not sure how to do this with ImageJ, I believe you can 'mask' the nucleus somehow, to analyze only cytoplasmic staining. http://www.cas.miamioh.edu/~meicenrd/ANATOMY/Ch14_IndependentInvestigation/Brief%20Instructions%20for%20ImageJ.html

however I do know that in a program called cell profiler, the program can automatically identify various objects (such as nuclei) based on the florescent channels, and then subtract out the dapi signal so that only cytosol staining is counted.

Create a mask or binary from each of the initial images and then perform and XOR to produce a third mask that will only show where one and only one signal exists.

Open ImageJ, start a new macro, paste this in with an image open and hit run.

run("Split Channels");

rename("DAPI");

run("Put Behind [tab]");

rename("GREEN");

selectWindow("DAPI");

run("Enhance Contrast", "saturated=0.35");

run("Apply LUT");

setAutoThreshold("RenyiEntropy dark");

//setThreshold(33, 255);

run("Convert to Mask");

imageCalculator("Subtract create", "GREEN","DAPI");

selectWindow("Result of GREEN");

run("Measure");

Thanks everyone for your super helpful feedback! I ended up kind of integrating everyone's suggestions and then made it into a macro (thanks Jordan R. Yaron for that tip!). I thresholded both channels, selected the DAPI channel as a ROI, applied it to the FTSC channel and then made the selection inverse and cut to remove the nuclear staining. I then selected the remainder of the FTSC channel as a ROI and applied it to an original, unthresholded version of the FTSC channel, and measured this selection. The macro is pasted below if anyone else is interested.

selectWindow("DAPI");

run("Z Project...", "projection=[Average Intensity]");

selectWindow("FTSC");

run("Z Project...", "projection=[Max Intensity]");

selectWindow("AVG_DAPI");

setAutoThreshold("Default");

//run("Threshold...");

setAutoThreshold("Default");

setOption("BlackBackground", false);

run("Convert to Mask");

selectWindow("MAX_FTSC");

setAutoThreshold("Default");

run("Convert to Mask");

selectWindow("AVG_DAPI");

run("Create Selection");

roiManager("Add");

roiManager("Select", 0);

roiManager("Rename", "DAPI");

selectWindow("MAX_FTSC");

roiManager("Select", 0);

run("Make Inverse");

run("Cut");

run("Create Selection");

roiManager("Add");

roiManager("Select", 1);

roiManager("Rename", "FTSC");

selectWindow("FTSC");

run("Z Project...", "projection=[Max Intensity]");

roiManager("Select", 1);

run("Measure");