Single-fibril Förster resonance energy transfer imaging and deep learning reveal concentration dependence of amyloid...

Amyloid fibril formation is a highly heterogeneous process as evidenced by polymorphism in fibril structure. It has been suggested that different polymorphs are associated with different diseases or disease subtypes. Detailed characterization of this heterogeneity is a key to understanding the aggregation mechanism and, possibly, the disease mechanism. In this work, we develop Förster resonance energy transfer (FRET) imaging of amyloid fibril formation in real time and investigate the concentration-dependent heterogeneous fibril formation of amyloid β 42 (Aβ42). We incubated a mixture of unlabeled and labeled (5% donor and 5% acceptor) Aβ42, followed aggregation, and characterized individual fibrils in terms of FRET efficiency, acceptor fluorescence lifetime, and stoichiometry of the donor- and acceptor-labeled monomers incorporated into the fibrils. By FRET efficiency, we found that there are two distinct species at a relatively low concentration, 2 μM. The high FRET species appears first, but the low FRET species becomes dominant at later times. On the other hand, the high FRET species dominates throughout aggregation at 4 μM. The broad FRET efficiency distributions are consistent with those calculated from various known fibril structures. In addition to the FRET efficiencies, different acceptor lifetimes at the two concentrations and broad acceptor density distributions indicate at least three structurally distinct fibril species exist at each concentration, which also differ between the two different concentrations. The distinct heterogeneity in fibril formation pathways depending on the monomer concentration highlights the importance of understanding heterogeneity in the context of the biologically relevant aggregation environment.