Suppose you wish to gather measurements from an image using real spatial units (µm, miles, etc), rather than just in "pixels". If your image data was in a format that contained spatial calibration meta data, and Fiji (maybe using bio-formats) was able to read it, then your image might already be spatially calibrated. You can see that in the info bar at the top of the image.
The process of spatial calibration involves calibrating a single image against known values, then applying that calibration to your uncalibrated image. This assumes, of course, that both images are at the same magnification. Here's an example.
- Open the stage micrometer image, and the uncalibrated test image. Select the stage micrometer image.
- Use the Line Selection tool to draw a selection line a known length on the stage micrometer image.
- Go to menu "Analyze - Set Scale"
- Select the stack to save, and go to menu "File - Save As - Gif"