Tool to distinguish between very approximate or overlapping spots

Hi folks,

I would like to know how to analyze the attached image to distinguish approximate or overlapping spots.

The attached image shows almost 20 spots (RGB and 32-bit images), but when I analyze the 32-bit image with Analyze particles tool and Threshold, this is only able to distinguish 13 spots (“Count” column in Summary).

Is there any other tool or process to be able to discern between very close or overlapping spots?

I could do manually, but it takes a lot of time.

All the best,

Luis

Hi
@Professor_OAT
Can you send us the original source image? The one on the left of your screenshot.

run("Convert to Mask");
run("Watershed");

Greetings

Hi @Mathew,

Thanks for reply me. I have attached another different two because unfortunately I did not save the posted image (my apologies…). As you can see, these images are similar than the previous image. One of them with more spots

530B.tif (1.7 MB) 46C.tif (3.3 MB)

Thanks again.

Greetings.

Luis

1 Like

Hi
@Professor_OAT

Here is what I get with this macro on this image. [Here they are transformed into jpeg.So do not use.]
For the second “vegetable” it will be necessary to “play” a little with the macro.

PS: You need the RGB to CMYK plugin.

macro "Distinguish between very approximate spots."
{
requires("1.52u");
setBackgroundColor(0,0,0);
setOption("BlackBackground",true);
img=getImageID();
// Start batch mode
setBatchMode(true);
selectImage(img);
run("Duplicate...", "title=1");
close("\\Others");
run("Duplicate...", "title=2");
// Start processing
run("Duplicate...", "title=1");
run("Duplicate...", "title=2");
run("Invert");
run("RGB to CMYK");
run("Stack to Images");
selectWindow("C");
setAutoThreshold("Shanbhag dark");
//run("Threshold...");
run("Convert to Mask");
run("Set Measurements...", "area add redirect=None decimal=2");
run("Analyze Particles...", "size=0-Infinity display exclude include add");
roiManager("Show All without labels");
selectImage("1");
roiManager("Show All");
roiManager("Set Color", "black");
roiManager("Set Line Width", 2);
close("\\Others");
// End of processing
// End of batch mode
setBatchMode(false);
roiManager("Show All without labels");
exit("All is done !");
}

Hi @Mathew,

Thanks for your help. I tried with 46C.tif to get the good distinct spots with the same picture than you and it did not work… I am trying the same with plugin Color transformer-Yuv and I am getting almost the same results than you but I still without get good results for overlapping spots…

All the best.

Luis