How to do an ImageJ area measurement

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One can measure each pixel for a precise area measurement of odd shapes (leaves, seeds, works with microorganisms too!) without bias.

I’ve included a practice image of an Arabidopsis stem


Paper ruler: Go to Vendian
ImageJ: Get ImageJ

Scan your target tissue (leaf, seed, stem*) on a paper ruler.  Use at least 300 dpi.  Over 600 dpi is overkill. 

Start ImageJ and drag in your image(s).

Select the line tool and draw a line from one point on the ruler to another

Try to make it as perpendicular to the mm markers as possible.
If you have a yellow line with 2 or 3 open squares you’re set.

Select Analyze --> Set scale.  Enter your known line length. In this case it’s 10 cm. At 300 dpi the pixel length is ~ 120 pixels.  Click global if you want to retain the calibration in memory for multiple images using the same ruler.

Choose Image --> Color Threshold

When you alter the hue, saturation and brightness you’ll cause the background to exclude the plant tissue.  In this case the green stem becomes distinct from the red background.   The background color choice is entirely up to you.

Now double click on the edge of your tissue.  The edge is the junction between the red background and the green tissue.   The edge is the junction between the red background and the green tissue.

It’s a little bit fiddly at times.  Don’t be frustrated if it doesn’t work right away.  It helps to zoom in (press the + key to zoom in).

Hit Ctrl+M or ⌘+M.  The area of your tissue is output to a table.