Signal from image #1 showing up on image #2 object analysis

I am new to CellProfiler, but have a lot of experience with FIJI. I modified one of the default pipelines to help me assess cytosolic signal vs nuclear signal of the protein stained in channel 2. I used channel1 (DAPI) to segment the nuclei and channel 3 (actin-don’t judge I didn’t prep the samples and the lab that did likely had outdated Phalloidin :-)) to segment the cell. My goal is to measure the nuclear and cytoplasmic mean intensity of channel 2, ultimately reporting a ratio of cyto:nuc. I thought my pipeline was working, but I appear to have layers/ghosting from previous samples on my images that are used as input to the object analysis. It seems the input image isn’t reset when going to a new image, I noticed this by getting more and more DAPI as I processed more images. DAPI signal that isn’t present in the original image not processed through cell profiler. Do I need to clear the objects, etc at the bottom of my pipeline somehow?

Attached is a screen shot using FIJI showing two images that were processed through Cellprofiler…top row processed first, bottom row processed 2nd. Second row shows residual nuclei from the first image.

cytoplasm_nuclear_ratio3.cpproj (141.2 KB)

Why is it that insomnia equals the best trouble shooting? It appears that the create make projection module was causing this error. Not sure if I did something wrong in setting up how the images were setup. Instead of getting the make projection module to work, I used my existing FIJI macros to make my make my max intensity projection images, split my channels and save as TIFF files and now things seem to be working as expected.

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Were you trying to make a projection and then use it in the same pipeline? Unfortunately you can’t do that, CellProfiler has no ability to “loop through” everything first and make the projections, then re-loop-through the projected images. It should work smoothly though as two separate pipelines!

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Good to know! Would that have to be two separate projects then? Still learning how Cellprofiler works.

Yep! You’d save two separate projects or pipelines. Sometimes I’ll number my projects to make it easier to remember what I’m doing, such as “1-ZProject.cpproj” and “2-SegmentMeasure.cpproj”.

Good luck!

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