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Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils.

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  • 1Department of Electrical and Systems Engineering, Washington University in St. Louis, Missouri 63130, USA.

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|August 25, 2020
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Summary

A new metric, variance upper bound (VUB), efficiently evaluates single-molecule orientation measurements for any point spread function (PSF). This method reveals precise Nile red binding on amyloid structures, offering insights beyond localization alone.

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Area of Science:

  • Biophysics
  • Chemical Physics
  • Molecular Imaging

Background:

  • Simultaneous single-molecule position and orientation measurements are crucial for understanding biological and chemical processes.
  • Existing methods using engineered point spread functions (PSFs) and the Cramér-Rao bound (CRB) are limited to evaluating performance at specific orientations.

Purpose of the Study:

  • To develop a novel performance metric, the variance upper bound (VUB), for efficient and comprehensive evaluation of single-molecule orientation measurement performance across all possible molecular orientations.
  • To utilize the VUB to identify optimal PSFs for orientation measurements, particularly near refractive index interfaces.
  • To apply the VUB-validated PSF to image Nile red (NR) molecules bound to amyloid aggregates, revealing binding modes and structural heterogeneities.

Main Methods:

  • Introduction of the variance upper bound (VUB) as a global performance metric for point spread functions (PSFs).
  • Development of an efficient computational method for VUB calculation, approximately 1000 times faster than average CRB calculations.
  • Application of a simple polarized standard PSF, identified as robust by VUB analysis, for super-resolution imaging of Nile red (NR) molecules.

Main Results:

  • The VUB provides a global maximum Cramér-Rao bound (CRB) for all molecular orientations, enabling rapid PSF performance assessment.
  • The VUB analysis identified a simple polarized standard PSF as optimal for precise orientation measurements near refractive index interfaces.
  • Super-resolution imaging using the optimized PSF revealed the primary binding mode of Nile red on amyloid fiber surfaces and structural variations within amyloid networks.

Conclusions:

  • The variance upper bound (VUB) offers a significant advancement in evaluating and optimizing single-molecule orientation sensing techniques.
  • The polarized standard PSF, validated by VUB, enables detailed structural and binding analysis of molecules like Nile red on amyloid aggregates.
  • This approach provides insights into molecular interactions and structural complexities not achievable with localization microscopy alone.