Of the three proteins that form amyloid deposits in AD and PD (Aβ, tau, α-Synuclein), more reported efforts have been made towards the development of fluorescent probes for Aβ oligomers, which can be partially attributed to the increased availability of structural models for varying Aβ species. We will first highlight the development of fluorescent probes that can detect all aggregated Aβ species, including oligomers. Many reports on these probes have speculated on potential binding motifs that may allow for targeting Aβ oligomers. Structures of various fluorescent probes that have been reported to detect Aβ oligomers are presented in Figure 2.

Most researchers who have reported on detecting soluble, oligomeric Aβ have employed computer-aided analysis to understand how their fluorescent probes can detect soluble Aβ species. Initial design principles for binding to Aβ oligomers, for instance, were based on structural analysis of fragments of monomeric Aβ. For example, Ran and coworkers hypothesized that interactions with the HHQKLVFF segment on the Aβ peptide would allow for binding to all forms of Aβ, including the more toxic oligomeric species. The HHQK fragment is a hydrophilic segment representative of soluble Aβ species, and the LVFF segment is a more sterically hindered hydrophobic segment. The authors reported on the ability of curcumin analogs, CRANAD-58 and CRANAD-3, to bind to all Aβ species in solution and in the brain of a transgenic mouse model (Zhang et al., 2013; Zhang et al., 2015). Molecular docking studies of CRANAD-58 suggest that the hydrophilic segment of the probe interacts with the hydrophilic part of Aβ allowing for the detection of oligomers, and the hydrophobic moiety of the probe interacts with the hydrophobic components of Aβ allowing for the simultaneous detection of aggregates (Figure 3). Focusing on interactions with an even smaller segment of Aβ, the Wu lab synthesized BF2-dipyrrolmethane fluorescence-imaging probe (NB) (Figure 2) that can detect different aggregation states of Aβ (Quan et al., 2023). The design of NB was based on the detection of diphenylalanine (FF), the smallest unit and core recognition motif of Aβ that plays a crucial role in AD pathogenesis (Görbitz, 2006; Ji et al., 2021).

The emergence of a trimeric Aβ model, Protein Data Bank (PDB) model 4NTR by the Nowick lab (which will be referred to as PDB 4NTR in the remainder of this review), significantly paved the way for developing fluorescent probes with improved specificity to Aβ oligomers (Spencer et al., 2014). A survey of the literature shows that many research groups utilized this model in their computational studies to rationalize the ability of their probes to detect oligomers. This structure is based on a synthetic peptide derived from Aβ_17-36 that crystallizes to form trimers and further aggregates to form other oligomers. This structure contains three β-hairpins that assemble triangularly and interlock to create a trimer, with each β-hairpin making up one side of the pseudo-equilateral triangle (Figure 4). In the center of this pseudo-equilateral triangle appears to be a potential binding pocket for small molecules with hydrophobic F19/V36 residues exposed to solvents. These residues are exclusively exposed to solvent in oligomers and are not present in Aβ fibrils; therefore, several groups have suggested that interaction with these hydrophobic residues can potentially be used to differentially target Aβ oligomers from Aβ fibrils (Lee and Ham, 2011; Teoh et al., 2015; Lv et al., 2016).

Due to the lack of clear, rational design principles for the development of oligomeric-specific Aβ probes, the Chang lab generated a diversity-oriented fluorescence library and performed high-throughput screening of 3,500 compounds (Teoh et al., 2015) to screen for compounds that bound to Aβ oligomers. This study resulted in the identification of BD-Oligo (Figure 2), which exhibited a fluorescence increase at early time points in an Aβ aggregation assay (where it is expected that oligomeric Aβ species will be most prevalent in solution) and a decrease in fluorescence signal at later time points when the solution was expected to contain a high fraction of amyloid fibrils. Transmission electron microscopy (TEM) experiments support the hypothesis that the fluorescence increase could be due to the binding of BD-Oligo to oligomeric species that formed at early time points in this assay. In contrast, the fluorescence decline of BD-Oligo followed the disappearance of oligomers and the formation of fibrils in the sample. Molecular docking studies with BD-Oligo in PDB 4NTR suggest that BD-Oligo adopts a conformational transition from a planar to twisted geometry to increase interactions with the Aβ trimer. In addition, docking studies indicated that the BODIPY ring and phenyl ring interact with F19/V36 (hydrophobic) residues, suggesting these interactions may play a dominant role in the specificity of BD-Oligo to recognize trimers (Lee and Ham, 2011). Despite showing promise for oligomer specificity in solutions containing synthetic Aβ, BD-Oligo was capable of staining Aβ plaques in brain slices from mature 18-month-old APP/PS1 mice, which presumably have mostly fibrillar species. It, therefore, remains to be seen whether BD-Oligo will exhibit the specificity for Aβ oligomers that could reveal new information than what is currently possible in a clinical setting.

