I have been using imagej for quantification of my fluorescence signal and found that while thresholding my images, one tissue sample will capture all of my signal while another will capture my slim to none of my signal. I know what my signal looks like compared to my background. I have determined my threshold value, by thresholding a negative control so no signal is detected. Unfortunately, this excludes weaker signal and has, therefore, does not give me the best and most accurate results. I have tried to subtract background, but that doesn’t fix my actual problem of normalizing my pictures so I can compare them. Thank you for any help.

I have attached a few of my images and I’d really appreciate any suggestion.

I am a bit confused here:

On 3/5/2019 at 10:40 AM, NKASHYAP said:

I know what my signal looks like compared to my background. I have determined my threshold value, by thresholding a negative control so no signal is detected. Unfortunately, this excludes weaker signal

So if using the background signal as threshold would eliminate your signal, it basically means that your signal is not above your background. Or to but it differently, your signal to noise ratio is too low. So how do you know that it is actually a weak signal and not just background? The other relevant question is whether your background noise is uniform in your image.