Comparing intensities between multiple images



Hey there,
First off, I’d like to say I’ve just starting using CP and it is amazing! Extremely easy and user-friendly interface. Thank you!!!

I am analyzing 8-bit grayscale images of E.coli cells. The cells are stained with DAPI and Alexa647. In my analysis, I want to measure Alexa647 intensities across multiple images (from the same experiment, under the same conditions). I built a pipeline which takes the DAPI image and identifies objects from it (=the cells), then overlays the objects on the Alexa647 image and measures intensities for each object.

When looking at the csv output file, under the “Intensity_MaxIntensity_Alexa647” column, I seem to have a value of “1” at least once (meaning at least for one object) in each image. This is odd seeing as I have images with relatively low signals and others with very high signals.

Under “select intensity range from”, I tried both options (from metadata/ from image bit-depth, although I think in this case the “image bit-depth” option would be the better one? All images are 8 bit images…) but both of these options give me the same exact results in the intensity output file. Am I missing something? When dividing the image intensity by bit-depth, this shouldn’t alter the actual values, right? Is this problematic when comparing intensities between images?

I’ve read countless topics regarding image intensity and I can’t seem to find an answer.
I’ve attached the pipeline, the csv output files for both intensity range options (metadata and image bit-depth) and some of my images as an example. Any help would be appreciated!

On an unrelated note, there is a warning under the “export to database” step saying that “DAPI is not in a 1:1 relationship with the other objects, which may cause downstream problems”. Should this concern me or can I ignore it?
Also, if there are any other unrelated suggestions regarding my pipeline I’d be very grateful to hear!

EditedObjects image bit-depth.csv (16.6 KB)
EditedObjects image metadata.csv (16.6 KB)
Ecoli_Analysis.cpproj (624.8 KB)


Hey guys,
I’m holding off on my analysis to have this issue solved…
Help please?