Absorption dipole effects on MINFLUX single molecule localization

Single-molecule fluorescence localization with minimum photon flux imaging (MINFLUX) can achieve localization precisions in the small nanometer range or better under suitable conditions. Potentially adverse conditions, such as a fixed fluorescence dipole or optical aberrations, that could cause systematic localization errors, have received little attention up to now. Here, we study these effects in simulation. We find that biases occur for fluorophores with a fixed absorption dipole tilted out of the imaging plane. These become larger (up to about 25% of the diameter of the circle spanned by the doughnut center positions), the larger the tilt angle gets. As a rule of thumb, the spread in bias is smaller than 5 nm in case the dipole orientation is less than 30° out of plane for the typical case of a doughnut probing circle of diameter 100 nm. For freely rotating dipoles, only the primary aberrations, astigmatism and coma, contribute to bias. This bias depends on the position of the fluorophore inside the circular probing area of MINFLUX and can be significantly larger than the localization precision. We show that increasing the number of measurements over the circle from a triangular to a hexagonal pattern is beneficial for reducing bias in all cases. Iterative shrinking of the probing area can eliminate the position-dependent bias completely, but a strong dependence on dipole orientation of the bias at the center of the probing area remains.