Introduction

Parkinson’s disease (PD) has emerged as the second major neurodegenerative disease that threatens people’s health in recent years. Its clinical manifestations are mainly motor symptoms such as resting tremor, bradykinesia, tonicity, and non-motor symptoms including olfactory loss, sleep disturbance, and gastrointestinal complications, which have a significant impact on the patient’s daily life [1, 2]. Since the pathogenesis has not been completely grasped and no efficient treatments for PD, all current preventive and therapeutic measures are merely helpful to delay the PD progression. Therefore, it is essential to seek strategies for PD early diagnosis and treatment.

In 1985, the discovery of amyloid-beta (Aβ) motivated researchers to investigate the molecular pathogenesis of AD proteins, which were discovered as the key proteins causing neurodegenerative diseases, along with microtubule-associated protein tau (Tau) and α-Syn. α-Syn is the main component of intracellular fibrillar Lewy bodies (LBs) and Lewy neuritis (LN). It can trigger nigral dopaminergic neuron degeneration and motor symptoms (Fig. 1a). Misfolded α-Syn can form protein oligomers, fibrils, and neuronal deposits with rich β-sheet structures and accumulate in patients’ cerebral cortex and hippocampus, causing memory and motor deficits. However, identifying the above pathological process in the early stages of PD is difficult [3, 4]. Therefore, the early detection of misfolded α-Syn is essential to control PD. Compared with changes in dopaminergic or vesicular monoamine transporter protein 2 (VMAT2), misfolded α-Syn has been illustrated that is the preferential evidence of PD before motor symptoms emergence [5, 6]. Currently, there have been numerous studies on α-Syn conformational changes and the pathogenesis and early diagnostic techniques of PD. In this review, we will summarize and prospect the relevant in vivo and in vitro studies to provide potential solutions for the early diagnosis and treatment of PD via real-time tracking of α-Syn (Scheme 1).

Fig. 1
figure 1

a α-Syn aggregates in neurons. b The primary sequence and three distinct regions of α-Syn

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Scheme 1
scheme 1

In vivo and in vitro detection for alpha-synuclein

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Physiological functions, structures, and characterization of α-Syn

α-Syn is a 140 amino acid (AA) presynaptic terminal protein encoded by SCNA genes. It is a soluble monomer in the physiological state and participates in cell membrane interactions, synaptic function, vesicle transport, and dopamine synthesis, storage, and release in the central nervous system (CNS) [2, 7]. α-Syn consists of three structurally distinct physiological functional regions (Fig. 1): the N-terminal region (AA1-60) has mutation sites and contains four incomplete repetitive sequences that can participate in membrane binding interactions and promote the formation of β-sheet structures; the central NAC hydrophobic region (AA61-95) is highly involved in the formation of β-sheet structures and the production of insoluble LBs and LNs; the C-terminal region (AA96-140) is full of proline, which is highly acidic and can engage in post-translational modifications (PTMs) of proteins to regulate their function.

α-Syn is a pathogenic biomarker of neurodegenerative illnesses such as PD, multiple system atrophy (MSA), and dementia with Lewy bodies (DLBs) and accounts for barely 1% of total brain protein content [6, 8,9,10]. The mechanism of α-Syn aggregation in the brain has not been clarified, and when intracellular homeostasis is disrupted, its non-structural monomer and sub-stable α-helical tetrameric forms will oligomerize, and form toxic oligomers, fibrils, and deposits with different intermediate conformations through PTMs, which are difficult to distinguish from other amyloid proteins [3, 8, 11, 12]. Studies have revealed that the preformed α-Syn conformational changes followed an S-shaped aggregation growth mechanism during the early stage of fibrillization [13], and self-conjugation interactions of α-Syn oligomers in the late stage of aggregation would accelerate the formation of fibrils and large-sized aggregates [9, 14]. Thioflavin-T (ThT) [11] exhibits different binding sites and binding patterns when generating fibrils with two polycrystalline α-Syn mutants (A30P, A53T), which indicates that there are non-single binding sites on the surface of α-Syn fibrils and lead to their conformational changes [8] (Fig. 2). This shows that it is difficult to identify α-Syn from other amyloid proteins because of its low content and a high degree of conformational plasticity in the brain. Therefore, the early dynamic detection of α-Syn conformation and content in vivo is necessary for early diagnosis and treatment of PD.

