Micro-emulsions analysis

Hello, Can somebody help me with my problem?
I have an issue analyzing micro-nano emulsion. Have already tried to do it as follows:

run("8-bit");
run("Subtract Background...", "rolling=50 light sliding");
run("Window/Level...");
run("Enhance Contrast", "saturated=0.35");
("Apply LUT");
setAutoThreshold("Default");
//run("Threshold...");
//setThreshold(0, 165);
setOption("BlackBackground", false);
run("Convert to Mask");
run("Fill Holes");
run("Watershed");

but this doesn’t seem to help me at all. I cant figure out how to include and separate smaller particles in the analysis.


Is there any known way to solve this problem?

Hey,

If you just want to count the numbers try that:

//open the image
title = getTitle();
print(title);
run("Duplicate...", "title="+title+"_analysis");
title2 = getTitle();
run("8-bit");
run("Subtract Background...", "rolling=250 light sliding");
run("Gaussian Blur...", "sigma=2");
run("Find Edges");
run("Threshold...");
setThreshold(20, 255);
setOption("BlackBackground", false);
run("Convert to Mask");
run("Fill Holes");
run("Watershed");
run("Analyze Particles...", "size=100-Infinity circularity=0.40-1.00 show=Masks");
selectWindow("Mask of " + title2);
run("Duplicate...", "title=Overlay");
run("Add Image...", "image="+title+" x=0 y=0 opacity=80");

This analyzes the image and creates an overlay so you can immediately see what is going on.

You will see that, with the image you provided, the area measurement will probably overestimate the true area, while the number measurement will give reasonable results (at least from what I can tell).

Let me know if it helps.
One question: Did you get a good separation on the droplet clusters (lower left of the image)?

P.S.: Don’t upload/use jpg files. The compression artifacts were really heavy for the small droplets and might have hampered their detection in the first place when you tried it. For TIF files, the Gaussian filter might not have been necessary…

Thanks, thank you for your time and help, but the problem is that you need to calculate exactly the area / diameter of each particle.

Thank you for your time and help, but the problem is that i need to calculate the exact area / diameter of each undividual particle.