In another study, the Pan lab developed JP-1 and JP-2, two curcuminoid derivatives (Figure 2) (Li et al., 2021). Molecular docking studies with PDB 4NTR suggest that interaction with Aβ trimers was due to π-π stacking interactions between the pyrimidine moieties and the benzene rings of three F19 residues in the binding pocket of Aβ trimers (Figure 5). Solution-based studies with synthetic Aβ and staining in a transgenic mouse model confirm the ability of JP-1 and JP-2 to detect all species of Aβ. Interestingly, the authors also observed the ability of JP-1 and JP-2 to inhibit fibril growth, which was confirmed with TEM.

In addition to studying hydrogen-bonding and π-π stacking interactions between probes and F19/V36 residues of PDB 4NTR, another related strategy for designing fluorescent probes that bind to Aβ oligomers is by also taking advantage of probe accessibility and steric hindrance of the Aβ oligomer binding pocket. Many groups hypothesized that creating fluorescent probes with steric bulk would trap or increase probe interactions with the central cavity/binding pocket of Aβ oligomers.

The Li and Wong labs published a study investigating three naphthylamine-based cyanines: DBAN-SLM, DBAN-SLOH, and DBAN-OSLM (Figure 2) (Wang et al., 2021). Of the three probes, DBAN-SLM showed selective fluorescent responses to Aβ monomers and oligomers. Molecular modeling studies with PDB 4NTR suggest interactions between the quinoline moiety of DBAN-SLM and hydrophobic F19/V36 residues. In addition, molecular docking supports that DBAN-SLM conforms to a slightly twisted geometry with the quinolinium ring freely entrapping in the hydrophobic pocket.

Previously, the Yang lab reported on a family of fluorescent probes with an amino naphthalenyl-2-cyanoacrylate (ANCA) motif specific for higher ordered Aβ aggregates (Chang et al., 2011). Drawing inspiration from ANCA and their previous work on spiropyran (SP) derivatives, the Sun and Yi labs designed AN-SP, which incorporated an SP moiety into the ANCA scaffold (Lv et al., 2016). AN-SP showed the most considerable fluorescence enhancement with Aβ oligomers, and very little fluorescence enhancement was observed in the presence of Aβ fibrils, amylin fibrils (an aggregation-prone peptide secreted with insulin), and prion fibrils (another amyloid associated with prion disease) (Luca et al., 2007; Colby and Prusiner, 2011). The authors hypothesized that this specificity towards oligomers might be due to the SP moiety providing rigidity and steric hindrance, precluding binding to fibrillar binding pockets. To test this hypothesis, the authors also synthesized AN, which replaced the SP moiety with a flexible chain, and found that AN showed no specificity between Aβ oligomers and fibrils. Molecular docking of AN-SP to PDB 4NTR suggested that the SP unit intercalates with the hydrophobic region of the Aβ trimer. Staining in a transgenic mouse line showed the ability of AN-SP to stain Aβ oligomers in the brain.

Similarly, the Li group reported on PTO-29 (Figure 2), which exhibited high selectivity to Aβ oligomers (Yang et al., 2020). The authors described the binding pocket of PDB 4NTR as “v-shaped” and hypothesized that a “v-shaped” wedge probe would allow for increased interactions with F19/V36 residues. Molecular docking studies of PTO-29 suggest that the phenyl rings of PTO-29 stacked between F19 and the N, N-dimethylbenzene inserts into the protein cavity, which suggests tighter binding affinity to trimers. To improve the biocompatibility of PTO-29, the same group modified PTO-29 to include a hydroxyethyl group as in PTO-41. Computational studies with PDB 4NTR suggested that the hydroxyethyl group is inserted into the binding pocket, presumably leading to increased affinity towards trimers compared to probes that lacked this hydroxyethyl group. In vivo imaging of PTO-41 in a 4-month transgenic mouse, presumably containing a higher proportion of Aβ oligomers, suggests that PTO-41 can detect Aβ oligomers.

In addition to studying interactions between probes and F19/V36 residues of the Aβ trimer, another strategy employed by researchers involved taking advantage of probe accessibility and differences in binding pocket sizes of Aβ species. A comparison of the binding pockets of Aβ oligomers (PDB 4NTR) with Aβ fibril models (PDB 2LMN) suggests that the size of the binding pocket for higher-ordered aggregates is narrower compared to the binding pocket of oligomers. For example, the Ran lab tuned for stereo-hindrance by introducing bulky functional groups that would prevent binding to the more sterically hindered Aβ fibrils and allow for the detection of oligomers. Building off previously reported CRANAD-3, the authors synthesized CRANAD-65, CRANAD-102, and CRANAD-75, presented in order of increased steric bulk, to test this strategy (Li et al., 2017) (Figure 6). The authors incorporated various phenoxy-alkyl chains at the 4-position to introduce steric bulk. CRANAD-75 showed no fluorescence enhancement with oligomers, and the authors hypothesize that the two isopropyl groups on the phenyl ring of CRANAD-75 were too large to access the β-sheets of the oligomers. Therefore, with CRANAD-102, the authors replaced the isopropyl groups in CRANAD-75 with methyl groups and showed a higher affinity for monomers, dimers, and oligomers than higher ordered Aβ species in solution.

While some progress has been made toward developing fluorescent probes that can detect oligomeric species of Aβ, fluorescent probes with improved specificity and biological properties are still needed. In addition, currently there is no clear identification of molecular determinants that dictate the binding of probes to Aβ oligomers. Recent developments suggest the utility of computer-aided design of molecules for improved predictability for fluorescent probes that preferentially bind to soluble Aβ species. While some research groups have focused on targeting fragments of the Aβ peptide sequence, studies suggest that using this approach is limiting for the development of oligomer-specific probes as these fragments are present in all Aβ species. While PDB model 4NTR has shown promise as a valuable structural model for developing probes that bind to oligomeric Aβ, we caution the readers that structures based on synthetic peptides or recombinantly expressed amyloids have been observed to produce different structures from those reported in patient-derived samples (Kollmer et al., 2019; Varshavskaya et al., 2022). Many research groups have highlighted the importance of probe interactions with F19/V36 on this structural model as these residues are not exposed in fibrils, allowing for potential specificity. In addition, groups have also implemented strategies based on steric hindrance/bulk and show that the increased size of the oligomer binding pocket compared to the binding pocket in Aβ fibrils can play a useful role in the potential design of an oligomer-specific probe.

Evidence in literature suggests that zinc contributes to tau toxicity by increasing tau phosphorylation and directly binding to tau (Boom et al., 2009; Sun et al., 2012; Huang et al., 2014; Fichou et al., 2019). Therefore, one approach that many research groups have attempted for detecting phosphorylated tau oligomers is to use a Zn²⁺ recognition motif.

The Hamachi group, for instance, reported the detection of soluble tau oligomers by developing fluorescent probes that targeted protein hyperphosphorylation (Ishida et al., 2009; Ojida et al., 2009). Ojida et al. reported on Probe 1 (Figure 7) with a BODIPY core and two Zn(II)-2,2′-dipicolyamine (DPA) complexes that serve as a binding site for phosphorylated amino acid residues. DPA contains nitrogen atoms that can coordinate with Zn²⁺ to form a chelate resulting in a change in the fluorescence properties of DPA probes. Probe 1 showed a high sensitivity for hyperphosphorylated full-length tau over non-phosphorylated tau and Aβ fibrils in solution. Histological staining showed that Probe 1 can bind to NFTs in brain sections from a confirmed AD patient; however, further studies are needed to test if Probe 1 can also detect phosphorylated tau oligomers in tissue. Ishida et al. later developed diazastilbene analogs 1-2Zn(II) and 2-2Zn(II) (Figure 7), which showed a fluorescence change when bound to phosphorylated tau peptides in solution. However, studies with 1-2Zn(II) and 2-2Zn(II) were limited to in vitro examination; therefore, it remains to be seen whether these probes can label phosphorylated tau oligomers in biological samples.

