I am interested in calculating signal-to-background ratios for my stained cells. For each picture, I adjusted the threshold manually to make sure all the cells were selected with minimal background. Then by measuring the particles. I used the max value divided by mean of the background to get a ratio. The threshold values are different among pictures as some of pictures are brighter than others. I am worried if I set the threshold number, some of the cells will be missed. I checked for literature, but most of them are talking about signal-to-noise ratio. What will be a standard way or any other way to calculate the signal-to-background ratio?