Fig. 2
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Confocal microscopy images of α-Syn amyloid fibrils. Reprinted with permission from ref. [8]

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Analytical methods commonly used to characterize α-Syn are mainly spectroscopy, microscopy, mass spectrometry (MS), electrophoresis, etc. Circular dichroism (CD) [15], fluorescence correlation spectroscopy (FCS) [16], Raman spectroscopy [17], atomic force microscopy (AFM) [18,19,20], and single-molecule total internal reflection fluorescence microscopy (TIRFM) [21] were used to monitor aggregation mechanism of recombinant α-Syn in vitro, allowing greater determination and sensitivity in samples such as cerebrospinal fluid (CSF) to obtain the α-Syn conformational changes and its secondary or tertiary structures. MS has high sensitivity and specificity for separating low molecular weight protein samples based on the mass-to-charge ratio upon vaporization and ionization. However, the method is less repeatable for protein separation with the same mass-to-charge ratio, poor hydrophobicity, and interrupted intermolecular interactions [22].

By using several MS methods, Rebecca’s group [23] has implemented a conformational analysis of α-Syn and apolipoprotein C-II (ApoC-II). Hydrogen–deuterium exchange mass spectrometry (HDX-MS), electrospray ionization mass spectrometry (ESI–MS), and ion mobility mass spectrometry (IM-MS) are sensitive to intrinsically disordered protein (IDPs) and can distinguish the disordered and structured proteins, confirm the disordered nature of α-Syn and ApoC-II, and reflect their conformational dynamics information. Payel et al. [24] detected different synaptic nuclear protein variants and quantified the α-Syn truncated at amino acid 119 and the acetylated N-terminal α-Syn from cingulate cortex and occipital cortex of control and PD patients by using liquid chromatography and mass spectrometry (LC–MS/MS) with differential solubilization, immunoprecipitation, and targeted proteomics approaches to analyze α-Syn. The results showed that the other α-Syn variants levels except for α-Syn103−119 (Ac-α-Syn1−6, α-Syn13−21, α-Syn35−43, α-Syn46−58, α-Syn61−80, and α-Syn81−96) in PD patient’s cingulate region and Ac-α-Syn1−6 in the soluble fraction were significantly higher than the controls, and whether acetylated α-Syn can be used as a potential biomarker for PD needs to be further investigated. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) combined with Western blotting and size exclusion chromatography (SEC) can detect protein aggregates based on antigen–antibody reaction and molecular sieve effect [25]. Furthermore, applying an electrical potential to the capillary, the capillary electrophoresis (CE) technique can efficiently separate the amyloid proteins such as Aβ and overcome the effects of SDS and gel smears. CE needs to be further improved to increase the resolution of amyloid protein aggregates and has the potential for early oligomers detection like Aβ and α-Syn [26]. Online nucleic acid aptamer affinity solid-phase extraction capillary electrophoresis–mass spectrometry (AA-SPE-CE-MS) [27] can improve the shortcomings of traditional CE-MS, which has a small injection volume, easily affected by sample concentration, and realize the purification, separation, and identification of α-Syn in blood by specific binding of single-stranded DNA aptamer with low detection limit and sensitivity to acidic and alkaline environment, which will be used in the clinical monitoring of α-Syn in PD patient’s brains. Microfluidic capillary electrophoresis (MCE) [25] and affinity capillary electrophoresis (ACE) [26] can improve the migration rate of proteins by using binding agents (antibodies, aptamers, serum proteins, etc.), further improving protein separation with the advantages of lower sample consumption and higher automation potential.

