Hello. I have been trying to use Fiji to get a measurement of fluorescence intensity.
Previously, my lab’s protocol has been to manually set the threshold but not apply it, to leave a red overlay instead of converting it to binary. Then, set the measurement to include “integrated density” and select “limit to threshold”, and finally measure.
This has allowed us to calculate the intensity from the integrated density and area so far. However, we would like to try using the “auto threshold” function to remove a source of bias, but that function converts directly to binary without having the same overlay as the standard threshold option. Naturally, this significantly changes the integrated density.
I have tried duplicating the photo before thresholding one copy and then redirecting to the original in “set measurements”, but that has not made any difference. Is there some way to set the measured region to the automatically thresholded area, but take the measurements from the original pixels? I have not managed to find any other discussions or instruction on using auto threshold with “limit to threshold” yet.
Thank you for any help. If there’s any more information needed to solve this problem, please let me know.