Table 1. Methods used to study amyloids in vitro and in vivo.

Method	Characteristics and peculiarities	Applicability				Limitations
		Detection of amyloids	Study of amyloid structure	Study of amyloid morphology	Study of aggregation kinetics
Immunohistochemistry and Immunochemistry	Immunohistochemistry is applied in pathology to visualise and localise protein aggregates and inclusions found in tissue sections of individuals with amyloidosis [39]. This technique is widely available and easily applicable in most pathology laboratories [40]. Immunohistochemistry uses antibodies for detection of amyloids. The availability of novel monoclonal antibodies targeting amyloidogenic precursors [41,42] and antibodies is directed against the amyloid fibrils [41,43]. Conformation-specific antibodies recognise soluble oligomers [44,45] or fibrils from many types of amyloid proteins [46–49], regardless of sequence. A small, bispecific antibody-based radioligand capable of crossing the blood–brain barrier can bind to intracerebral Aβ, allowing for in vivo visualisation [50]. This technique also uses luminescent–conjugated oligothiophenes as a unique class of amyloid dyes [51,52,53].	Yes	No	No	No	Commercially available antibodies for various proteins are often suboptimal for the identification of the same proteins in an amyloid fibril conformation [54]. This is caused by the proteins adopting different conformations and a variety of modifications during fibril formation, such as N- or C-terminal truncation [54]. To overcome this limitation, novel conformation-specific antibodies were designed [44–47]. At present, they are not yet widely used and require additional testing. Novel techniques that use luminescent–conjugated oligothiophenes [51–53] and the antibody-based radioligand [50] also need to be tested further. These novel developments are primarily used to detect Aβ fibrils and occasionally α-synuclein fibrils [46].
Staining with CR	The binding of CR to amyloids in vitro induces a characteristic increase in CR absorption leading to a red shift of its absorbance peak from 490 to 512 nm and the presence of a unique shoulder peak at approximately 540 nm [55,56]. Amyloid is detected by the increased optical anisotropy after CR binding [57], which is called the ‘apple-green birefringence’ (under crossed polarisers) [58]. ‘Apple-green birefringence’ is used to detect amyloid deposits in tissues and in in vitro studies of amyloids [15,57,58]. Today, this method is commonly used in histopathology laboratories because it is simple and cost-effective. There are different modifications of the method, including CR fluorescence (CRF) [59,60].	Yes	No	No	No	There exists some limitations (additional information is given in the main text).
Staining with Thioflavin Т/S	Thioflavin fluorescence is a classical method for detecting and analysing amyloids in tissue samples [61]. Thioflavin T is selectively localised to amyloid deposits, thereupon exhibiting a dramatic increase in fluorescent brightness [61]. This staining is also used in in vitro studies [62,63]. Upon binding to amyloid fibrils, Thioflavin T gives a strong fluorescence signal at approximately 482 nm when excited at 450 nm [64]. The fluorescence intensity scales linearly with amyloid fibril mass (e.g., with the number of available binding sites). Based on increasing fibril mass, the concentration of amyloid is calculated [65]. Reductions in Thioflavin T emission intensity are often interpreted as an indicator of fibril growth inhibition [66].	Yes	No	No	Yes	Staining with Thioflavin T/S is easy to perform, but the requirement of fluorescence microscopy limits the usefulness of this staining method [61]. Other tissues, such as cartilage, elastic fibres and mucopolysaccharides, can also be stained with ThT [67–69]. Sometimes, even at substoichiometric dye/monomer ratios, ThT undergoes substantial self-quenching that results in a non-linear relation between its binding and emission properties [63]. In this regard, without preliminary experiments, the use of this method for the study of fibril formation kinetics is problematic. ThT fluorescence is not generally a suitable tool for the detection of oligomeric intermediates during amyloid fibril growth [62]. Nevertheless, this dye binds to some oligomers [70–72].
Circular dichroism	This method is used to determine the secondary structures of proteins and peptides in amyloid aggregates [73,74]. This technique operates by the differential absorption of the left- and right-handed components of circularly polarised light by chiral molecules in solution [75].	No	Yes	No	No	This technique can be used only for determination of changes in secondary structure and is sensitive to aromatic compounds present in the sample. It can be employed only in in vitro studies.
Fourier transform IR spectroscopy	Fourier transform IR spectroscopy is an absorption spectroscopy in which the transitions detected are those arising from vibrational modes of bonds involving heteroatoms [77,78]. The presence and relative abundance of β-sheet structure in peptides and proteins can be assessed by this technique [76] also for amyloid studies [79,80]. Methodologies exist to acquire spectra from proteins in any physical state, including crystals, powders, thin films, and aqueous solutions, as well as from membrane-bound proteins [76].	No	Yes	No	No	There is water interference [76]. High protein concentration is necessary [76]. This technique can be employed only in in vitro studies.
NMR	Molecular and supramolecular structures of amyloid fibrils can be probed by various solid-state NMR techniques [24,82–89].	No	Yes	Yes	Yes	This technique is expensive and difficult [81]. Analysis is limited to small proteins [81]. It can be employed only in in vitro studies.
