 positive object, but at the same time I need to have the value of dapi intensity for each identified object. Is it possibile? I tried with different approaches, but I not had good results. In fact I obtained an intensity of the all dapi objects, that are different compare to the intensity value of dapi object identified in 488 positive cells.



Let’s try to work it out together:

  • Let’s first identify the nuclei by IdentifyPrimaryObjects, using DAPI channel, let’s call them objects “Nu”, for e.g. we now have 1000 objects “Nu”.
  • Then we measure the DAPI signal (MeasureObjectIntensity) in every Nu, now we have 1000 values.
  • Then we measure the 488 signal (MeasureObjectIntensity) in every Nu, we still have 1000 values, but about 700 of these values are high for 488-positive Nu, and 300 of these values are low for 488-negative Nu.

This way, the values of DAPI signals are independent from being 488-positive or 488-negative.

(Do NOT identify your objects by using 488 signal)

Hope that helps.


Thanks Minh.
I tried as your suggestions and I obtained this excel data:

In same case, I obtained very high values (as you can see in the highlighted line): how can interpret them? At the end of this analysis I want to represent every single object in a graph with a scale from 0 (negative 488 object) to 1 (100% 488 positive) , where the 0 object are the negative for 488, 0.5 an object with a average positivity, etc…so I need also the 488_positive intermediate values.
I need also some suggestions to put a threshold to define the 488-negative objects ( I obtained only 2 objects that are 0.0, but a lot of them had a 0.000XXX value, in fact the 488 positive objects are 101 on 113 total Dapi objects).
Foxg1_488.cpproj (863.6 KB)

Thanks for your helpful advices.


In Excel, you can try to select the 2 columns Intensity_MeanIntensity_DAPI and Intensity_MeanIntensity_488, then make a scatter plot out of them. You will then see a distribution of 488-positive/negative to decide where is the threshold.

Hope that helps.


The intensity should always be a number between 0-1; I’m wondering if somehow the values got corrupted opening the CSV and that should have been 7.40 x 10^-11? Can you look at that nucleus and tell if it’s pretty dim in that channel?


Thanks for your help