Single-molecule fluorescence spectroscopy and imaging of heterogeneous amyloid β aggregation

Protein aggregation is a complex process involving a variety of intermediate states along multiple pathways of fibril formation. It is extremely difficult to characterize this heterogeneity using conventional ensemble measurements. In this paper, we introduce single-molecule Förster resonance energy transfer (smFRET) spectroscopy and fluorescence imaging techniques to effectively characterize oligomeric species and fibril formation and growth, with a particular focus on amyloid β (Aβ) aggregation. We describe the procedures for bacterial expression, purification, and dye labeling of Aβ peptides and how to perform various single-molecule fluorescence experiments.