Difficulty "Limiting to Threshold" with "Auto Threshold"

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.

@schuylerkogan

No worries! I see what you mean… Auto-Threshold is the way to go! So stick with that - much more reproducibile. So in this case - all you have to do is generate that binary mask. Then use “Create Selection” or “Analyze Particles” functions to then add those selection(s) to the ROI Manager. Once you have those selections saved to the ROI Manager… you can then open your original image and overlay them on it to do the measurements you need (can click/toggle the Show All button on the ROI Manager for example).

Hope this helps! If not - ask more questions… we’ll get you squared-away.

Thank you very much. This seemed to help part of the way. I can definitely get the ROI from the threshold over the original photo with these steps, but when I go to measure it I’m still getting very unusual results, which are making me think that it’s still somehow taking the measurement from the thresholded image. This happens even when I select or deselect “limit to threshold” or redirect to the original photo.

@schuylerkogan

So you were able to add your ROIs to the ROI Manager then? If so… then you can even close the mask window - just close it to be sure it’s gone. Open the ORIGINAL image… all the while leaving the ROI Manager open with your ROIs saved there… then activate the original image window (ie - just click on it). And then click the Show All button on the ROI Manager window (near the bottom). You should see your ROIs being displayed on your image… and then all measurements are made on that image window.

This is also gone over step-wise in the Segmentation workshop:

Start at 1:32:42 for the part that shows this.

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It took me a while to figure out all the details (it seems like it only limits the measurement when the specific ROI is selected in the manager, for instance), but this has finally gotten the process we needed. Thank you very much for your help.

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ok. Good! I’m glad you figured out a solution… If you have more questions - post again. We are here to help.