Quantify the volume fractions of 2 components in gel matrix, CLSM z-stack

Hello friends,

I’m working on a series of gel samples with 2 different components. I used the CLSM and got a few z-stack 3D images. I’m pretty new on the image analysis, or specifically ImageJ. Please help me have a look at my problem, really appreciate your help to the beginners.

I looked on the topics on CLSM stack, then I tried 3D object counter. However, the images on the different positions of one stack have different intensities. Therefore when I choose the proper threshold for the image in the middle of the sample, then the threshold of the image on top or bottom is too high. So after the calculation, the volume fraction of Green is 47.89%, Red is 4.96%. The sum of them should be 100%, but obviously it is far away from 100%.

12%MS+2%PPI gel, z-stack.zip (5.3 MB)

Any comments/help/alternative methods will be appreciated! Many thanks!