[31] Electron microscopy of prefibrillar structures and amyloid fibrils

Several techniques, such as X-ray crystallography, light scattering, fluorescence spectrometry, size exclusion chromatography, atomic force microscopy, and transmission electron microscopy, have been employed in studies of structural intermediates of fibril formation and fibrillar assembly of amyloid proteins. Electron microscopy, with a resolution of approximately 2 nm, offers a useful technique for the ultrastructural characterization of preprotofilaments, protofilaments, and mature fibrils formed during in vitro fibrillogenesis. For contrast enhancement of specimens, negative staining is applied. This chapter outlines the methodology used in laboratories for electron microscopic examination of negatively stained prefibrillar structures and amyloid fibrils. The Aβ-peptide used influences the kinetics of the fibril formation. This chapter uses Aβ1–42 (Bachem, Bubendorf, Switzerland), which forms fibrils within a few hours of incubation at 37°. In order to decelerate the fibril formation enabling it to investigate early intermediates and prefibrillar structures, this chapter has performed the in vitro studies at low concentrations of Aβ1–42 (170 μg/ml), as the kinetics of fibril formation is highly concentration dependent.