Fluorescence Measurement: GFP nucleus vs. cytoplasm

Hi I have a technical question: I have a series of pics that include DAPI, GFP and RFP. DAPI is used to set edge of nucleus and RFP for cell shape. I need to compare GFP signal intensity between nucleus and cytoplasm. They are in separate frames.
My method is as below: 1. Set nucleus by"Identify Primary Objects" using the DAPI frame, and using the similar method to identify cytoplasm. Then I am able to compare.
But the problem is how can I overlay the two into one single frame?
And how can I do the GFP comparison technically?
Thanks very much!


One preliminary comment: I assume that you are using IdentifySecondaryObjects to identify the cell body in the RFP image, using the nuclei as inputs, correct. If you’re not, you probably should be. You can then use IdentifyTertiaryObjects to identify the cytoplasm.

Once that’s done, you can then use MeasureObjectIntensity to make measurements from the GFP image using the nuclei and cytoplasm objects. Then you can use CalculateMath to do a ratio between the intensities between the cytoplasm and nuclei (or whatever arithmetic operation you deem suitable).

You can see an example of this workflow on our examples page; search for “translocation assay” on that page.

Hope this helps!