In vitro approaches for the detection of α-Syn

Immunohistological results of autopsy brain sections can be used to diagnose and reflect the severity of PD [9, 28]; the method is often difficult to accept by patients’ families and is commonly characterized by sampling errors. Therefore, a more cost-effective sampling method to detect α-Syn is required. Braak et al. proposed a hypothesis [7, 29,30,31,32] that microorganisms of the gastrointestinal can conduct immunological and endocrine signaling through the gut-brain axis; it was suggested that α-Syn could spread from the enteric nervous system (ENS) to CNS when exposed to environmental and genetic damage. Studies showed that α-Syn has been found in the tissues of patients’ skin [33,34,35,36,37,38,39,40], gastrointestinal tract [41, 42], retina [43, 44], and body fluids such as blood [45, 46], CSF [47,48,49], saliva [50,51,52], and urine [53] (Fig. 3). In recent years, some analytical methods for detecting α-Syn and its conformation in the actual human biological samples have been developed, which will facilitate the early diagnosis and prevention of PD.

Fig. 3
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The biological samples with α-Syn

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Detection of actual human biological samples

Immunofluorescence (IF) staining based on antigen–antibody recognition, enzyme-linked immunosorbent assay (ELISA), and electrochemical sensors are commonly used to detectα-Syn in human biological samples such as skin tissues and body fluids. Giorgia et al. [35] performed immunofluorescence analysis on skin tissue from PD patients’ ankle, thigh, and neck; the results revealed that α-Syn formed positive deposits with 5G4 antibodies in each tissue’s dermal nerves and the degree of neck skin degeneration increased with the duration of the disease. Sharon’s team [54] detected total α-Syn of 16.7 ± 5.9 ng/ml and 55.9 ± 10.3 ng/ml in CSF and saliva samples by using phospholipid ELISA, whereas the traditional sandwich ELISA recorded 1.2 ± 0.4 ng/ml in CSF samples and could not achieve α-Syn detection in saliva. This approach was more sensitive than sandwich ELISA. For most peptides and proteins, IF and ELISA have the advantage of rapid analysis speed, low radiation, and high specificity, but lack international standard dyeing, and are vulnerable to personnel operation skill, body gland secretion, type and quantity of protein, protease inhibitors, and category of kits, restricting their accurate detection of α-Syn in vitro. Currently, there are electrochemical sensors mainly to detect Aβ, and fewer available to detect α-Syn, such as polyethylene glycol gold surface electrode sensors [55] based on antigen–antibody recognition, graphene oxide sensors [56], nucleic acid aptamer sensors [57, 58], indium tin oxide electrode sensors [59], etc. [60,61,62] (Fig. 4), have lower detection limits and higher specificity and sensitivity for α-Syn in vitro and are simple, portable, and mass-produced. However, their further clinical applications are restricted by the variety of expensive electrode materials, complex structural modifications, and serious biological contamination.

Fig. 4
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Electrochemical biosensors for α-Syn detection. a Schematic illustration of graphene oxide–modified biosensor to detect α-Syn autoantibodies. Reprinted with permission from ref. [56]. b Schematic illustration of the electrochemical aptamer sensor to detect α-Syn oligomer. Reprinted with permission from ref. [57]. c Schematic representation of the indium tin oxide electrode sensors to detect α‐Syn. Reprinted with permission from ref [59]

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Detection of recombinant α-Syn in vitro

