Single-molecule orientation localization microscopy II: a performance comparison

TL;DR

This paper compares the performance of various advanced and standard orientation localization microscopy techniques, identifying optimal methods for different conditions and highlighting simple modifications that often outperform more complex setups.

Contribution

It provides a comprehensive performance comparison of multiple microscopy methods against fundamental limits, guiding optimal technique selection for specific experiments.

Findings

Simple modifications to standard microscopes can outperform complex methods.

Optimal imaging strategies depend on sample geometry and experimental conditions.

Certain state-of-the-art methods approach fundamental measurement limits.

Abstract

Various techniques have been developed to measure the 2D and 3D positions and 2D and 3D orientations of fluorescent molecules with improved precision over standard epifluorescence microscopes. Due to the challenging signal-to-background ratio in typical single-molecule experiments, it is essential to choose an imaging system optimized for the specific target sample. In this work, we compare the performance of multiple state-of-the-art and commonly used methods for orientation localization microscopy against the fundamental limits of measurement precision. Our analysis reveals optimal imaging methods for various experiment conditions and sample geometries. Interestingly, simple modifications to the standard fluorescence microscope exhibit superior performance in many imaging scenarios.