Assessing amyloid fibrils and amorphous aggregates: A review

Protein misfolding and aggregation play a central role in the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's. These aggregates manifest either as structured amyloid fibrils enriched in β-sheet conformations or as irregular amorphous aggregates with diverse morphologies. Understanding their formation, structure, and behavior is critical for deciphering disease mechanisms and developing targeted diagnostics and therapeutics. This review presents an integrated overview of both conventional and advanced techniques used to detect, distinguish, and structurally characterize these protein aggregates. It covers a range of spectroscopic and spectrometric tools, such as fluorescence, Raman, and mass spectrometry that facilitate aggregate identification. Microscopy methods, including atomic force and electron microscopy, are highlighted for morphological analysis. The review also discusses in situ detection strategies using fluorescent dyes, conformation-specific antibodies, enzymatic reporters, and real-time imaging. Separation methods like centrifugation, electrophoresis, and chromatography are outlined alongside structural analysis tools such as X-ray diffraction. Furthermore, the growing utility of computational approaches and artificial intelligence in predicting aggregation propensities and integrating biological data is emphasized. By critically evaluating each method's capabilities and limitations, this review provides a practical and forward-looking resource for researchers studying the complex landscape of protein aggregation.