Analyzing output pics from SaveImages in the same pipeline that generated them

Hi everybody!

I am new here, so please excuse me if I forget some parts of the etiquette, I’ll try to stick to it and I’ll be glad to accept any advice!

I have immunofluorescence images with DAPI staining for the nuclei and another staining for GM2 (see the attached example KO-GM2_channel2.tiff (1.0 MB) WT-GM2-DAPI.tif (2.0 MB) ). My aim is to compare the average GM2 signal intensity per cell in two cell populations (wt and ko).

I created a pipeline (attached Create PPI images without background intensity.cpproj (212.6 KB) ) that hopefully:

  1. identifies nuclei
  2. identifies cells as secondary objects from GM2 signal (with a smoothing factor and an intensity threshold)
  3. measure GM2 signal intensity from the cells
  4. subtracts from this intensity its lower quartile to eliminate background (this step is necessary because of aspecific staining which could not be reduced diluting the antibody)
  5. saves the images resulting from point 4, which I called “PPI”
  6. calculates mean image intensity (just to double check and compare, you can ignore this part).

To measure total GM2 signal intensity from cells (i.e. secondary objects) from PPI images (i.e. the processed images, without background intensity) do I have to create another pipeline or is there a way to re-input the output of SaveImages in the same pipeline that created it?
At the moment I am using another pipeline (attached IF Obtain total GM2 intensity of output PPI images.cpproj (886.2 KB) ), manually re-inputting the processed images.

Sorry for the long post and thank you so much for your time!
best,
LFS

Welcome!
SaveImages does not create any new images, it only takes images from prior steps and saves them to the computer disk (otherwise, the module that made the image just displays the image, doesn’t save it).

So, your step 4 creates the PPI images you want to measure? If so, you can just feed those PPI images directly into a Measure module.

Hope that helps!

Thank you so much, that helps a lot, I do not know why PPI images would not show up in the list of images to measure the first time I tried with a Measure module!

Thanks again!

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