α-Syn aggregates

Currently, there are many emerging techniques applied to detect α-Syn such as fluorescence imaging techniques and real-time quaking-induced conversion (RT-QuIC). Fluorescence imaging techniques [63,64,65] can better target the protein and generate strong fluorescence responses, as a potential technique for finding amyloid in vivo and in vitro without using any radionuclides, with lower injury, higher sensitivities, lower costs, detection limits, and faster elimination in vivo. ThT (Table 1) is a benzothiazole molecule based on the intramolecular charge transfer (TICT) mechanism to form a rigid molecular structure upon binding the β-sheet structure of amyloid proteins and generates fluorescence based on the energy released by the n-π* transition. Although the ThT method is the “gold standard” for detecting amyloid fibrils in vitro and a tool for recognizing their aggregation and restraint mechanisms [66, 67]. ThT is difficult to realize α-Syn detection because it lacks specificity and low sensitivity, has susceptibility to interference by sample background fluorescence signal and inability to penetrate the blood–brain barrier (BBB), and easily quenches after aggregation. Therefore, it is necessary to further develop other types of fluorescent probes to detect α-Syn aggregation.

Table 1 The structures and characteristics of ThT, 2,6-ANS, 2,6-TNS, bis-ANS, bis-TNS, C15H15N3 (C1), C16H16N3O2 (C2), RB1, RB2, ThX, PAP_1, curcumin, EGCG, ASCP, Anle138b, 11b, SL-631, SH-299, DCVJ, TPE-TPP, JC-1
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Thomas M’s group presented four N-arylaminonaphthalene sulfonates (NAS) derivatives, 2,6-ANS, 2,6-TNS, bis-ANS, and bis-TNS, as universal probes for the detection of amyloid protein; bis-ANS and bis-TNS are the most sensitive probes to amyloid proteins. Compared with ThT (Kd = 14.9 M), the structure of NAS was more susceptible to α-Syn, with Kd values of 8.8, 11.7, 8.6, and 11.6 M, their charged properties and emission wavelength preventing them from being used as in vivo imaging probes for α-Syn [68]. Two fluorescence probes based on the benzothiazole structure, C15H15N3(C1) and C16H16N3O2(C2), can detect α-Syn at submicromolar concentrations. C1 and C2 do not interfere with α-syn aggregation and have lower detection limits and higher sensitivity than THT. They tend to bind α-Syn aggregates in SH-SY5Y cells with little cytotoxicity, so they are potential probes for detecting α-Syn [69]. Gorka et al. [70] found that the trochanteric thiazole molecules RB1 and RB2 have an extended π system, the molecular size of RB1 is between RB2 and ThT, and it has a higher affinity with α-Syn aggregate. Compared to ThT, the maximum absorption of RB1 combined with α-Syn aggregates was red-shifted at 76 nm, and the fluorescence intensity was increased by 112 times. So water-soluble RB1 could as a potential probe detect α-Syn aggregates in living cells, which can overcome strong background signals in living cells and stain the cytoplasm of HeLa and SH-SYY cells.

Needham’s group [71] created a new generation of probe ThX with methoxy and dimethylamino groups and PAP_1 based on the ThT. ThX contains a higher density of electron clouds surrounding the C (sp3)-N bond, restricted rotation, and improved lipophilicity. ThX is suitable to detect α-Syn aggregates in high-background biological samples such as plasma and cerebrospinal fluid, because it can recognize α-Syn oligomers that ThT is unable, and the binding affinity and brightness of wild reconstituted α-Syn aggregates are increased by 7 times and 5 times. Although the PAP_1 probe was 100 times less brighter than ThT, it had a greater binding affinity for α-Syn aggregates and could image single recombinant α-Syn aggregates in brain tissue [72, 73]. Real-time quaking-induced conversion (RT-QuIC) [62, 74,75,76] can combine with ThT to rapidly detect α-Syn with low concentrations in vitro. However, due to the influence of sample size, concentration, temperature of α-Syn, and other conditions, the sensitivity and specificity of this method in the clinical application need to be further verified.

