Limit to Threshold setting isn't giving me area of thresholded pixels on FIJI

I’m trying to determine the fraction of overlapping pixels between two channels. I thresholded my two images, and generated a new image with just the overlapping pixels using the image calculator. Now I want to get the area or pixel number from each image but something weird is going on.

I’m just trying to get an area measurement of the white pixels by doing Analyze>Measure after having check the “limit to threshold” box in measurement settings. But it is just giving the area of the whole image, can anyone tell me what I’m doing wrong?

Screen Shot 2020-01-09 at 4.16.51 PM

Hi @scor!

Perhaps your are missing a final thresholding step of the final image:

See the second result, after thresholding. Both measurments have “Limit to threshold” set, but only the second one has a threshold in place.

Hope this helps,
Cheers,
Nico

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So I need to re-apply my threshold before doing Analyze>Measure? When I do that I do get different numbers on area (thank you!)… but the area calculated seems to be black pixels. I can invert to get the actual value I care about, but I thought that white pixels were what would be counted here.

The actual color you’re thresholding depends on the actual LUT that’s on the image. If you look closely, the image example that I posted has an inverting LUT (which comes by default after creating a mask from a threshold). In that image, white means 0, and black 255.

Also, you can check wether the threshold is set with the option “Dark background” checked, which will invert the criteria for the thresholded region.

In any case, the area that’s measured is the one shown in read when thresolding.

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