Hello I am rather new to Image J Macros. However, over the last couple of weeks I have developed a macro for fluorescence imaging analysis. I want to be able to obtain area and mean from two images (each from different channels). For the images, I have conducted background subtraction and adjusted threshold. A brief look at the total area and they look reasonable, as Image 1 is highly expressed (area=20,699) in this region whereas Image 2 is sparsely expressed (area=1,958).
I come across a problem when I get the co-localization Image 1 and Image 2. I use the following function:
imageCalculator("AND create 32-bit stack", imgImage2, imgImage1);

Afterwards, I adjust threshold
setThreshold(14, 122);

But the total area equals 7,237, which is greater than the Image 2 expression :(

Does this seem reasonable or should I be worried about the order I place the images when using the Image Calculator function? Or whether I should be converting the image to 32 bits.