New to Cellprofiler, need help with pipeline

Hi, I am new to Cellprofiler and want to know if an automated solution for analysis is possible. Let me tell you a little bit about my IHC assay. I am doing a co-localization assay using 10-12 different proteins of interest. We stain the tissue sections, scan the slides, destain and remove the antibodies, then repeat for the next set of proteins. The hard part is manually aligning the images. So far I have thought about applying the ColorToGray module on each image and then performing an alignment on the greyscale images, but this would mean performing an alignment on ~30 images and I’m not sure how to do this.

Sorry for the confusing explanation. I would appreciate any help.

The Align module might be able to do what you what. At minimum, you can provide two images as input, and the second will be aligned to the first.

If you have multiple images beyond these two, then in the same module, you can either align each of them independently with respect to the first (i.e, the alignment is computed for each, so they may all be different), or the offset computed from the first two is simply applied to the others. Keep in mind, the alignment is an x/y translation; no rotation is incorporated.

Hypothetically, the module could handle 30 images with the above approach, but the module settings will look ugly :smile:

Mark, thank you for all of your help. I used the align module and for the aligned images, it says “X offset: 0, Y offset: 0”. Does this indicate that no alignment occurred?

Yes, that’s right.

If that’s not the desired result, try changing the alignment method; sometimes, just that produces a different result.