How to perform a colocalization (with %area output) around nuclei?

Hello!

I am analyzing mouse vein grafts on the presence of types of macrophages. The image shows a cross section of the vein graft. In red nuclei are visible and the colors blue and green show two specific macrophage markers. Both markers are present around the nuclei, when observing both the blue and green channel separately the channels have the shape of a hollow convex lens/stretched oval - hollow because the nuclei are normally present inside of this shape.

I am trying to measure what the intensity is of blue around red (so of the specific marker around one nuclei, for all the nuclei present in the picture) and of green around red in exactly the same way. In this I have succeeded, having results in area and %area per nuclei. However, I am interested in knowing the intensity of blue and green simultanously (colocalized) around red. I don’t know how I can script this. I have tried some colocalization plugins (Coloc2,JaCoP, colocalization colormap) on the whole image, but I do not know how to incorporate it with the measurement of intensity per nucleus. I also do not know how to get the output in %area while doing such a colocalization.

This is the macro for just the blue - red measurement:
currentTitle = getTitle();
run(“Split Channels”);
redTitle = currentTitle +" (red)";
selectWindow(redTitle);
setAutoThreshold(“Default dark”);
setOption(“BlackBackground”, true);
run(“Convert to Mask”);
run(“Close-”);
run(“Watershed”);
run(“Analyze Particles…”, “size=12-Infinity show=Overlay clear summarize”);
run(“Dilate”);
run(“Dilate”);
run(“Watershed”);
run(“Analyze Particles…”, “size=20-Infinity show=Overlay clear add”);
blueTitle = currentTitle +" (blue)";
selectWindow(blueTitle);
run(“Enhance Contrast”, “saturated=0.40”);
roiManager(“Combine”);
roiManager(“multi-measure measure_all append”);
run(“Summarize”);

Thank you!
9068375_40X_12_5adjusted_m00.tif (12.9 MB)

So this isn’t really going to help directly, but I wanted to point out a few things about the image.
The nuclear red stain is very weak, and all of the bright red spots appear to be red blood cells (though maybe your min size threshold in the analyze particles step deals with those?).

There is a bit of a balance artifact to the tiling where the left side of the tile is fainter than the rest.


The cytoplasmic means reflect the shading issues, as seen by the vertical stripes.

Maybe if you elaborate with some examples of what you want to find, someone will be better able to help you within FIJI. I am not really clear on what % area per nuclei would mean biologically. If you have a standard dilation, for some nuclei you will run into other cells, and for other nuclei you will expand into empty space unless you keep the dilation very small. But if you keep the dilation small, your % area doesn’t really reflect much about the cell.

Also, macrophages are very, very oddly shaped in terms of their cytoplasms, so dilations are definitely not going to capture the total shape of the cell.

In that case you might be better off looking for single or double positive cells, and avoid the colocalization issue entirely. That would be a much “easier” problem.