Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking

Many single-particle tracking and localization-based superresohition imaging techniques use the width of a single lateral fluorescence image to estimate a molecule's axial position. This determination is often done by use of a calibration data set derived from a source adhered to a glass-water interface. However, for sources deeper in solution, aberrations will change the relationship between the image width and the axial position. We analyzed the depth-varying point spread function of a high numerical aperture objective near an index of refraction mismatch at the water-glass interface using an optical trap. In addition to the well-known focal shift, spherical aberrations cause up to 30% relative systematic error in axial position estimation. This effect is nonuniform in depth, and we find that, although molecules below the focal plane are correctly localized, molecules deeper than the focal plane are found to be lower than their actual positions.