Hopefully, I’m understanding things correctly…
I think that this is why the 1st two images need to be aligned first, and then save that result as a template first, before running through the rest of the images. In short, you’ll need to initialize the template with images 1 and 2, and then run the alignment procedure with images 3 onwards, and so on with 1st template image. You may need separate pipelines, (1) for the initialization step and (2) the full alignment step.
So your pipeline for (1) would be something like:
LoadImages (for images 1 and 2)
SaveImages (to initialize the template)
and the pipeline for (2) would be:
LoadImages (for images 3 onwards)
LoadSingleImage (the first cycle, it will load the template for 1 and 2)
SaveImages (to save the new template)
The trick is to start pipeline (2) at image 3, which you may be able to do using the filename exclusion setting in LoadImages in the latest version of CellProfiler.
Hopefully, this makes sense. Is this something like what you already tried and it wasn’t working?