I am currently working with human cancer cell images from a three channel (RGB) acquisition performed on a 96-well plate. My pipeline includes:

CorrectIlluminationCalculate (x 3)
CorrectIlluminationApply (x 3)
IdentifyPrimaryObjects (nuclei)
IdentifySecondaryObjects (a new marker that I am trying to quantify)
Crop (x 4)
Tile (x 4)
SaveImages (x 8)

Everything works fine, and I obtained processed images and csv files just fine. My question is what are the units of my intensity measurements (Mean, Median, Max, Min, StDev)? Are they integrals of the pixel intensities? Can someone please explain this to me?

And also, what is MADIntensity…?

Thank you in advance for replying, I appriciate it!

To answer this question, it’s important to keep two things in mind:

  • Since microscopes are not calibrated to an absolute scale, the pixel values will always be in terms of arbitrary florescence units, depending on your hardware.
  • CellProfiler scales the pixel values of all images from 0 to 1 by dividing all pixels in the image by the maximum possible intensity value, so this should be kept in mind when making intensity measurements.

For MeasureImageIntensity, all measurements are computed from the aggregate of all the pixels in the image using these scaled values. E.g., the Mean is the average intensity value of all the pixels in the image.

“MAD” stands for “median absolute deviation” and is defined as the median of the absolute deviations from the data’s median; see for more details. It can be considered to be a more robust form of the STD that is more resistant to outliers.


Thank you very much!