I change the name of your file in Archive 2.zip. 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.
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.
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.
Shrink the cell. You can adjust the size (pixel number) of shrinkage.
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.
Susana.zip (12.1 MB)