Separating Channels from ND2 Files




I’m new to CP and imaging analysis in general, so bear with me.

My goal is to quantify different types of macrophages (different stains) on an image, but I’m hoping to use the original .ND2 file or a four channel .tiff file. However, regardless of how I save these images, CellProfiler isn’t recognizing the channels that should be in the image’s metadata.

Just to provide more context, I’m looking at an image that has four channels resulting from the different stains used (DAPI (blue), CY5 (orange), etc.).

When I upload the images to ImageJ, I can see the correct channels. However, when I try to convert the image to grayscale, CP seems to only recognize RGB and Alpha channels. Is this to be expected? Does anyone have any suggestions?



I use ND2 files in CellProfiler a lot and haven’t had this issue.

Do you have:

  • In the NamesAndTypes module the image type set to “Color Image”.
  • A ColorToGray module set to split the image into your 4 channels. So conversion method set to “Split” then image type to “Channels”.

If you have these settings as I’ve described and are still having trouble it might be useful to upload your pipeline and an example file.