Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T

Area of Science:

• Biophysics
• Chemical Biology
• Materials Science

Background:

• Conventional fluorescence microscopy, including polarization imaging, is diffraction-limited for amyloid fibril structural analysis.
• Amyloid fibrils, implicated in neurodegenerative diseases, possess nanoscale structures (10-100 nm) beyond the reach of diffraction-limited techniques.

Purpose of the Study:

• To evaluate the photoswitching capability of Thioflavin T (ThT) when bound to insulin amyloid fibrils.
• To enable super-resolution imaging and polarized orientational analysis of amyloid fibril ultrastructure.

Main Methods:

• Modulating the redox environment to control ThT photoswitching properties.
• Utilizing stochastic super-resolution imaging based on ThT on-off fluorescence duty cycles.
• Performing polarized imaging of single ThT molecules bound to insulin amyloids.

Main Results:

• ThT fluorescence emission under optimized buffer conditions facilitates stochastic super-resolution imaging with ~20 nm localization precision.
• Adequate signal-to-noise ratios permit polarized orientational imaging of individual ThT molecules.
• Revealed ultrastructural signatures of protofilament twisting within amyloid fibrils.

Conclusions:

• Photoswitchable ThT offers a pathway to overcome diffraction limits in amyloid fibril imaging.
• This approach provides nanoscale structural insights into amyloid formation and organization.
• Polarized single-molecule imaging of ThT reveals novel details about fibril protofilament twisting.