Overlaying channels


I am looking to study the relationships of fluorescence in three different channels. I was wondering if it was possible to overlay two channels and find the points of co-localisation, and add only these points onto a third channel. The trouble at the moment is that I want to see the relationship of a third channel compared to sites of co-localisation of the other two, and I can never get a clear picture of this, as sites where the first two channels don’t overlap just confuse the image.

Sorry if this description isn’t very clear, I can possibly clarify it further if needs be.


You might be able to use IdentifyPrimaryObjects to identify two sets of objects, one for each of the two channels of interest. In this module, you can retain the outlines of the detected objects, and then use OverlayOutlines to overlay the two sets of outlines in different colors onto the third channel, so you can see the result.

Alternately, if you are more interested in analysis than simple visualization, you can use MaskObjects to mask objects from channel 1 with objects from channel 2, to get the regions of overlap (the co-localized region) as a new set of objects. Again, you can retain the outlines of the co-localization objects and overlay them on the 3rd channel, or perform more sophisticated operations like obtain the intensity against the 3rd channel or measure their shape.

Hope this helps!

Hi Mark,
Okay, I will try this then.

Thank you for your help, and for Cell Profiler!