Single-particle Fluorescence Research Articles

Ensemble and Single Particle Fluorescence Characterization of Dye-Labeled Cellulose Nanocrystals.

Cellulose nanocrystals (CNCs) have been covalently labeled with both fluorescein and rhodamine and studied by a combination of UV-vis absorption spectroscopy and ensemble and single molecule fluorescence spectroscopy. For all samples, the fluorescence anisotropy and lifetimes were consistent with effects expected for covalently bound dye molecules. Low dye loading levels (∼0.1 dye/particle) were estimated for the fluorescein-labeled CNC which coupled with the strong pH dependence make this a less suitable fluorophore for most applications. Rhodamine-labeled CNCs were prepared from both sulfated and carboxylated CNCs and had loading levels that varied from 0.25 to ∼15 dye molecules/CNC. For the sulfated samples, the absorption due to (nonfluorescent) dimeric dye increased with dye loading; in contrast, the carboxylated sample, which had the highest rhodamine content, had a low dimer yield. Single particle fluorescence studies for two of the rhodamine-labeled CNCs demonstrated that individual particles are readily detected by their stepwise blinking/bleaching behavior and by polarization effects. Overall, the results indicate the importance of understanding the effects of loading on dye photophysics to select an optimal dye concentration to maximize sensitivity while minimizing the effect of the dye on the CNC behavior. The results also demonstrate that CNCs with relatively low dye loadings (e.g., ∼1 dye/particle) are readily detectable by fluorescence and should be adequate for use in fluorescence-based biological assays or to probe the distribution of CNCs in composite materials.

Time-resolved monitoring of polycyclic aromatic hydrocarbons adsorbed on atmospheric particles.

Real-time monitoring of individual particles from atmospheric aerosols was performed by means of a specifically developed single-particle fluorescence spectrometer (SPFS). The observed fluorescence was assigned to particles bearing polycyclic aromatic hydrocarbons (PAH). This assignment was supported by an intercomparison with classical speciation on filters followed by gas chromatography-mass spectrometry (GC-MS) analysis. As compared with daily averaged data, our time-resolved approach provided information about the physicochemical dynamics of the particles. In particular, distinctions were made between background emissions related to heating, and traffic peaks during rush hours. Also, the evolution of the peak fluorescence wavelength provided an indication of the aging of the particles during the day.

Plasmonics Enhanced Smartphone Fluorescence Microscopy

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Cathepsin S increases tau oligomer formation through limited cleavage, but only IL-6, not cathespin S serum levels correlate with disease severity in the neurodegenerative tauopathy progressive supranuclear palsy.

Limited cleavage promotes the aggregation propensity of protein tau in neurodegenerative tauopathies. Cathepsin S (CatS) is overexpressed in brains of patients suffering from tauopathies such as Alzheimer's disease (AD). Furthermore, CatS serum levels correlate with survival in the elderly. The current study investigates whether limited cleavage by CatS promotes tau aggregation, and whether CatS serum levels may correlate with disease severity in tauopathies. Oligomer formation of fluorescently labeled protein tau was monitored by single particle fluorescence spectroscopy after coincubation with CatS. Tau cleavage patterns were investigated by SDS-PAGE. For serum analyses, samples were collected from 42 patients with probable progressive supranuclear palsy (PSP) according to NINDS-PSP criteria. Disease severity was assessed by PSP rating scale (PSP-RS), PSP staging system (PSP-S) and Schwab and England Activities of Daily Living (SEADL). CatS, cystatin C (CysC) and interleukin 6 (IL-6) serum levels were determined by ELISA, ECLIA and turbidimetry, respectively. SDS-PAGE demonstrated a distinct cleavage pattern of protein tau after coincubation with CatS. Furthermore, tau oligomer formation was increased 2.4-fold (p<0.05) after limited cleavage. Serum CatS and CysC levels did not correlate with disease severity in PSP. Of note, IL-6 correlated with PSP-S (r=0.41; 95% CI 0.11-0.65; p=0.008), SEADL (r=-0.37; 95% CI -0.61 to -0.06; p=0.017) and the history and gait/midline subdomains of the PSP-RS. While CatS facilitates tau aggregation in vitro, serum levels of CatS appear not to correlate with disease severity. The observed correlation of IL-6 with disease severity warrants further investigation of inflammatory markers in PSP.

Fluorescence calibration method for single-particle aerosol fluorescence instruments

Abstract. Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan–ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310–400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420–650 nm). The relationship between fluorescence and mass for the mixed tryptophan–ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

Crosslinked polymer nanoparticles containing single conjugated polymer chains

Conjugated polymer nanoparticles are widely used in fluorescent labeling and sensing, as they have mean radii between 5 and 100 nm, narrow size dispersion, high brightness, and are photochemically stable, allowing single particle detection with high spatial and temporal resolution. Highly crosslinked polymers formed by linking individual chains through covalent bonds yield high-strength rigid materials capable of withstanding dissolution by organic solvents. Hence, the combination of crosslinked polymers and conjugated polymers in a nanoparticulated material presents the possibility of interesting applications that require the combined properties of constituent polymers and nanosized dimension. In the present work, F8BT@pEGDMA nanoparticles composed of poly(ethylene glycol dimethacrylate) (pEGDMA; a crosslinked polymer) and containing the commercial conjugated polymer poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) were synthesized and characterized. Microemulsion polymerization was applied to produce F8BT@pEDGMA particles with nanosized dimensions in a ∼25% yield. Photophysical and size distribution properties of F8BT@pEDGMA nanoparticles were evaluated by various methods, in particular single particle fluorescence microscopy techniques. The results demonstrate that the crosslinking/polymerization process imparts structural rigidity to the F8BT@pEDGMA particles by providing resistance against dissolution/disintegration in organic solvents. The synthesized fluorescent crosslinked nanoparticles contain (for the most part) single F8BT chains and can be detected at the single particle level, using fluorescence microscopy, which bodes well for their potential application as molecularly imprinted polymer fluorescent nanosensors with high spatial and temporal resolution.

Preparation and Fluorescence Properties of Perylenediimide Nanodispersions Having a One-Dimensional π-Stacked Structure.

We prepared stable nanodispersions of a fluorescent perylenediimide (PDI) derivative having long alkyl chains by nanosecond laser fragmentation of its microcrystalline powder in acetonitrile (ACN). The nanoparticles had cube-like or rod shapes with a mean size of 100 nm, and they dispersed stably for longer than 1 month. The prepared nanobricks exhibited absorption and fluorescence spectra characteristic of one-dimensional aggregates with cofacial stacking of PDI planes. Single-particle fluorescence measurements demonstrated that nanobricks had a well-aligned structure of one-dimensional columns of PDI. The aqueous dispersions were also fabricated by redispersing the prepared nanobricks, utilizing lipophilic interactions of surfactants having long alkyl chains. We examined the fluorescence properties of nanoparticles dispersed in ACN and in water, and observed amplified fluorescence quenching by the surface-adsorbed dye.

TiO2 mesocrystals composited with gold nanorods for highly efficient visible-NIR-photocatalytic hydrogen production

TiO2 mesocrystals (TMC) have efficient charge transport properties and long-lived charges which cause high photoconductivity and photocatalytic activity. However, TMC have no absorption in the region of visible and near-Infrared (NIR) light. The optical resonance of Au nanorods (NRs) depending mainly on their length and width (aspect ratio) can be used to design panchromatic absorbers, covering most of the useful solar spectrum. Therefore, in this article, Au NRs and TMC composites were prepared by the ligand exchange method to show highly efficient H2 production (924µmolh−1g−1) under visible-NIR light irradiation. The efficient H2 production is explained by surface plasmon resonance (SPR) of Au NRs, electron injection to TMC, efficient charge transport in TMC due to the superstructure of TMC and water reduction on TMC. Single-particle confocal fluorescence microscopy and time-resolved diffuse reflectance measurements confirmed the efficient hot electron injection and charge separation from Au NRs with strong SPR to the superstructure of TMC.

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release.

Phycobiliproteins: A Novel Green Tool from Marine Origin Blue-Green Algae and Red Algae.