Monomers, oligomers, and fibrils of α-Syn

Compared with other conformations, α-Syn oligomers have a stronger neurological damaging effect on the brain and can cause neuronal injury and death. Therefore, it is essential to detect α-syn oligomers and inhibit production. Curcumin [77] may hasten the transition of α-Syn from oligomers to fibrils and decrease neurotoxicity by reducing the number of oligomers. Epigallocatechin gallate (EGCG) [78] can preferentially bind α-Syn fibrils and form them into novel amorphous and smaller aggregates. As a result, probes or inhibitors with curcumin, EGCG, and Anle138b [79] structures can intervene early in the α-Syn aggregation process. BE01, BE02, mBE01, and mBE02 [80] are four highly sensitive bifunctional fluorescence probes with higher fluorescence intensity than ThT (Fig. 5a) and simultaneously detect α-Syn and H2O2. The non-methylated structure of BE01 and BE02 binds α-Syn with lower bonding constants, allowing the detection of a single α-Syn fibril as shown in Fig. 5b. In addition, phenothiazine derivative 11b showed a high affinity for α-Syn fibrils and will be expected as a radioactive ligand to target α-Syn in vivo after further characterization [81].

Fig. 5
figure 5

a Synthetic scheme for the dyes of BE01, BE02, mBE01, and mBE02. b Schematic illustration of the fluorescence intensities of the BE, mBE, and ThT probes in PBS buffer, with H2O2, with α-Syn aggregates and with concomitant H2O2 and α-Syn aggregates. Reprinted with permission and adapted from ref. [80]

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A few fluorescence probes with different phenyl, alkyl, and halogen substitutions and a large aromatic conjugated system can bind α-Syn monomers and oligomers, be used as its in vitro potential imaging probes, and accumulate experience for further development of in vivo imaging probe technology. By selecting target α-Syn oligomers and fibrils, respectively, the aromatic dyes pentamethyl anthocyanine SL-631 and trimethyl anthocyanine SH-299 [82] further enhance the detection limit of α-Syn fibrils. DCVJ probe [83] binds α-Syn oligomers with 2–4 times higher fluorescence intensity than that of bind monomers or fibrils. JC-1 carbocyanine [84] can distinguish α-Syn monomers and fibrils by emitting lights of different colors and has a lower detection limit for α-Syn monomers.

Additionally, aggregation-induced emission (AIE) probes and dyes [85, 86] were developed and applied for α-Syn detection. The methylpyridine and dimethylamine in the ASCP structure [87] improve the ability of electron-π conjugate expansion, share the ThT binding site, and have a larger Stokes shift (145 nm), which can replace ThT to research α-Syn fibrils growth in vitro. TPE-TPP [88] is a sensitive probe for identifying α-Syn monomers, oligomers, and fibrils with higher affinity, fluorescence intensity, and lower signal-to-noise ratio. It can compete with ThT on the binding site of α-Syn fibrils; however, its selectivity and affinity for other proteins are unidentified. Currently, the emerging fluorescence probes and RT-QuIC methods only achieved the detection of in vitro recombinant α-Syn; it is necessary to improve these methods to realize the detection of α-Syn in actual human samples and in vivo.

Compared with in vitro recombinant α-Syn fibrils, various α-Syn conformations in the human brain and actual human biological samples are significantly different in composition and cellular environment; thus, the recombinant α-Syn cannot completely simulate the in vivo aggregation of α-Syn. Therefore, it is necessary to develop in vivo detection techniques for α-Syn.

In vivo methods for the detection of α-Syn

At present, compared with in vitro detection techniques, radiographic positron emission tomography (PET), single photon emission computed tomography (SPECT), and non-radiographic magnetic resonance imaging (MRI) can achieve in situ detection of α-Syn in vivo. These methods accurately reflect abnormal metabolic changes of the brain and anatomical information to achieve early diagnosis and identification of brain diseases such as PD.

Radiative PET ligands for imaging α-Syn

PET and SPECT are less invasive and have higher specificity, sensitivity, and spatial resolution, allowing labeling biological samples such as enzymes or proteins by radionuclides (20 and 110 min) with short half-lives and high safety including 11C, 18F, 123I, or 99Tc and tracking imaging at the nanoscale [89, 90].

