Optimizing fluorophore density for single virus counting: a photophysical approach

Chatterjee, Swarupa; Molenaar, Robert; Tromp, Leroy; Wagterveld, R.M.; Roesink, H.D.W.; Cornelissen, Jeroen J. L. M.; Claessens, Mireille Maria Anna Elisabeth; Blum, Christian

doi:10.1088/2050-6120/abd8e4

Cited by 10 publications

(14 citation statements)

References 40 publications

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“…Considering that of the total αS concentration of 20 nM, only half was labeled and ignoring the possible fluorescence quenching due to fluorophore–fluorophore interactions that have been observed in other protein systems under certain conditions, this indicates that on average, 3 to 4 αS proteins are present in an αS/N-protein complex. 29 Note that although this indicates that N-proteins accumulate αS, the FCS experiment was performed in excess of N-protein. The aggregation experiments were performed in excess of αS; accumulation of even higher numbers of αS on N-protein is therefore likely.…”

Section: Resultsmentioning

confidence: 99%

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation

Semerdzhiev

Fakhree

Segers-Nolten

et al. 2021

ACS Chem. Neurosci.

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105

108

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First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinson’s disease (PD) have been reported. Currently, it is unclear if there is also a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of α-synuclein protein into amyloid fibrils characteristic for PD, we investigated the effect of the presence of SARS-CoV-2 proteins on α-synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 spike protein (S-protein) has no effect on α-synuclein aggregation, while SARS-CoV-2 nucleocapsid protein (N-protein) considerably speeds up the aggregation process. We observe the formation of multiprotein complexes and eventually amyloid fibrils. Microinjection of N-protein in SH-SY5Y cells disturbed the α-synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and α-synuclein as molecular basis for the observed correlation between SARS-CoV-2 infections and Parkinsonism.

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Section: Resultsmentioning

confidence: 99%

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation

Semerdzhiev

Fakhree

Segers-Nolten

et al. 2021

ACS Chem. Neurosci.

Self Cite

105

108

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“…Fluorophore interactions that are responsible for self-quenching include the formation of dark fluorophore aggregates and the transfer of excitation energy to these dark aggregates [ 15 , 33 , 34 , 35 ]. Self-quenching of fluorescence not only manifests as a lower particle brightness than expected, but it additionally results in a shift of the emission peak to higher wavelengths [ 36 ]. Although generally a nuisance in labeling strategies, self-quenching may be exploited in applications.…”

Section: Resultsmentioning

confidence: 99%

“…Although generally a nuisance in labeling strategies, self-quenching may be exploited in applications. For self-quenching to occur, the fluorophores do not have to be on the same protein, it is also observed in protein assemblies like viruses [ 36 ]. When the total number of fluorophores on the protein assemblies is much smaller than the total number of virus proteins, individual proteins typically contain less than one fluorophore.…”

Section: Resultsmentioning

confidence: 99%

Exploiting Complex Fluorophore Interactions to Monitor Virus Capsid Disassembly

et al. 2021

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Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.

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“…The accessible range of particle concentrations is in good agreement with our earlier work on counting labelled viruses. 20 It is possible to access lower concentrations with the outlined single particle tracking and counting strategies. Note that at the lowest concentrations the average number of particles in the detection volume is <1 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Particle counting and tracking experiments were performed on a custom built setup. 20 In short: an inverted microscope (Nikon TE-2000U) was equipped with a custom in-coupling for a multimode 520 nm 1.2 W laser diode (Laserland, A-G1000F-C). Through the microscope backport excitation light Environmental Science: Nano Paper was focused on the objective's back focal plane (Nikon, CFI PlanApo 60× NA1.2 Wi).…”

Section: Fluorescence Microscopy Setupmentioning

confidence: 99%

Nanoplastic Sizes and Numbers: Quantification by Single Particle Tracking

Molenaar¹,

Chatterjee²,

Claessens³

et al. 2020

Preprint

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<p>Plastic particles have been found almost everywhere in the environment, in oceans, terrestrial water bodies, sediments and air. The extend of this unwanted contamination is difficult to fully capture. Existing quantification methods focus on the detection of millimeter to micrometer sized plastic particles, while plastic breakdown processes continue to smaller, nanometer sized, particles. For these nanoplastics methods that are inexpensive and can be (semi-) automated for high throughput analysis of dilute nanoplastic particle suspensions, are lacking. Here we combine sensitive fluorescence video microsopy, NileRed staining of plastic particles, and Single Particle Tracking (SPT) to count and size nanoplastics. With this approach we show that particle diameters as low as 40 nm can be extracted, mixing ratios can be recovered, and number concentrations as low as 2·10<sup>6</sup> particles/ml can be determined. These results indicate that this approach is promising for the quantification of sizes and concentrations of nanoplastics in environmental samples.</p>

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Optimizing fluorophore density for single virus counting: a photophysical approach

Cited by 10 publications

References 40 publications

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation

Exploiting Complex Fluorophore Interactions to Monitor Virus Capsid Disassembly

Nanoplastic Sizes and Numbers: Quantification by Single Particle Tracking

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