The Bai lab reported on CyDPA0, CyDPA1, and CyDPA2 (Figure 7). These probes have one or two DPA-Zn(II) complexes and are designed based on indocyanine green, a near infra-red dye (Kim et al., 2013). Of the three probes, CyDPA2 showed the best affinity to recombinant phosphorylated tau. In gel staining of CyDPA2 with homogenized samples from an AD patient, a transgenic mouse that overexpresses tau, and recombinant phosphorylated and non-phosphorylated tau proteins supported the ability of this probe to detect hyperphosphorylated tau oligomers (Ramsden et al., 2005; Guerrero et al., 2009). Ex vivo staining of brain homogenates from a tau transgenic mouse and a confirmed AD patient with CyDPA2 supported fluorescence signals corresponding to only tau species and showed a reduction in fluorescent signals with the addition of pyrophosphate (ppi), a phosphate inhibitor.

Fluorescence lifetime imaging (FLIM) has gained increasing attention as a new microscopy technique used in biological research. When a fluorophore is excited with light, there is a time delay before these probes relax to the ground state and release a photon and this time delay is called the fluorescence lifetime (τ). FLIM images of phosphorylated tau by a cyanine probe (τ-p-tau, Figure 7) was reported by the Tian lab (Ge and Tian, 2019). Similar to the previous groups, the authors used two DPA units to recognize Zn²⁺ ions. τ-p-tau was shown to be selective for phosphorylated tau over solutions of monomeric tau, Aβ monomer, and Aβ fibril. FLIM imaging of a single neuron from a mouse model showed that τ-p-tau was able to monitor (via changes in lifetime) the increase in the concentration of phosphorylated tau in live cells that were pre-incubated with Okadaic acid (OA), which induces hyperphosphorylation (Figure 8). However, evidence that this probe can detect tau oligomers specifically was not reported.

In addition to attempts at targeting tau oligomers using a Zn²⁺ recognition motif, another design strategy involves modifying fluorescent probes previously reported to bind to NFTs. The Nilsson lab reported on a series of oligothiophene-based fluorescent ligands that bind to Aβ and tau (Klingstedt et al., 2013). These analogs were designed with minor variations in their chemical structure to restrict or increase the conformation flexibility of the conjugated backbone to allow for the detection of a variety of amyloids. Of the probes reported, pFTAA (Figure 7) detected both NFTs and Aβ aggregates in AD patient samples. While pFTAA did not exhibit any apparent selectivity for various aggregate forms of Aβ and tau (soluble or insoluble), the Aigbirhio lab drew inspiration from pFTAA and made chemical modifications to develop pentathiophene-trifluoroethanol (pTP-TFE, Figure 7), a fluorescent probe that showed selectivity towards soluble, aggregated tau (Y. Zhao et al., 2020). The authors hypothesized that anionic dyes such as pFTAA interact with the positively charged flexible polyelectrolyte brush, or fuzzy coat, that is found on mature tau fibrils. Therefore, to minimize interactions with tau fibrils, the authors replaced the carboxylic acids on pFTAA with 2,2,2,-trifluoroethan-1-ol groups to generate pTP-TFE. pTP-TFE showed preferential binding to early soluble tau aggregates in solution, which was supported by TEM imaging experiments. The authors then tested the ability of pTP-TFE to bind to soluble tau in a confirmed AD patient and a confirmed patient with progressive supranuclear palsy (PSP), another ND that involves tau, and found that pTP-TFE colocalized with an antibody that detects phosphorylated tau in both patients (Figure 9).

The Yu lab designed tau-selective and aggregation-induced-emission probes, JL-3 and JL-6 (Figure 7) (Ji et al., 2023). JL-3 and JL-6 have a triphenylamine (TPA) electron-donating unit conjugated with -CN and N, N′-dimethylaminobenzene groups to form a Donor-Acceptor-Donor scaffold. Both probes showed the ability to discriminate between tau and Aβ aggregates; however, JL-3 and JL-6 are not oligomer-specific and bind to tau fibrils as well.

While universal rational design principles for targeting tau NFTS do not currently exist, the Kim lab showed that extending the π-network of Aβ probes enabled binding to tau NFTs (Verwilst et al., 2017). Tau 1 and Tau 2 (Figure 7) showed preferential binding to tau NFTs over Aβ fibrils in a transgenic mouse model. Molecular docking studies suggested that these probes interacted with amino acid residues on tau along the crystallographic structure of hexapeptide PHF6 fragment ³⁰⁶VQIVYK³¹¹ (Fitzpatrick et al., 2017). This hexapeptide was identified as responsible for nucleation of tau and was used in computational studies to study binding to NFTs. To target phosphorylated tau, the Boffi lab drew inspiration from Tau 1 and further extended the π conjugation on position 3 of the BODIPY core to synthesize a family of fluorescent probes (Figure 7) (Soloperto et al., 2022). Docking to a published structure of aggregated PHF6 fragment showed that all probes interacted with tau and were docked into the central cavity of the binding site, suggesting a preference for lipophilic regions of tau. Of the probes that were designed, BT1 showed the ability to bind to hyperphosphorylated and oligomeric tau in OA-treated human-induced Pluripotent Stem cells (hIPSC) derived neurons.

