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?

Hi Alvah,
Looking at all of your images, they aren’t dim at all- they all have at least some saturated pixels, so if they were “originally dim” I think however you got them from the microscope rescaled them (or they got rescaled at some point between when you exported them and uploaded them). The measurements are accurate, so I’d go back to try to track down the raw data.

You were right, my export was incorrect, I was accidentally exporting “qualitative” images, not “quantitative”. Thank you!