Quantification of 4 channels fluorescence intensity



Hi, I am Jessica and I am a new user of CellProfiler !

I would like to create an easy pipeline to analyze 4 channels of fluorescence : (1) dapi (nucleus), (2) CY3 : membrane, (3) CY5 (membrane) and (4) FITC (cytoplasm). The output file can be an excel file with the number of the cell and the 4 intensities associated. I do not have so much time firstly to develop a more sophisticated pipeline firstly because my deadline is very short.

Is there a pipeline to answer to my question ? Or an existing pipeline than a can custom ?

Many thanks !





Welcome to the community! Have you tried searching the forum yet, or checking out our example pipelines yet? ExampleHuman is a good place to start for simple pipelines.


Many thanks , I will try this pipeline to begin.
I would like to known in what order I have to put my modules :

  1. Identify an object (example nucleon)
  2. MeasureObject intensity
  3. Identify an object (membrane)
  4. MeasureObjectIntensity
    Or Identify all my objects first (step 1 and 3) and then Measure Intensity (2 and 4) ?

Many thanks :slight_smile:



Identifying all objects first is definitely most intuitive, and will work. I guess your order can also work.



Thanks for your message. That’s what I did :slight_smile:
I have a cell line control FITC positive in the cytoplasm and another cell line (FITC negative) CY5 positive in the membrane. I have two questions !
(1) I made a custom pipeline to quantify the 3 channels (dapi, FITC, CY5) but I have a doubt about the fluorescence intensity measure of the membrane (CY5-w4). I used the “identifysecondaryobject” for FITC and CY5 channels but I am not sure for CY5 channel. I don’t have how to identify the membranes …
(2) When I look to my excel file, some CY5 positive (4 in total) are also FITC positive (in attachment). These are FITC false-positives. I think this problem is related to a failure about the proximity of FITC- positives cells ?
Many thanks ! :slight_smile:
Analysis_Fluo_Image_V2.cpproj (740.1 KB)
<a class=“attachment” href="/uploads/imagej/original/3X/9/8/98a4779571d6b14MyExpt_Cells_V2.zip (96.8 KB)
2eb29fcb3326b682aa094d07e.cpproj">Analysis_Fluo_Image_V2.cpproj (740.1 KB)


I would guess you need to optimize your segmentation. Use the ‘test mode’ to progress stepwise through your pipeline, and then try to adjust the parameters for the IdentifySecondaryObjects modules. Perhaps the threshold needs to be adjusted.


Is it possible to identify the cell membrane ? My CY5 antibody is localized on the membrane.

Ok, thanks, i will try to change my threshold :slight_smile:


Cell membrane only? Not as far as I know. You can identify the nucleus and then the whole cell, and then subtract the nucleus (in the IdentifyTertiaryObjects module) to define the cytoplasm including cell membrane.