Merging brightfield and fluorescent channels to create a composite image



Hi CellProfiler Team,

I’m trying to merge three greyscale images (DAPI, Cherry and Brightfield) into one composite image, but I keep getting a green background.

I’ve tried using the GrayToColour module (composite), setting DAPI to blue, Cherry to red and Brightfield to grey. I’ve also tried using GrayToColour to merge DAPI and Cherry and then ImageMath to add the merged image to Brightfield, but the result is the same. I found an earlier post on this forum that included a RescaleIntensity step, which I also tried adding in prior to ImageMath, but the background still had a weird green tinge.

Is there a way to merge two coloured images with a grey brightfield image in CellProfiler?
I’m using the 3.0.0 Windows version.

Thank you!!



Have you exported this image, is the green background truly there? I suspect it may just be a normalization artifact in CP’s depiction in the window- if that is indeed the case, can you make sure your normalization is set to “raw” rather than “normalized” (either in individual subplots by right clicking on them or by changing your defaults in the Preferences window).


Hi Beth,

You’re right!
Without rescaling, I can’t view the image when I change the normalisation setting to "normalized’.
However, when I rescale both the fluorescent images and the brightfield image prior to ImageMath, the green tinge disappears when I set the normalisation to “raw”, and the exported images look as expected.

Thanks for your help!