Long-Term Super-Resolution Imaging of Amyloid Structures using Transient Binding of Standard Amyloid Probes

Abstract

Oligomeric amyloid structures are crucial therapeutic and diagnostic targets in Alzheimer's disease and other amyloid diseases. However, these oligomers are too small to be resolved by conventional light microscopy. We have developed a new tool to image amyloid structures on a nanometer scale using standard amyloid dyes such as Thioflavin T (ThT), without the need for covalent labeling of the amyloid protein or staining via fluorescently labeled antibodies. Transient amyloid binding (TAB) imaging is compatible with epifluorescence and TIRF microscopies and uses 488 nm cw laser excitation to excite ThT molecules that are bound to amyloid structures. Dynamic binding and unbinding of ThT molecules generate photon bursts (‘blinking’) that are used for single fluorophore localization at nanometer resolution. Thus, photobleaching cannot degrade either the number or brightness of blinking events, which enables us to image the same structure for multiple days without loss of image quality. As proof-of-principle experiments, we imaged oligomeric and fibrillar structures formed during different stages of amyloid-β aggregation as well as the structural remodeling of amyloid fibrils by the anti-amyloid compound epi-gallocatechin gallate (EGCG). TAB promises to directly image native amyloid in cells and tissues using standard probes at nanometer resolution and at the same time record amyloid dynamics over time scales of minutes to days.