In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging

Abstract

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

“…It is also extendable to 3D orientations measurements, in a version that would require more than two emission polarization states (31,32), at the expense, however, of lower signal-to-noise ratio conditions. Ultimately, this technique can serve as a quantitative indicator for local structural properties at the nanoscale in more complex filament organizations, such as the hierarchical assembly of amyloid protofilaments (38) or the dynamic actin filaments reorganization involved in cell shape changes (39).…”

Secondary nucleation of monomers on fibril surface dominatesα-synuclein aggregation and provides autocatalytic amyloid amplification

“…Two-color super-resolution microscopy using dSTORM Two-color super-resolution microscopy was performed on an inverted total internal reflectance fluorescence microscope which was custom-built for dSTORM imaging, as described previously (Kaminski Schierle et al 2011;Pinotsi et al 2014). Alexa Fluor 647 and 568 dyes were excited using a 640 nm diode laser (Toptica) and a 561 nm DPSS laser (Oxxius), respectively with an irradiance between 1 and 5 kW cm −2 , and a 405 nm laser diode was used as a reactivation source.…”

Thioflavine-T and Congo Red Reveal the Polymorphism of Insulin Amyloid Fibrils When Probed by Polarization-Resolved Fluorescence Microscopy

“…Although it does not reach the power of structural analysis techniques, fluorescent microscopy is an established technique, especially for diagnostic purpose, because it allows to observe easily the formation of amyloid fibrils in tissues or in vitro 7,8,9,10,11 . In this context, much work has been carried out in order to synthesize efficient fluorescent probes that bind specifically to the amyloid structure 12,13,14 .…”