The misfolding and self-assembly of proteins into β-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo TEM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-β, tau, α-synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo TEM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo TEM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimer's disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo TEM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a potent tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. This study should not only facilitate the profiling and characterization of amyloids for structural studies by cryo TEM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM