Resolving the Orientations of and Angular Separation between a Pair of Dipole Emitters

TL;DR

This paper demonstrates the limitations of polarization-sensitive imaging in distinguishing coincident dipole emitters and proposes a combined modulation method that significantly improves measurement accuracy of their orientations and separation.

Contribution

It introduces a novel combined modulation technique that enhances the discrimination and measurement precision of dipole pairs in fluorescence imaging.

Findings

Improves centroid orientation measurement precision by 50%.

Enhances angular separation measurement by 2- to 4-fold.

Shows that existing methods perform poorly for dipole pairs.

Abstract

We prove that it is impossible to distinguish two spatially coinciding fluorescent molecules from a single rotating molecule using polarization-sensitive imaging, even if one modulates the polarization of the illumination or the detection dipole-spread function (DSF). If the target is known to be a dipole pair, existing imaging methods perform poorly for measuring their angular separation. We propose simultaneously modulating the excitation polarization and DSF, which demonstrates robust discrimination between dipole pairs versus single molecules. Our method improves the precision of measuring centroid orientation by 50% and angular separation by 2- to 4-fold over existing techniques.