did I get it right that this module should generate an overlay image of my microscopy image (Nucleus) and outlines (e.g. PrimaryOutlines from module IdentifyPrimaryObjects). I gave this overlay the name OrigOverlay and tried to save with SaveImages.
However, in the display window I just get the outlines displayed in an image next to the microscopy image (no overlay), and my saved image only contains the outlines.
Is this the normal behaviour? (maybe it’s a stupid question, but I haven’t fould an answer by myself so far). If it’s not the normal behaviour, are ther any clues what might go wrong?


Hi Michael,

A couple of questions:

  • Are you saving the overlay image and not
    the outline image in SaveImages? - On the OverlayOutlines display window, if you right-click the overlay image, and select Image contrast > Log normalized
    , does the microscopy image show up under the outlines?


Hi Mark,

thank you!
The saving was done correctly.
Setting to log normalized helped - the window that opened during processing of the images was not set correctly.

In the meanwhile I have realized, that when I open the saved image via CellProfiler the nuclei appeared (apparently log normalized set by default). When I wrote the message I had looked with ImageJ, where the nuclei in the original 16bit image were clearly visible. In the processed image the intensities of the nuclei were so low, that I didn’t realize they were actually there when I tried to adjust brightness/contrast.

Thank you for your help - and thanks to the whole CellProfiler Development team for creating such a nice tool (even if this won’t be my last question).

Beat regards,

This sort of thing commonly occurs if you have 12-bit images that are saved as 16-bit; the upper 4 bits which would otherwise represent the higher intensities aren’t used, and everything looks dark.

If this is the case, and you are looking to rescale the images so things look better, you can insert a RescaleIntensity module to convert your images from 12-bit to 16-bit and do the overlay on those. Use “Choose specific values to be rest to the full intensity range” and enter [0, 0.0625] for the intensity range of the input image.