Measuring membrane intensity




I am trying to measure the membrane intensity of a protein that localizes in the cytoplasm and membrane. I have 2 color images with red channel being another protein that localizes to the whole cell (but not enrich at the membrane) and the YFP channel with the protein enriched at the membrane.
I am tried a couple of different possibilities to get to the detection of the membrane object.
the enhance edge function works pretty well but i am not sure how to use it to convert to


Do you want to take a quick look at a tutorial we have for colocalization? It describes a couple of ways to do it, whether pixel-level or object-level. Once you’ve taken a look it might give you some ideas (or more questions to ask, in which case stop back here!)

p.s. if that’s not what you aim to measure, feel free to reply with a deeper description and especially upload the images and pipeline you have so far.


Hi Anne

Thanks so much for your quick reply.
I am not interested in colocalization but instead in quantifying the intensity of the tagged protein at the membrane. My problem is how to define membrane as an object but i do not have any membrane staining.
I have tried to upload images but i cannot upload them - there is an error saying it is unable to determine the size of the image. Is there any other way I can upload the images?


Can you make a zip archive perhaps? Others might know other tips.

#5 (10.7 MB)

It works! Thanks.
There are images: Cherry and Venus.
The Venus image shows the protein I would like to quantify the intensity at the membrane.

Thanks so much




Can you please tell me the the staining for each image? 171130_U2OS_newR19_21hDoxC1 for Cherry and 171130_U2OS_newR19_21hDoxC2 for Venus?

My second question: when you mention “The Venus image shows the protein I would like to quantify the intensity at the membrane.” I do not know the membrane is the outline of cell or the whole cell. If you mean the outline of cell, it is hard to define.

If you want to measure the intensity on membrane per cell, probably you need to stain the nuclei. It is important. Use nuclei as the primaryobject and Cherry as the secondaryobject, thus the identification of cells can be made.



171130_U2OS_newR19_21hDoxC1 for Cherry = Erk
171130_U2OS_newR19_21hDoxC2 for Venus = Ras

Second question: I want to measure the outline of the cell. the enhanceEdge function does a good job at detecting the outline but then I do not know how to use that to measure intensities.

I am attaching a second batch of images and in this case the Erk is in the nuclear.



Archive (11.2 MB)



I change the name of your file in Archive ch00 is 171129_U2OS_newMAPK_R20_4hDox.nd2 - C=0; and ch01 is 171129_U2OS_newMAPK_R20_4hDox.nd2 - C=1

To make things simple, let us just regard the ch01 and ch00 are nuclei and Venus. The goal is to measure the intensity on the membrane.

Please see the pipeline attached.

  1. Identify the nuclei. The signal of nuclei is too weak. To identify the nuclei better, I use RescaleIntensity module to remove the background and enhance the image. I also use the Gauss Filter to help identify the nuceli.

  2. Identify the cell. Use IdentifySecondaryObject, we can identify the cell by ‘Propagation’. The result is not that bad. Next time, please increase the intensity of your image when you doing the experiment.

  3. Shrink the cell. You can adjust the size (pixel number) of shrinkage.

  4. Measure the intensity of im_cell in the space of obj_cell and obj_cell_shrinke.

I guess there is a module in the Cellprofiler which can reduce the area in obj_cell_shrinke from obj_cell. But I do not find it yet. Anyway, it dose not matter. You need to substract the intensity of obj_cell from obj_cell_shrinke. Is it what you want? As far as I know, you cannot use a ‘line’ to define the membrane. But you can use a limited space, which is very close to membrane to define the membrane.

You can export the data into excel via ExportToSpreadsheet or via MATLAB. I prefer using MATLAB. (12.1 MB)


Hi jedyzdc

Thank you very much for the pipeline you built. It works pretty well. I am going to run in more images and adjust as needed but it is definitely promising!

Have a nice weekend