I want to calculate the real distance of object during one frame changing.
How can I estimate it from the camera pixel coordinate?
(If the camera coordinate (0,0) in 3th frame and (1,1) in 4th frame, the camera distance is root 2 and what is the real distance of the object motion?)
Are there any needed information that I calculate it?
If so, could you let me know about it?
You need to calibrate your image acquisition process using a reference object that is in the same optical plane as your moving object. You can use a known object in the scene, or use a separate image acquired under the same conditions in which such an object exists. You can then set your image’s pixel-to-distance scale using the “Analyze>Set Scale…” command. This command is explained here in the User Guide: https://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.8