Impact of optical aberrations on axial position determination by photometry

Abstract

Recently, Franke et al.introduced a way to estimate the axial position of single molecules (temporal radial-aperture-based intensity estimation (TRABI)). To this end, they compared the detected photon count from a TRABI estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found that the photometric ratio is ~0.7–0.8 at points close to focus and decreases as the distance from the focal plane increases. Here we explain this reported but unexplained discrepancy and, furthermore, show that the photometric ratio as an indicator for axial position is susceptible even to typical optical aberrations.