Abstract
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.
Highlights
Amyloid aggregates, which originate from protein misfolding as a starting point for aggregation and plaque formations, are known to be at the origin of crucial processes responsible for neurodegenerative diseases
We report the use of Thioflavin T (ThT) for super-resolution imaging of amyloids, with the goal to benefit from its intrinsic intercalating nature that provides a high degree of order, potentially reporting the local ultra-structure of fibril without profound modification of its function
Super-resolution Direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging relies on the measurement of localization of isolated molecules with high precision, followed by image reconstruction based on a collection of a high number of detection events[13]
Summary
Amyloid aggregates, which originate from protein misfolding as a starting point for aggregation and plaque formations, are known to be at the origin of crucial processes responsible for neurodegenerative diseases. We report the use of ThT for super-resolution imaging of amyloids, with the goal to benefit from its intrinsic intercalating nature that provides a high degree of order, potentially reporting the local ultra-structure of fibril without profound modification of its function.
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