Cell profiler intensity measurement

Hello,

I am using CellProfiler sofware to measure the intensity of NEF and set up Dapi as Primary object. However. I am getting two excel file at the end, one called Nuclei and other one NEF, but both excel files contained NEF and Dapi intensities, but with different value.
Which file should I choose to measure NEF intensity?

Many Thanks for your help

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Welcome! I suspect it will be easiest to help if you can post your pipeline and images.

To explain the fundamental principle: you use Identify module to identify regions of interest (sounds like nuclei) then you use MeasureObjectIntensity to measure the amount of NEF within the nuclei.

At least, if I understand your goals correctly… Feel free to elaborate!

Thank you for your prompt reply!

NEF staining is mostly cytoplasmic (neurofilament). Is it still ok to use the nuclei spreadsheet instead of NEF spreadsheet?

Please find attached the segmentation overlay and my pipeline screenshot

Many thanks for your help

Ah! Ok, then after identifying nuclei with IdentifyPrimaryObjects, add an IdentifySecondaryObjects module to expand out from the nucleus region a reasonable distance to define the cell borders. You can even add IdentifyTertiaryObjects to define the cytoplasm region alone. Then use Measure Intensity for whichever compartment you like.

This blog post elaborates: https://blog.cellprofiler.org/2016/12/21/help-what-are-these-three-different-modules-to-identify-objects/

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