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As a step toward absolute calibration of optical tweezers, a first-principles theory of trapping forces with no adjustable parameters, corrected for spherical aberration, is experimentally tested. Employing two very different setups, we find generally very good agreement for the transverse trap stiffness as a function of microsphere radius for a broad range of radii, including the values employed in practice, and at different sample chamber depths. The domain of validity of the WKB (“geometrical optics”) approximation to the theory is verified. Theoretical predictions for the trapping threshold, peak position, depth variation, multiple equilibria, and “jump” effects are also confirmed. [ABSTRACT FROM AUTHOR]