Help using CellProfiler to analyze 3D image stack


I am new to image analysis and have a few basic questions. I would like to measure total nuclear DNA content and levels of DNA synthesis of each nucleus in my sample, which is a sac-like organ consisting of a single layer of cells surrounding a lumen. I am learning to use CellProfiler and have modified the pipeline 3D Segmentation of Tumor Cells ( to do this. I would like to know if my pipeline and analysis are valid.

First, I am not sure if the pipeline I created is doing what I think it is doing. After rescaling intensity and running a median filter on the DAPI images, I segmented nuclei from background using Otsu threshold and ran Watershed to separate clusters of nuclei. The result looks good to me.

Can I go directly to MeasureObjectIntensity from here, selecting the images of DAPI and EdU and specifying watershed as the objects to measure?

How do I match the measurements from the data exported to Excel to specific nuclei in the image? In 2D, DisplayDataOnImage does this. Is there an equivalent for 3D?

Images and the pipeline can be found here: