During camera calibration you can only calculate the ratio of the focal length to the pixel size, i. e. the focal length in pixels. You cannot calculate the focal length in world units and the pixel size separately. The best you can do is look up the pixel size from the manufacturer's spec, and then calculate the focal length in millimeters.
The magnification of a positive lens is M=f/(f-v) (see wikipedia), with f the focal length and v the distance of the center of the lens to the object. So if you can find the magnification with your pixel difference and know distance to the lens, you can find it with this formula.
It is also written on the lens in 99% of the cases.
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