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Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy
Matthew D Lew1, Mikael P Backlund, W E Moerner
1Departments of Chemistry and ‡Electrical Engineering, Stanford University , Stanford, California 94305, United States.
Nano Letters
|January 31, 2013
Summary
Single-molecule dipole emission asymmetry limits super-resolution microscopy accuracy. Rotational mobility significantly impacts mislocalization, with errors bounded only for molecules rotating widely.
Area of Science:
- Optics and Photonics
- Biophysics
- Microscopy
Background:
- Localization-based super-resolution fluorescence microscopy relies on precise single-molecule position determination.
- The dipole emission pattern of single molecules (SMs) is inherently asymmetric, posing challenges for accurate localization.
- Understanding the impact of molecular dynamics on localization accuracy is crucial for advancing super-resolution techniques.
Purpose of the Study:
- To investigate how the asymmetric emission patterns of single molecules affect localization accuracy in super-resolution microscopy.
- To determine the relationship between single-molecule rotational mobility and position determination errors.
- To explore the consequences of varying rotational mobility on the quality of super-resolution reconstructions.
Main Methods:
- Utilized theoretical simulations to model single-molecule dipole emission.
- Analyzed the influence of rotational mobility, quantified by cone half angle (α), on localization precision.
- Evaluated the impact of different rotational mobility regimes on simulated super-resolution image quality.
Main Results:
- The asymmetric dipole emission pattern of single molecules inherently limits localization accuracy.
- Mislocalization errors are strongly dependent on the rotational mobility of single molecules.
- Localization errors are bounded to ≤10 nm only when single molecules rotate within a cone half angle α > 60°.
- Simulations revealed that both low and high rotational mobility can lead to resolution degradation or distortion in super-resolution reconstructions.
Conclusions:
- The rotational dynamics of single molecules are a critical factor influencing the precision of super-resolution fluorescence microscopy.
- Strategies to control or account for single-molecule rotational mobility are necessary to overcome inherent limitations in localization accuracy.
- Accurate super-resolution imaging requires careful consideration of single-molecule photophysics and dynamics.
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