To find the real size of an object can I use this formula
Objects original size (in m) = Objects size on sensor (in mm) * distance between camera and object (in m) / focal length (in mm)
knowing these informations
size of object: 400 pixels
working distance: 31 mm
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Yes, that is the thin lens equation. Do you have all those quantities? Note that the size of the object is in mm, not pixels. So you have to know what length 400 pixels corresponds to on your sensor. And 31 mm is awfully close - are you sure the thin lens equation holds in that kind of "macro focus" situation? I have doubts. And if you use a good lens, for things very close (not at infinity) then the thin lens equation is not so clear. The working distance (scene-to-front of lens) is not the same as the scene-to-front principal plane for a thick lens.
This seems like the hard way of doing it though. Why not just measure something of a known length to get a spatial calibration factor?
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