Calculate signal-to-background ratios for fluorescent imaging


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?

Many thanks.

Hello there, @djonair
I think what you mentioned can be used to calculate SNR. I normally only divide the mean intensity values of the ROI with the mean intensity of the background. Moreover, when you set a threshold, try not to click the ‘Apply’ button as this creates a binary image. You might want to have a look here - for other ways to check SNR.


SNR can also be defined as the mean of the foreground divided by the standard deviation of the background.

Hello Praveen,

I noticed if I adjust the threshold, the mean intensity values changes. Higher threshold, higher mean intensity values. How should I set the threshold to minimize its effect?


Hmm. I am really not sure about that. I always set my threshold so that my ROI is always covered (the lowest possible threshold).