Marine species are comprising about a half of the whole global biodiversity; the sea offers an enormous resource for novel bioactive compounds. Several of the marine origin species show multifunctional bioactivities and characteristics that are useful for a discovery and/or reinvention of biologically active compounds. For millennia, marine species that includes cyanobacteria (blue-green algae) and red algae have been targeted to explore their enormous potential candidature status along with a wider spectrum of novel applications in bio- and non-bio sectors of the modern world. Among them, cyanobacteria are photosynthetic prokaryotes, phylogenetically a primitive group of Gramnegative prokaryotes, ranging from Arctic to Antarctic regions, capable of carrying out photosynthesis and nitrogen fixation. In the recent decade, a great deal of research attention has been paid on the pronouncement of bio-functional proteins along with novel peptides, vitamins, fine chemicals, renewable fuel and bioactive compounds, e.g., phycobiliproteins from marine species, cyanobacteria and red algae. Interestingly, they are extensively commercialized for natural colorants in food and cosmetics, antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective agents and fluorescent neo-glycoproteins as probes for single particle fluorescence imaging fluorescent applications in clinical and immunological analysis. However, a comprehensive knowledge and technological base for augmenting their commercial utilities are lacking. Therefore, this paper will provide an overview of the phycobiliproteins-based research literature from marine cyanobacteria and red algae. This review is also focused towards analyzing global and commercial activities with application oriented-based research. Towards the end, the information is also given on the potential biotechnological and biomedical applications of phycobiliproteins.

Hot electron-driven hydrogen evolution using anisotropic gold nanostructure assembled monolayer MoS2.

Plasmonic nanostructures attracting particular interest in plasmon-induced highly energetic electrons, also known as hot electrons, play a fundamental role in photocatalysis for solar energy conversion. Plasmon-induced hot electron excitation, relaxation, transport, and injection to two-dimensional semiconductors are necessary to clearly understand the efficient plasmon-induced chemical reaction. Herein, we use a plasmonic photocatalyst composed of anisotropic gold nanostructures as the electron donor assembled on two-dimensional molybdenum dichalcogenide, monolayer MoS2, as the electron acceptor in order to unveil the plasmon-induced interfacial hot electron transfer for the hydrogen evolution reaction (HER). Single-particle confocal fluorescence microscopy, computational calculation of finite-difference-time-domain (FDTD) simulation, and time-resolved transient absorption measurements revealed that anisotropic gold nanostructures with strong plasmon resonance exhibit interfacial hot electron transfer to monolayer MoS2, giving the charge separated state with a long lifetime of 800 ps which is responsible for efficient HER. This is the first example to show the plasmon-induced interfacial hot electron transfer from anisotropic Au nanostructures to two-dimensional materials.

Full-colour carbon dots: integration of multiple emission centres into single particles.

The single particle emission behaviours of our previously reported excitation-dependent full-colour carbon dots (F-C dots) have been analyzed by a single-particle fluorescence imaging technique. The co-localization of the F-C dots excited with different wavelengths shows that single F-C dots can also be excited with multiple energies. The co-localization of the F-C dots that emit at different colour regions under the same excitation wavelength or different excitation wavelengths shows that single F-C dots have a broad emission band from blue to red, but the emission intensities in different colour regions vary from one particle to another. So this study concretely proves that the full colour emissions are single particle behaviours; they are different from the other type of excitation dependent full-colour emission carbon dots whose full-colour behaviour originates from the large heterogeneity in both particle size and the structures of the ensemble. Then the origination of the full-colour emission at the single dot level was further studied by comparing the emission properties of the F-C dots and the small molecular byproducts, and it is found that the emissions of the dozens of molecular byproducts can also cover the full visible regions. And the emission positions of F-C dots are very similar to those of the byproducts at the same excitations, but they show different lifetimes. So a mechanism for the full colour emissions of F-C dots is proposed to originate from the hybridization of multiple small emissive molecules on the emissive carbon cores. This single particle level understanding of full-colour emission properties will pave the way towards the development of single dot imaging or tracking.

Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

Exploring the HYDRAtion method for loading siRNA on liposomes: the interplay between stability and biological activity in human undiluted ascites fluid.

