Automatic Particle Sizing Using IMAGEJ

I have been on holidays for a while so it’s time to go back to work. Today I’m going to show an easy way of counting/measuring particles using a free software. The image analysis software is called ImageJ.

imagej-logo

ImageJ can be downloaded and used for free. The first thing that appears once installed is the toolbox.

toolbox

To load your image just File>Open. In my case, I’m going to use an SEM image where a few nanoparticles where split. (Thanks Nimkahl for the image). If you want to practice with it, just right click and download it.

MNP

Once the image is loaded into the program, the first thing is to get the scale of the image. For doing that, simply focus on the scale bar Ctr + Up Arrow. Then use the rectangular bottom to drag a rectangle that fits into the scale bar. Now Analyze>Measure to show the width and height of the rectangle. In this way we will know the relation between size and pixels. In our case 10 micronmeters is the same as 237 pixels.

scale

Now Analyze>Set Scale and use the numbers we got. 

scaleadjust

Hit Ok and the scale will be set. To make sure it is correct, if you try now to measure again the rectangle it will have 10 micrometers in length.

The next step now is to turn the image into grayscale (it is almost now but it will make your life easier). Simply Image>Type>8-bit. And now using the threshold you might be able to pick only the nanoparticles. Image>Adjust>Threshold. Make sure the nanoparticles look like red dots.

threshold

Once you apply the threshold it should look similar to this.

spots

Now it’s the moment to do nanoparticle counting. Because this image has edges it will not work very well, so I will limit the detection to a particular area. Analyze>Analyze Particles. In the menu that appears you can select many options. Since I know the nanoparticles are around 1 micrometer^2 in area I’m going to limit the Size. Circularity doesn’t have too much effect here because I limit the area where to look for nanoparticles. Include holes is very important because usually nanoparticles appear as a black ring.

area

When you run this analysis the output looks like this. It will show what was identified as nanoparticles, the summary of the results and also each individual nanoparticle.

dots

According to this the area of the nanoparticles is around 0.982 micrometers^2, good result. And it identify 27 nanoparticles. These results can now be exported into excel files, just in each window File>Save As and save the excel table, or can be plot. In the Results table Results>Distribution and select to use the round. It will appear something like this indicating the most of the nanoparticles are close to be round with a few of them differing quite a lot from round shape.

distribution

Hope you find it interesting.

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