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

Thioflavin T (ThT) is standardly used as a fluorescent marker to detect aggregation of amyloid fibrils by conventional fluorescence microscopy, including polarization resolved imaging that brings information on the orientational order of the fibrils. These techniques are however diffraction limited and cannot provide fine structural details at the fibrils scales of 10–100 nm, which lie beyond the diffraction limit. In this work, we evaluate the capacity of ThT to photoswitch when bound to insulin amyloids by adjusting the redox properties of its environment. We demonstrate that on-off duty cycles, intensity and photostability of the ThT fluorescence emission under adequate buffer conditions permit stochastic super-resolution imaging with a localization precision close to 20 nm. We show moreover that signal to noise conditions allow polarized orientational imaging of single ThT molecules, which reveals ultra-structure signatures related to protofilaments twisting within amyloid fibrils.