X-ray diffraction	The method is used to examine the structures of insoluble amyloid fibres [90,91]. With this method, it is possible to reveal the presence of the cross β-sheet structure by detection of the 4–5 Å equatorial and 10–12 Å axial reflections that correspond to the inter-chain distance and to the face-to-face separation of β-sheets, respectively [92,93].	No	Yes	No	No	This method can only be used when the sample is in the crystallised form. It is difficult to employ this method to study intermediates at the initial aggregation stages. This technique can be employed only in in vitro studies.
Small-angle X-ray scattering	This technique can be used to investigate the structure, folding and conformational dynamics of globular proteins, including multidomain and multisubunit proteins [94]. Small-angle X-ray scattering is a powerful and flexible technique for the characterisation of structural variations in amyloid fibrils [95–98].	No	Yes	Yes	No	This technique is expensive and difficult. This technique can be employed only in in vitro studies.
Cryoelectron microscopy	Cryoelectron microscopy technology allows for high resolution (less than 5 Å) determination of the atomic structures of amyloid fibrils in vitro [99–101]. Also in this technique, it is possible to explore amyloids extracted from tissues (ex vivo) [102,103].	No	Yes	Yes	No	It is difficult or impossible to study amorphous amyloid aggregates using this technique. The process of sample preparation is very complicated.
TEM	Since amyloid fibrils have unique electron microscopy characteristics [104,105], electron microscopy is routinely used in the analysis of kidney biopsies in the United States and other countries [104]. All types of amyloid deposits seen in different tissues, are mainly composed of bundled, not branched, straight fibrils, ranging from 6 to 13 nm in diameter (average 7.5–10 nm) and 100–1600 nm in length [106]. However, other morphological characteristics can also exist [107]. To increase the diagnostic significance of the method, immunogold electron microscopy, a technique that combines immunohistochemistry with electron microscopy, can be used [108]. With this technique, it is possible to explore both amyloids extracted from tissues and amyloids generated in vitro [106].	Yes	No	Yes	No	In some cases, this technique is not suitable to diagnose amyloidosis. For instance, immunotactoid glomerulopathy and fibrillary glomerulonephritis can be misdiagnosed as immunoglobulin light and heavy chain amyloidosis [109].
Atomic force microscopy	This technique can be used to investigate the size and morphology of protein aggregates in solution [110]. This method is also used to probe intrinsic properties of amyloid fibrils, such as mechanical strength and Young’s modulus [111].	No	No	Yes	Yes	When contact mode is used, high shear forces cause damage to the fibrils and may require immobilisation strategies [112]. The tapping-mode in air requires a ‘dried’ sample, so it cannot probe fibril processes directly; as a result, potential artefacts from dehydration (e.g., salt crystals, fibril damage) may be observed, and the degree of hydration is not known and cannot be controlled [112]. For the tapping-mode in liquid, current scan speeds are too slow to image rapid processes, and often, samples (fibrils) need to be well-adhered to a surface. Other atomic force microscopic techniques, including the use of support surfaces (e.g., lipid bilayers) are often difficult to use [112].
Dynamic light scattering	Dynamic light scattering is a laser scattering technique capable of unbiased analysis of size distributions for diffusing particles in the nanometre to micrometre size range [113]. The ability to resolve multimodal size distributions and make absolute size measurements makes dynamic light scattering a powerful technique for systems with heterogeneous species. It has been used to quantitatively study fibril formation in a range of systems from different proteins [114–117].	No	No	No	Yes	A hydrodynamic radius of the particle is measured, but this can differ from the true radius. As a result, determination of the real size and shape of macromolecules is difficult.
Fluorescence correlation spectroscopy	This highly sensitive analytical technique is used to measure dynamic molecular parameters, such as diffusion time (from which particle size can be calculated), conformation and concentration of fluorescent molecules [118,119]. It has been particularly powerful for characterising size distributions in molecular associations (e.g., dimer/multimer and fibril formation) both in well-behaved thermodynamically equilibrated systems in vitro and in more complex environments in vivo. Fluorescence correlation spectroscopy could therefore be used as a highly sensitive and specific competition assay to identify potential inhibitors of fibril formation [118].	No	No	No	Yes	This technique requires the use of fluorescent tags to label amyloid samples. This makes it difficult and rarely used.
Analytical size-exclusion chromatography	The method is used to separate a diverse range of differently sized particles by passing a solution containing the particles through a partially permeable gel medium. It is used to analyse intermediates (mainly, oligomers) and identify soluble aggregates in tissue [120,121].	No	No	No	Yes	It is impossible to study aggregates with high molecular weight. This method is inefficient for scale-up because size-exclusion chromatography performs poorly on large liquid volumes.
Analytical ultracentrifugation	This technique is based on the sedimentation velocity analysis used to determine the size, shape, and hydrodynamic behaviour of soluble macromolecules, including amyloid fibrils, as well as to study the process of amyloid aggregation [122–125].	No	No	No	Yes	This technique is used only for specific tasks.