Quantitative Transmission Electron Microscopy Analysis of Amyloid Fibril Morphology

Abstract

Amyloid fibrils are increasingly regarded as versatile protein-based nanomaterials whose functional properties depend not only on their molecular cross-β architecture but also on their supramolecular morphology. The present study was undertaken to compare the mesoscale organization of albumin, insulin, and lysozyme fibrillar assemblies using quantitative transmission electron microscopy. Representative TEM images were analyzed by a standardized image-processing workflow based on segmentation, skeletonization, orientation analysis, and connected-object morphometry. The resulting descriptors included projected fibril/aggregate area fraction, skeleton length density, apparent junction/crossover and endpoint densities, local width, object density, Feret length, aspect ratio, and orientation anisotropy. Insulin displayed the highest projected area fraction, skeleton length density, object density, endpoint density, and apparent junction/crossover density, indicating a compact and highly fragmented architecture dominated by numerous short rod-like elements. In contrast, lysozyme fibrils exhibited the greatest median Feret length and aspect ratio, consistent with a stronger contribution of longitudinal elongation and the formation of more anisometric structures. Albumin showed an intermediate morphology characterized by comparatively sparse, curvilinear fibrils and lower densities of discrete objects and endpoints. Orientation distributions were broad for all three systems, and the corresponding anisotropy indices remained low, demonstrating predominantly isotropic rather than globally aligned organization. These findings indicate that the three protein systems occupy distinct regimes of amyloid assembly, ranging from fragmentation-dominated architectures to more elongated fibrillar networks. The quantitative framework established here provides a basis for relating amyloid morphology to surface accessibility, connectivity, mechanical behavior, and the design of fibril-based hydrogels, biosensors, drug-delivery platforms, and nanocomposites.