At present, only the US Food and Drug Administration (FDA)–approved PET probes [18F]Florbetapir [91], [18F]Florbetabeben [92, 93], and [18F]Flutemetamol [94,95,96] can be used to detect Aβ, while fewer PET ligands can target α-Syn, and have not been imaged in vivo. Although the ligands [11C]PiB [97], [18F]BF227 [98, 99], and [11C]-PBB3 [100] are selective for α-Syn recombinant fibrils in vitro, more easily bound to Aβ and Tau fibrils, which are highly abundant in human brain homogenates and have similar β-sheet structure rather than α-Syn fibrils [101]. Therefore, it is crucial to develop high-sensitivity PET ligands that target α-Syn to detect α-Syn fibrils in vivo at low concentrations. Studies demonstrated that the phenothiazine derivatives as [125I] SIL23[102], [11C]-SIL5, [11C]2a, and [18F]2b [103], indole diene derivatives [18F]WC-58a and [18F]46a[104, 105], and thiazole derivatives Anle253b [106] and [18F]DABTA-11 [12, 107, 108] were highly selective for in vitro reconstituted α-Syn fibrils. Among them, ligands [11C]-SIL5, [11C]2a and [18F]2b, Anle253b, and [18F] DABTA-11 can penetrate BBB of healthy cynomolgus monkeys or rats and achieve rapid brain elution, and these structures are anticipated to be used for in vivo α-Syn imaging after further optimization [12, 102]. However, ligands [18F]WC-58a, [18F]46a, and [18F]DABTA-11 are unable to participate in the imaging of α-Syn fibrils in human brain homogenates due to their lipophilicity. Currently, the drawbacks of existing ligands structures may be improved by enhancing the affinity with α-Syn fibrils and reducing their lipophilicity, in order to provide a possibility for PET ligands (Table 2) to target human α-Syn imaging and as a potential diagnostic tool for neurodegenerative diseases like PD.

Table 2 The structures and characteristics of [18F]Florbetapir, [18F]Florbetabeben, [18F]Flutemetamol, [11C]PiB, [18F]BF227, [11C]-PBB3, [125I]SIL23, [11C]-SIL5, [11C]2a, [18F]2b, [18F]WC-58a, [18F]46a, Anle253b, and [18F]DABTA-11
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Non-radiative MRI to image α-Syn

MRI can provide information about brain anatomy, function, and metabolism [116, 117] based on the longitudinal (T1) and transverse (T2) relaxation signals generated by magnetized water protons in brain tissues. For the detection of PD, MRI-based conventional methods are mainly used to identify the secondary PD caused by structural lesions in the brain. In addition, the development of diffusion-weighted imaging (DWI), susceptibility-weighted imaging (SWI), and other MRI imaging technologies has further improved the reliability of PD diagnosis in clinical practice [90, 118].

