A common framework for single-molecule localization using sequential structured illumination

TL;DR

This paper introduces a unified mathematical framework for single-molecule localization using sequential structured illumination, enabling fair comparison and guiding the development of improved localization methods like orbital tracking and raster scanning.

Contribution

It provides a common framework for evaluating and designing sequential structured illumination localization techniques, including two novel methods.

Findings

Benchmarking of existing methods shows their relative performance.

The proposed framework facilitates comparison and optimization of localization strategies.

New methods demonstrate competitive localization precision.

Abstract

Localization of single fluorescent molecules is key for physicochemical and biophysical measurements such as single-molecule tracking and super-resolution imaging by single-molecule localization microscopy (SMLM). Recently a series of methods have been developed in which the localization precision is enhanced by interrogating the molecular position with a sequence of spatially modulated patterns of light. Among them, the MINFLUX technique outstands for achieving a ~10-fold improvement compared to wide-field camera-based single-molecule localization, reaching ~1-2 nm localization precision at moderate photon counts. Here, we present a common mathematical framework for this type of measurement that allows a fair comparison between reported methods and facilitates the design and evaluation of new methods. With it, we benchmark all reported methods for single-molecule localization using sequential structured illumination, including long-established methods such as orbital tracking, along with two new proposed methods: orbital tracking and raster scanning with a minimum of intensity.