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The focus of an oil-immersion microscope objective is shifted because of the refractive-index mismatch between the cover glass and the aqueous sample. We present a procedure with which to determine the focal shift by use of an inverted microscope equipped with optical tweezers. As the position of the sample chamber is scanned vertically, we measure the axial displacement of an optically trapped bead; the relative motion of the bead with respect to the surface supplies the effective focal shift. Measurements of this quantity deviate from electromagnetic calculations of the focal shift, a discrepancy attributable to the depth-dependent decrease in axial trap stiffness that arises from spherical aberration.

Measurement of the effective focal shift in an optical trap