Robust and bias-free localization of individual fixed dipole emitters achieving the Cram\'{e}r Rao bound

TL;DR

This paper presents a method combining astigmatic imaging and dipole orientation information to achieve bias-free, high-precision localization of single emitters in microscopy, reaching the theoretical Cramér-Rao limit.

Contribution

It introduces a novel approach that eliminates localization bias caused by residual defocus, enabling nanometer precision in single molecule localization microscopy.

Findings

Achieves ~1nm localization precision

Removes bias from residual defocus effects

Reaches the Cramér-Rao bound in localization accuracy

Abstract

Single molecule localization microscopy has the potential to resolve structural details of biological samples at the nanometer length scale. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of ~1nm, thereby reaching the corresponding Cram\'{e}r Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.