Imagej How To Set Scale. Just fill in the fields. Then bring up the set scale dialog enter the known distance and unit of measurement then click ok.
This could be a ruler or any other object in the image you know the exact size of. In the analyze tools menu select scale bar. If your image is already scaled pixels are displayed in µm values then skip to part 2.
If you check the global checkbox this spatial calibration will be applied to all open image windows.
The scale bar dialog will open and a scale bar will appear on your image. If you check the global checkbox this spatial calibration will be applied to all open image windows. This could be a ruler or any other object in the image you know the exact size of. You need an object of known size to be able to set the scale in imagej.