This section discusses recent progress and attempts at the rational design of probes that detect various tau species, including hyperphosphorylated tau oligomers. A survey of the literature suggests that there are still no clear molecular features that can be used for designing probes that specifically target soluble tau oligomers. However, one common approach for developing probes that can detect tau oligomers includes a Zn²⁺ recognition motif such as DPA, which allows for the detection of all tau species, as non-pathological tau and NFTs are also phosphorylated to a various extent. Another strategy for designing oligomeric tau targeting fluorescent probes involves making chemical modifications on probes previously reported to target NFTs; however, to the best of our knowledge, the specific detection of oligomeric species of tau in tissue has yet to be achieved and it remains to be seen whether these probes will show specificity in tissue. As with developing probes that target Aβ oligomers, we hypothesize that incorporating computer-aided design would allow researchers to pre-screen probes in silico, which could help advance the field in developing tau oligomer-specific fluorescent probes. While protein models for higher-ordered tau aggregates from AD patients exist, a protein model of hyperphosphorylated tau oligomers has not yet been reported (Fitzpatrick et al., 2017). A model for phosphorylated tau oligomers would be of great utility; however, until one is available, a pre-screening of molecules that can bind to the published structure of aggregated PHF6 fragment of tau may represent a reasonable starting point.

Numerous probes have been developed to detect α-synuclein deposits in tissue (Xu et al., 2020; Haque and Maity, 2023). Although most of these probes show improved binding affinity for α-Synuclein compared to the micromolar affinity of ThT, they do not distinguish the different aggregation states among α-Synuclein species (e.g., monomer vs. oligomer vs. fibril). Despite the lack of aggregation specificity, these probes remain valuable as the foundation for the development of future α-Synuclein oligomer-specific probes. For more information regarding α-Synuclein probe development in general, we refer readers to other reviews that summarize reported α-Synuclein binding fluorescent probes (Xu et al., 2020; Haque and Maity, 2023). In this section, we will review the recent α-Synuclein fluorescent probe development, with a highlight on oligomer-binding probes.

Due to recent revelations about α-Synuclein oligomer toxicity and aforementioned difficulties in developing oligomer-specific α-Synuclein probes, only a few papers in recent years have reported on probes that bind to α-Synuclein oligomers. Most of these probes are phrased as aggregation monitoring probes, which means they bind to diverse α-Synuclein aggregate species as their photophysical properties change during the monomer to fibril aggregation process. One such example was developed by the Jovin group where they screened derivatives of the hydrophobicity-sensitive fluorescent probe N-arylaminonaphthalene sulfonate (NAS). (Celej et al., 2008). As α-Synuclein aggregates, the polarity of binding sites putatively changes; therefore, a polarity-sensitive probe can monitor changes in aggregation states. By incubating NAS derivatives with monomeric α-Synuclein, the researchers found several molecules that exhibited fluorescence enhancement during aggregation. One such molecule, bis-TNS (Figure 10), also showed a shift in its emission. Other photophysical properties were also measured, and they found two molecules, bis-TNS and bis-ANS, that report changes in fluorescence lifetime (τ) as the aggregation process proceeds. The researchers claim that such changes in emission wavelength and τ could be used to show different aggregation states and binding to diverse α-Synuclein aggregation species. The Jovin group further explored probes that have different properties during aggregation and developed a 3-hydroxychromones (3HC) probe AS140-MFC (Yushchenko et al., 2010). AS140-MFC is prepared by introducing sensor molecules with covalent adducts of Ala-to-Cys mutants of α-Synuclein with a thiol-reactive maleimide probe (MFC) (Figure 10). AS140-MFC probe has two excited states, and when incubated with α-Synuclein, the ratio of these two states changes due to polarity differences which reflect the transition of aggregation states. A similar approach for finding α-Synuclein oligomer probes through enhancing hydrophobic interactions has been adopted by other groups as well. The Yarmoluk group published a series of tri- and pentamethine cyanine probes with bulky phenol and alkyl groups and tested binding with different α-Synuclein aggregates in solution (Kovalska et al., 2012). Of the reported probes, SL-631 and SH-299 (Figure 10) showed strong fluorescence enhancement when bound to α-Synuclein oligomers and less enhancement for fibrils in solution. Despite these two probes showing promise for detection of α-Synuclein oligomers, it remains to be seen if they can detect α-Synuclein oligomers specifically in tissue.

