Absorption dipole effects on MINFLUX single molecule localization

Area of Science:

• Super-resolution microscopy
• Single-molecule imaging
• Biophysics

Background:

• Minimum photon flux imaging (MINFLUX) offers nanometer-scale localization precision.
• Systematic errors from fixed dipoles or optical aberrations in MINFLUX are not well understood.

Purpose of the Study:

• To investigate the impact of fixed and rotating fluorescence dipole orientations and optical aberrations on MINFLUX localization accuracy through simulations.
• To identify strategies for mitigating systematic localization errors in MINFLUX.

Main Methods:

• Computational simulations were employed to model MINFLUX imaging under various conditions.
• Analyzed localization bias caused by fixed and freely rotating dipoles with varying tilt angles.
• Evaluated the effect of optical aberrations (astigmatism, coma) on localization accuracy.
• Assessed the impact of different measurement sampling patterns (triangular, hexagonal) and iterative probing area shrinking.

Main Results:

• Fixed dipoles tilted out-of-plane introduce significant localization bias, increasing with tilt angle (up to ~25% of probe diameter).
• For freely rotating dipoles, astigmatism and coma cause position-dependent bias exceeding localization precision.
• Hexagonal sampling patterns and iterative shrinking of the probing area reduce bias.
• A residual bias dependent on dipole orientation persists at the center even with iterative shrinking.

Conclusions:

• Fluorophore dipole orientation and optical aberrations are critical factors affecting MINFLUX accuracy.
• Optimized sampling strategies can significantly reduce MINFLUX localization bias.
• Further investigation is needed to address residual orientation-dependent biases.