Table 1.

In vitro models of Aβ amyloid fibril formation and the associated biophysical techniques, based on the reviews of Malmos et al. (2017), Bruggink et al. (2012), Siddiqi et al. (2019), and Kumar et al. (2017) [34,37,38,39].

Aβ Fibrillization Models and Associated Techniques
Associated Methods	Examples	Advantages	Pitfalls	References
Additive models
Dye-based methods	Congo Red (CR) dye	Historical dye. Can also be used on tissues.	The short beta-sheet structures are not bound. The oligomers and protofibrils are not detected.	[32]
	Thioflavin T (ThT) assays	Best tool to study amyloid fibril formation: does not affect fibril formation, linearity, availability, robustness; easy to use.	ThT does not bind specifically to fibrils but also to DNA, cyclodextrin and SDS micelles. Need to use protein-pure samples. Cannot detect early aggregates (oligomers and protofibrils). The binding affinity depends on the fibril type. Need to use complementary techniques to confirm the results.	[33,34,40,41,42,43]
	ANS dye	Useful to characterize protein folding and aggregation intermediates.	Not specific to fibrils. Any protein with a hydrophobic region folded in the protein core has a fluorescent intensity.	[35,36,38]
Antibody-based methods	Time-resolved fluorescence (HTRF) immunoassay	Aβ peptide quantification. Sensitivity. Simple, rapid and robust method. Real-time kinetic study.	This technique requires specific antibody non cross-reacting with the different Aβ peptides.	[44,45]
	Surface plasmon resonance (SPR)	Real-time method. Study short-term or long-term aggregation kinetics (from second to hours). Study of aggregation modulators and potential drug inhibitors.	Need to know precisely which oligomer species or fibrils are bound by the antibody.	[46,47,48,49]
Microscopy and spectroscopy	Fluorescence microscopy and Fluorescence correlation spectroscopy (FCS)	Sensitivity. Real-time imaging. Small samples are sufficient. Can be used with fluorophore-coupled antibody (specificity gain). Also used to observe samples stained with Thioflavin T or ANS dyes.	Labeling can change aggregation. Autofluorescence interference.	[37,50,51,52]
Pure models
Microscopy and spectroscopy	Time-resolved emission spectra (TRES)	Non-invasive and label-free technique. Nanosecond timescale and nanometer spatial resolution.	Difficulties for data treatment and interpretation.	[53,54]
	Turbidity, multiangle laser light scattering (MALLS), dynamic light scattering (DLS)	Label-free methods. Light scattering is very sensitive. Real-time detection.	Turbidity is not a very reliable technique. Cannot differentiate oligomer intermediates. Low resolution of light scattering techniques.	[37,38,55,56]