Mean Gray Value under Roi

Hiya,
I’m trying to quantify the fluorescence of a plasma membrane using regions of interest. I can find the plasma membrane fairly well, but I can’t get a good method of getting the mean gray value of the plasma membrane which is outlined by the ROI manager. If there anyway to get mean gray value underneath the roi outline rather than the mean gray value for the whole selection?
Thanks

Edit: Image for clarity


Edit2: More image examples + further explanation


So, here the plasma membrane was marked by hand, the thick line, and the mean gray value underneath that mark was able to retrieve in the ROI manager because it was not an enclosed region, My question is if there is a way to do this with an enclosed ring, like the ones produced by analyze particles.

Hi @mdl54,

Welcome to the forum.

Do you mean the ring-shaped area between your two lines? I’m struggling to understand the use of underneath…

Hi Hiya,

I guess you want to measure a Ring.

The ROIs in the ROI Manager are not really capable to understand the “concept” of “holes”. Since ROIs are just outlines as far as I understood.

A similar Ring ROI problem was discussed in this thread:

Two workarounds were discussed here, I use the second workaround and it gives me good results:

Cheers,
Christopher

Sorry for not being clear enough lemme show another example


So, here the plasma membrane was marked by hand, the thick line, and the mean gray value underneath that mark was able to retrieve in the ROI manager because it was not an enclosed region, My question is if there is a way to do this with an enclosed ring, like the ones produced by analyze particles.

That’s overall idea, my current method is to outline the cell using analyze particles and then shrink that region and subtract to get just the Plasma membranes mean gray value. But that way is not very direct, so I’m hoping to be able to get the mean gray value underneath lines, like when there is no enclosed region.

Yes if you want to do that you just:

You need a mask of this ring however… This does not work with the ROI manager.

No problem!

I think you probably do want to see the solution that @schmiedc is talking about but just to add two selection uses that might help you depending on the specifics of your project.

In your original post where you seem to have two ROIs that represent the inside and outside of your desired area you can end up with the donut shape. What you can do is select them both and go to the More >> button on the ROI Manager and select the “XOR” function you will end up with a selection representing the ring shape between the lines. If you then click Add on the ROI Manager then the newly added ROI will represent the ring shape so measurements with that ROI selected will be measuring what you want.

Another option if you have a way to end up with the inner boundary is to use Edit > Selection > Make Band to try and roughly capture the plasma membrane by setting the number of pixels you need to measure.

Good luck!

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Thanks, the XOR solution worked well.

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