I haven’t seen the data so I am not sure, but I have a feeling the “last” slice of your Z stack would be very difficult to use well. With the center of your spheroid likely not passing light accurately, plus depth decay, you will likely find a lot of variability in that measurement due to variations in spheroid size.
Some of the answers will depend on more specifically what your question is. Should the value you detect go up if there is more volume in the external areas (showing greater expansion)? This would be similar to CTFC, except for the whole, or some defined part of, the spheroid.
Should the value be the same no matter what the volume as long as the amount of protein per cell is the same? How heavily influenced do you want to allow the measurement to be by the morphology of the spheroid (as all spheroids are not spherical. Or even most of them usually.) This measurement would ideally be a bunch of CTFC measurements, or a mean area intensity of some sort.
You mostly can’t, as DAPI itself tends to be very… variable. Or at least it has been in the hands of the people I have seen imaging. The closest I could think of right off was my mention of using the depth TO the spheroid, which may influence the measurements.
If you consistently have a solid core (ok, solid sphere with an empty core? spherical doughnut?), you might be able to segment that dense DAPI signal as a single object, and then measure the intensity in your other channel Xum away in all outward directions. Something like create the mask in analyze particles after processing, fill the hole, save that ROI, Dilate a certain amount, measure the fluorescence in that Dilated area.
This would have upsides and downsides. Upside, no need for bias in the segmentation of your green signal as that would only be measured a certain distance from your DAPI core. Downside, your bias might come from how far from the core you decide to measure, where you take the measurement (Z heights), and whether that expansion ever extends out into “empty” matrix thus reducing the mean intensity. Median might be another option as a measurement, but that no longer estimates the protein content.
If you can define the problem more precisely, someone might be able to help further