Delivery of small interfering RNA (siRNA) is recently gaining tremendous attention for the treatment of ovarian cancer. The present study investigated the potential of different liposomal formulations composed of (2,3-dioleoyloxy-propyl)-trimethylammonium (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) encapsulating siRNA (hydration method) for their ability to knockdown luciferase (Luc) activity in human ovarian cancer SKOV-3 cells. Fluorescence single particle tracking (fSPT) and fluorescence correlation spectroscopy (FCS) in human-undiluted ascites fluid obtained from a peritoneal carcinomatosis patient revealed that cationic hydra-lipoplexes (HYDRA-LPXs) and HYDRA-LPXs decorated with stable DSPE-PEG (DSPE HYDRA-LPXs) showed high stability during at least 24h. HYDRA-LPXs decorated with sheddable C8 and C16 PEG-Ceramides (Cer HYDRA-LPXs) resulted in rapid and premature release of siRNA already in the first hours. Despite their role in preventing aggregation in vivo, liposomes decorated with stable PEG residues resulted in a poor transfection compared to the ones decorated with sheddable PEG residues in reduced serum conditions. Yet, the transfection efficiency of both Cer HYDRA-LPXs significantly decreased following 1h of incubation in ascites fluid due to a drastic drop in the cellular uptake, while DSPE HYDRA-LPXs are still taken up by cells, but too stable to induce efficient gene silencing.

Tracking single fluorescent particles in three dimensions via extremum seeking.

The ability to track single fluorescent particles in three-dimensions with sub-diffraction limit precision as well as sub-millisecond temporal resolution has enabled the understanding of many biophysical phenomena at the nanometer scale. While there are several techniques for achieving this, most require complicated experimental setups that are expensive to implement. These methods can offer superb performance but their complexity may be overwhelming to the end-user whose aim is only to understand the feature being imaged. In this work, we describe a method for tracking a single fluorescent particle using a standard confocal or multi-photon microscope configuration. It relies only on the assumption that the relative position of the measurement point and the particle can be actuated and that the point spread function has a global maximum that coincides with the particle's position. The method uses intensity feedback to calculate real-time position commands that "seek" the extremum of the point spread function as the particle moves through its environment. We demonstrate the method by tracking a diffusing quantum dot in a hydrogel on a standard epifluorescent confocal microscope.

Design of smart GE11-PLGA/PEG-PLGA blend nanoparticulate platforms for parenteral administration of hydrophilic macromolecular drugs: synthesis, preparation and in vitro/ex vivo characterization

Active drug targeting and controlled release of hydrophilic macromolecular drugs represent crucial points in designing efficient polymeric drug delivery nanoplatforms. In the present work EGFR-targeted polylactide-co-glycolide (PLGA) nanoparticles were made by a blend of two different PLGA-based polymers. The first, GE11-PLGA, in which PLGA was functionalized with GE11, a small peptide and EGFR allosteric ligand, able to give nanoparticles selective targeting features. The second polymer was a PEGylated PLGA (PEG-PLGA) aimed at improving nanoparticles hydrophilicity and stealth features. GE11 and GE11-PLGA were custom synthetized through a simple and inexpensive method. The nanoprecipitation technique was exploited for the preparation of polymeric nanoparticles composed by a 1:1weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9±5.0nm). In vitro cellular uptake in EGFR-overexpressing cell line (A549) demonstrated an active internalization of GE11-functionalized nanoparticles. GE11-PLGA/PEG-PLGA blend nanoparticles were loaded with Myoglobin, a model hydrophilic macromolecule, reaching a good loading (2.42% respect to the theoretical 4.00% w/w) and a prolonged release over 60days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30days in physiological saline solution at 4°C and for 24h in pH 7.4 or pH 5.0 buffer at 37°C respectively, giving indications about potential storage and administration conditions. Furthermore ex vivo stability study in human plasma using fluorescence Single Particle Tracking (fSPT) assessed good GE11-PLGA/PEG-PLGA nanoparticles dimensional stability after 1 and 4h. Thanks to the versatility in polymeric composition and relative tunable nanoparticles features in terms of drug incorporation and release, GE11-PLGA/PEG-PLGA blend NPs can be considered highly promising as smart nanoparticulate platforms for the treatment of diseases characterized by EGFR overexpression by parenteral administration .

