Spatial calibration of an image using FIJI

Suppose you wish to gather measurements from an image using real values (µm, miles, etc). The process involves calibrating a single image against known values, then applying that calibrated image to your unknown image. This assumes, of course, that both images are the same magnification. Here's an example.

Open your original image as well as a calibrated image. Here you see (right) an image of a microscope stage micrometer. It's markings are 100µm and 10µm apart.

Use the Line Selection tool to draw a selection line a known length. Here we've drawn a 400µm line:

With the selection line present, select "Set Scale" from the Analyze menu.

This brings up the following dialog box:

Just fill in the fields. If you check the "Global" checkbox, this spatial calibration will be applied to all open image windows. Click OK.

The select your unknown image, generate ROIs, and Analyze Particles.

In this example ImageJ calculated the Area of the selected ROIs in the original image. The Results window (R) shows the area in square µm (our original calibration scale).