A common strategy to develop fluorescent probes for protein aggregates is aggregation-induced emission (AIE). These molecules show weak fluorescence when unbound in solution but become fluorescent upon interacting with targets (Hong et al., 2009; Z; Zhao et al., 2020). Tetraphenylethene tethered with triphenylphosphonium (TPE-TPP, Figure 10) was designed based on known π-stacking and hydrophobic interactions between ThT and higher-ordered amyloid aggregates. TPE-TPP was incubated during α-Synuclein aggregation and showed fluorescence enhancement at earlier time points compared to ThT, indicating potential binding with α-Synuclein oligomers. Comparison with immunoblotting of TPE-TPP with an α-Synuclein oligomer antibody also confirmed oligomer binding. This probe was later tested with other aggregated proteins derived from α-lactalbumin, κ-casein, and hen egg white lysozyme (HEWL), showing potential to be an aggregation monitoring probe for various amyloidogenic proteins in addition to α-Synuclein (Kumar et al., 2017).

Since ThT is a traditional amyloid dye to detect aggregates, researchers have used it as inspiration for developing oligomer-specific probes. One such ThT derivative, ThX, was shown to bind to α-Synuclein oligomers by showing early fluorescence enhancement during aggregation of α-Synuclein in solution. (Yap et al., 2011). Further studies suggested ThX might bind to heterogeneous α-Synuclein oligomers not detected by ThT.

Few reports have been made towards detection of α-Synuclein oligomers in biologically relevant conditions such as tissue or cell culture. Inspired by GFP, a widely used genetically encoded fluorescence protein, the Zhang group created probes based on GFP’s fluorescent chromophore 4-hydroxybenzylidene-imidazolinone (HBI, Figure 10). (Liu et al., 2018) The Zhang lab synthesized derivatives of HBI to improve aggregated protein detection by increasing π conjugation, restricting bond rotation, and substituting the phenol group with electron-donating substituents. The probes were incubated in solution with α-Synuclein and fluorescence intensity increase was detected early in the aggregation process, which the authors suggest is due to binding to α-Synuclein oligomers. The authors then incubated the HBI derivative probes in cell culture and visualized Huntingtin exon 1 protein (Htt) and superoxide dismutase 1 (SOD1) aggregation in vivo. Although the ability of HBI derivatives to detect α-Synuclein was not tested in cell culture, this work showed the potential of fluorescent probes to visualize and study the function of oligomers in a living system.

The development of fluorescent probes for oligomeric α-Synuclein has gained attention from many researchers in recent years as studies show that α-Synuclein oligomers are more toxic to neurons and are important in the pathology of many synucleinopathies. Currently, most design routes are based on existing probes for higher-ordered α-Synuclein aggregates, and the focus has been to monitor the α-Synuclein aggregation process rather than an oligomeric-specific probe. These aggregation-monitoring probes bind to both oligomeric and higher-ordered α-Synuclein aggregates but can also respond earlier during the aggregation process or show slightly different photophysical properties when bound to oligomers versus fibrils in solution. These probes have advanced our understanding of α-Synuclein oligomers, but they still lack binding specificity and significant contrast between different states of α-Synuclein aggregate species. Furthermore, most of the probes reported have only been tested with in vitro assays with minimal validation of the presence or characterization of α-Synuclein oligomers in these experiments.

Moving forward, many directions can be taken for developing α-Synuclein oligomer-specific probes. Chemical modifications to probes that detect higher-ordered α-Synuclein aggregates represents an actionable starting point. Furthermore, chemical modifications in tandem with computer-aided design and pre-screening of fluorescent probes can further advance the field. While models for α-Synuclein oligomers do not currently exist, structures of α-Synuclein fibrils have been reported and may allow for development of rational design principles for targeting α-Synuclein in general (Guerrero-Ferreira et al., 2018). In addition, hydrophobicity differences between various aggregation states of α-Synuclein may allow for new design principles for chemical probes that specifically detect α-Synuclein oligomers (Lee et al., 2018).