Khairnar et al. [119] used diffusion kurtosis imaging (DKI) with mean kurtosis (MK) as the assessment index to scan α-syn overexpressing TNWT-61 mice and the method overcame the environmental limitation of water quality subanisotropy to achieve the detection of mouse brain microstructural changes, which could be an effective clinical diagnosis tool for early PD. Studies found that 3-month-old mice showed increased MK values in striatal and thalamic regions with insignificant changes in substantia nigra, hippocampal regions, and sensorimotor cortex, whereas 6-month-old TNWT-61 mice showed increased MK values in substantia nigra regions and behavioral assessments showed some age dependence. This suggests that the α-syn accumulation may be manifested in higher-density dopaminergic nerve terminals and that the young mouse model lacks dopaminergic neuronal degeneration; LB and brain atrophy is only applicable to imaging of early prodromal symptoms in PD patients. In view of the role of the glymphatic system and the meningeal lymphatic system in excreting toxic macromolecular protein metabolites and maintaining the metabolic balance of the central nervous system, Xue et al. [120] used dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to evaluate the basal meningeal lymphatic flow in patients with idiopathic PD (IPD) or atypical PD (APD); the results showed that meningeal lymphatic vessels have an ability to clear α-Syn. The impaired function can delay meningeal lymphatic drainage and aggravate the pathological reaction of α-Syn, indicating that this method is helpful for the early differentiation between IPD and APD, and meningeal lymphatic vessels are expected to be key targets to delay the progression of PD. MRI is an important method for the diagnosis of early PD brain structural imaging, which can demonstrate the functional and structural changes of α-Syn pathological areas in the brain through image differences. However, MRI is an expensive instrument with drawbacks of standardized tissue quantification methods, diagnostic indicators, and prognostic treatment strategies; it needs to overcome the extremely low chemical shift dispersion of α-Syn and improve high resolution in 3D structure detection and is unable to achieve α-Syn in situ imaging.

Conclusions and perspectives

In this paper, we reviewed the associated methods to detect α-Syn in vivo and in vitro. Currently, traditional methods based on antigen–antibody reactions to detect human actual samples such as autopsy, tissues, and biological fluids are specific for α-Syn, but with highly invasive, lower sensitivity, and are difficult to detect different forms of α-Syn. Although in vitro analytical detection and in vivo MRI can obtain information about secondary and tertiary structures of α-Syn and structural pathologies in the brain with high resolution, and realize the size quantification and image, it is difficult to realize in situ α-Syn detection and widespread use for clinical patients because of lack standardized quantitative methods and indicators, low resolution, and environmental influence. The abovementioned in vivo and in vitro methods for α-Syn detection have made considerable progress in illustrating the pathogenesis of PD and the dynamics of α-Syn aggregation, but they still lack effective methods for α-Syn in vivo detection and intervention.

The improvement of PET ligands and fluorescence imaging probes are potential methods and research hotspots to realize α-Syn in situ detection, early diagnosis, and therapy of PD. In situ α-Syn with lower abundance, complex intracerebral environment, and PTMs lead to different forms and binding sites that are difficult to be detected compared with Aβ, Tau, and in vitro recombinant α-Syn models. At present, fluorescence probes that target α-Syn have slow development compared to PET ligands. Only a few fluorescence probes can target in vitro and intracellular recombination α-Syn but have not yet achieved α-Syn in vivo imaging due to poor selectivity, elution ability, and high lipophilicity. A few α-Syn PET ligands are still in the primary stage compared to Aβ and Tau ligands. Although PET ligands can target different forms of α-Syn in vitro and penetrate the BBB in rodent animal models, its limited emission wavelengths are unsuitable for human studies, and no clinically approved PET ligands for in vivo α-Syn. Therefore, fluorescence probes and PET ligands for α-Syn in vivo imaging should have the following characteristics: (1) small molecular weight, low lipophilicity and cytotoxicity, long fluorescence lifetime, and good elution capacity after penetrating the BBB by passive diffusion; (2) highly sensitive to α-Syn, high selectivity and affinity, and low detection limit; (3) suitable emission wavelength to achieve α-Syn in situ imaging. Therefore, in further development and application of α-Syn in situ imaging technology, it is necessary to evaluate the fidelity of the recombinant α-Syn models in vitro and simulate its aggregation environment in the brain, considering the influence of the difference distribution and concentration of the target proteins and non-target proteins. Using structural modification strategies like substitution and enhanced conjugation improves the abovementioned features of new fluorescence probes and PET ligands. Meanwhile, microscopy, MS, and computer molecular docking technology could be used to clarify the process of aggregation dynamics, detect α-Syn conformation and size, and predict the binding sites. The development of potential probes and ligands for targeting various α-Syn conformations in vitro is anticipated to further apply for α-Syn in situ imaging.