  Nanoscale localization of point emitters is critical to several methods in
optical fluorescence microscopy, including single-molecule super-resolution
imaging and tracking. While the precision of the localization procedure has
been the topic of extensive study, localization accuracy has been less
emphasized, in part due to the challenge of producing an experimental sample
containing unperturbed point emitters at known three-dimensional positions in a
relevant geometry. We report a new experimental system which reproduces a
widely-adopted geometry in high-numerical aperture localization microscopy, in
which molecules are situated in an aqueous medium above a glass coverslip
imaged with an oil-immersion objective. We demonstrate a calibration procedure
that enables measurement of the depth-dependent point spread function (PSF) for
open aperture imaging as well as imaging with engineered PSFs with index
mismatch. We reveal the complicated, depth-varying behavior of the focal plane
position in this system and discuss the axial localization biases incurred by
common approximations of this behavior. We compare our results to theoretical
calculations.