Deciphering dynamics of clathrin-mediated endocytosis in a living organism

Current understanding of clathrin-mediated endocytosis (CME) dynamics is based on detection and tracking of fluorescently tagged clathrin coat components within cultured cells. Because of technical limitations inherent to detection and tracking of single fluorescent particles, CME dynamics is not characterized in vivo, so the effects of mechanical cues generated during development of multicellular organisms on formation and dissolution of clathrin-coated structures (CCSs) have not been directly observed. Here, we use growth rates of fluorescence signals obtained from short CCS intensity trace fragments to assess CME dynamics. This methodology does not rely on determining the complete lifespan of individual endocytic assemblies. Therefore, it allows for real-time monitoring of spatiotemporal changes in CME dynamics and is less prone to errors associated with particle detection and tracking. We validate the applicability of this approach to in vivo systems by demonstrating the reduction of CME dynamics during dorsal closure of Drosophila melanogaster embryos.

Nanoprecipitation of Fluorescent Conjugated Polymer onto the Surface of Plasmonic Nanoparticle for Fluorescence/Dark-Field Dual-Modality Single Particle Imaging.

In this work, a hybridized nanoparticle with fluorescence/dark-field dual-modality imaging capability was prepared by nanoprecipitation of fluorescent conjugated polymer onto the surface of silica-coated rod-shape plasmonic nanoparticle. According to the spectroscopic and microscopic characterizations, the fluorescence intensity of conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo(2,1',3)-thiadiazole)] (PFBT) could be enhanced around 2-fold after assembling onto the silica-decorated metal nanorod surface compared with the fluorescence intensity of regular PFBT polymer dots without the metal core. The in situ nanorod etching experiment further confirmed this result at the single particle level. In addition to the fluorescence enhancement effect, improved fluorescence stability was obtained from the single particle fluorescence intensity characterizations. As a consequence, this self-assembled functional nanoparticle could be extensively applied to biological imaging such as cellular labeling and single particle tracking owing to the novel and unique optical features, for example, the superior optical stability and specific identification capability from the scattering and fluorescence domain, respectively. Furthermore, the amendable peripheral polymer surface of this nanostructure will promote its applications in biological sensing and imaging-guided functional molecule delivery in the future.

Functionalization of emissive conjugated polymer nanoparticles by coprecipitation: consequences for particle photophysics and colloidal properties

The functionalization of polyfluorene (PFO) nanoparticles by coprecipitation of the conjugated polymer with an amphiphilic comb polymer, consisting of a hydrophobic polystyrene backbone with hydrophilic, carboxylic acid-terminated polyethylene oxide side-chains (PS-PEG-COOH), is investigated. The comb polymer affects the properties of the formed hybrid nanoparticles. Non-functionalized particles are typically larger (28 nm) than functionalized ones (20 nm); peak molar extinction coefficients are found to differ in a similar trend. Zeta potentials are negative, consistent with negative surface charge on PFO particles due to chemical defect formation, with additional charge on functionalized particles due to the pendant carboxylic acid groups. Emission quantum yields of functionalized particles are typically larger, consistent with lower efficiency of energy transfer to quenchers in smaller particles and weaker PFO interchain interactions due to chain dilution. The trend in per-particle fluorescence brightness values, as confirmed by single particle fluorescence imaging, reflects the nanoparticle extinction coefficients. Photostability studies on aqueous dispersions of hybrid particles indicate mild photobrightening under continuous illumination while PFO particles exhibit slow exponential emission decay. Functionalized particles are also resistant to aggregation during exposure to adenocarcinoma cells. Generally, the hybrid particles exhibit more favorable time-, pH- and medium-dependent stabilities, likely due to steric and electrostatic stabilization by PEG-carboxylic acid functionalities. Overall, the functionalized particles exhibit attractive properties: Reasonably small size, tight size distribution, high absorption cross section, radiative rate and emission quantum yield, excellent brightness and photostability, and good colloidal stability.

Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation.