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Updated: May 5, 2026

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy
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Absorption dipole effects on MINFLUX single molecule localization.

Sjoerd Stallinga, Wenxiu Wang, Bernd Rieger

    Optics Express
    |May 4, 2026
    PubMed
    Summary
    This summary is machine-generated.

    Minimum photon flux imaging (MINFLUX) can be biased by fluorophore dipole orientation and optical aberrations. Simulations show these biases can exceed localization precision, but improved measurement patterns can mitigate errors.

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    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.