You’ve told it to set files containing ‘ch4’ as your DNA image but your experiment only has files ending in ‘ch1’, ‘ch2’, and ’ch3’.One of the sets of rules you’ve used to identify a channel isn’t set correctly – for example:.You turned on ‘Groups’ but never gave it a piece of metadata to group by (in which case the ‘Metadata category’ is still set to ‘None’).Check the output of the Metadata module to ensure all of your images have the information that they need. A piece of extracted metadata is missing (e.g., you remembered to extract the Plate metadata from your ‘regular’ images but not from your illumination correction functions).You forgot to hit ‘Update’ in the Metadata module when using the ‘Extract from image file headers’ option.Your image files have been moved to a different location on your computer, but CellProfiler is still looking for them in their original location.But here are some common triggers for this kind of error. If you want to learn more, we recommend reading the help sections for each module, and everything under ‘Help->Creating A Project’ is a fantastic resource as well (we’ve included a table from it below that’s a great cheat sheet to understanding these modules). These errors simply mean that CellProfiler doesn’t know how to sort the images you’ve given it, and it can’t process them until it knows how they should be organized. If you have mistakes in any of these modules, you may run across the dreaded errors ‘The pipeline did not identify any image sets’ or ‘Sorry, your pipeline doesn’t produce any valid image sets as currently configured’. But it’s sometimes confusing what each one does, and it’s not always obvious which ones you need for your experiment. Incoming images are configured in the first 4 modules of CellProfiler – Images, Metadata, NamesAndTypes, and Groups – which offer lots of flexibility. Defining the input to CellProfiler can be the hardest part of getting your pipeline set up and your analysis underway.
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