# Birefringent Fourier filtering for single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation

## TL;DR

This paper introduces a Fourier filtering technique that combines polarization and phase control to simultaneously measure 3D position, orientation, and wobbling of single molecules with high precision in super-resolution imaging.

## Contribution

It presents a novel method integrating polarization control into Fourier filtering for unambiguous 3D localization and orientation measurement of single molecules.

## Key findings

- Achieves nanometer-scale positional accuracy
- Provides degree measurements within a few degrees
- Successfully applied to actin filament imaging

## Abstract

Super-resolution imaging based on single molecule localization allows accessing nanometric-scale information in biological samples with high precision. However, complete measurements including molecule orientation are still challenging. Orientation is intrinsically coupled to position in microscopy imaging, and molecular wobbling during the image integration time can bias orientation measurements. Providing 3D molecular orientation and orientational fluctuations can offer new ways to assess the degree of alignment of protein structures, which cannot be monitored by pure localization. Here we demonstrate that by adding polarization control to phase control in the Fourier plane of the imaging path, all parameters can be determined unambiguously from single molecules: 3D spatial position, 3D orientation and wobbling or dithering angle. The method, applied to fluorescent labels attached to single actin filaments, provides precisions within tens of nanometers in position and few degrees in direction.