Deposition of anionic conjugated poly(phenylenevinylene) onto silica nanoparticles via electrostatic interactions — Assembly and single-particle spectroscopy

2011 ◽  
Vol 89 (3) ◽  
pp. 385-394 ◽  
Author(s):  
An T. Ngo ◽  
Kai L. Lau ◽  
Jeffrey S. Quesnel ◽  
Robert Aboukhalil ◽  
Gonzalo Cosa
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Electrostatic Interactions ◽  
Emission Spectra ◽  
Fluorescent Nanoparticles ◽  
Oxygen Scavenger ◽  
Conjugated Polyelectrolyte ◽  
Exciton Migration ◽  
Silica Bead ◽  
Single Particle Spectroscopy

Fluorescent nanoparticles were prepared via adsorption of the conjugated polyelectrolyte poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) onto 50 and 100 nm aminosilane functionalized silica beads. The particles were investigated via ensemble and single-molecule or -particle spectroscopy techniques to quantify the effect of the silica bead core on the exciton migration efficiency within the polymer. Ensemble emission spectra and ensemble fluorescence quenching studies with methyl viologen are consistent with good exciton migration along the polymer in the polymer-coated bead. The silica nanobead scaffolding preserves the sensitivity of the free polymer and provides a controllable architecture that minimizes nonspecific interactions. Single-particle spectroscopy studies were conducted on particles immobilized onto the positively charged surface of glass cover slips. Particle immobilization enabled us to monitor the effect of oxygen scavenger solutions on individual particles by changing the surrounding solution. The intensity–time trajectories of individual beads provide a mechanism of signal transduction with potential applications in multiplexing studies. Hundreds of individual beads can be imaged in a rapid parallel fashion.

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

2021 ◽  
Author(s):  
Anders K Engdahl ◽  
Oleg Grauberger ◽  
Mark Schüttpelz ◽  
Thomas Huser
Keyword(s):  
Single Molecule ◽  
Fluorescent Dyes ◽  
Sodium Sulfite ◽  
Super Resolution ◽  
Oxygen Scavenger ◽  
Dark State ◽  
Alexa Fluor ◽  
High Illumination ◽  
Human Osteosarcoma Cells ◽  
Super Resolution Microscopy

Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells.

scholarly journals Probing the variability in oxidation states of magnetite nanoparticles by single-particle spectroscopy

2018 ◽  
Vol 6 (4) ◽  
pp. 875-882 ◽  
Author(s):  
A. Fraile Rodríguez ◽  
C. Moya ◽  
M. Escoda-Torroella ◽  
A. Romero ◽  
A. Labarta ◽  
...  
Keyword(s):  
Oxidation State ◽  
Absorption Spectroscopy ◽  
Magnetite Nanoparticles ◽  
Single Particle ◽  
Cation Distribution ◽  
Oxidation States ◽  
Magnetic Response ◽  
X Ray ◽  
Single Particle Spectroscopy ◽  
X Ray Absorption

Single-particle X-ray absorption spectroscopy reveals that the oxidation state and cation distribution of individual magnetite nanoparticles may be largely heterogeneous even when the macroscopic structural and magnetic response of the ensembles is uniform.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Single particle ICP-MS-based absolute and relative quantification ofE. coliO157 16S rRNA using sandwich hybridization capture

The Analyst ◽  
2019 ◽  
Vol 144 (5) ◽  
pp. 1725-1730 ◽  
Author(s):  
Xiaomin Xu ◽  
Jiyun Chen ◽  
Bangrui Li ◽  
Lijuan Tang ◽  
Jianhui Jiang
Keyword(s):  
16S Rrna ◽  
Single Molecule ◽  
Single Particle ◽  
Inductively Coupled Plasma ◽  
Inductively Coupled ◽  
Hybridization Capture ◽  
Sandwich Hybridization ◽  
Icp Ms ◽  
Magnetic Capture ◽  
Plasma Mass Spectrometry

Herein, a novel 16S rRNA detection platform was achieved by combining a sandwich hybridization reaction, a single-molecule magnetic capture, and single particle-inductively coupled plasma mass spectrometry amplification.

scholarly journals Automated single particle detection and tracking for large microscopy datasets

2016 ◽  
Vol 3 (5) ◽  
pp. 160225 ◽  
Author(s):  
Rhodri S. Wilson ◽  
Lei Yang ◽  
Alison Dun ◽  
Annya M. Smyth ◽  
Rory R. Duncan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Image Data ◽  
Ground Truth ◽  
Detection Algorithm ◽  
Large Datasets ◽  
Single Particle Tracking ◽  
Synthetic Image ◽  
Particle Detection ◽  
Very Large Datasets

Recent advances in optical microscopy have enabled the acquisition of very large datasets from living cells with unprecedented spatial and temporal resolutions. Our ability to process these datasets now plays an essential role in order to understand many biological processes. In this paper, we present an automated particle detection algorithm capable of operating in low signal-to-noise fluorescence microscopy environments and handling large datasets. When combined with our particle linking framework, it can provide hitherto intractable quantitative measurements describing the dynamics of large cohorts of cellular components from organelles to single molecules. We begin with validating the performance of our method on synthetic image data, and then extend the validation to include experiment images with ground truth. Finally, we apply the algorithm to two single-particle-tracking photo-activated localization microscopy biological datasets, acquired from living primary cells with very high temporal rates. Our analysis of the dynamics of very large cohorts of 10 000 s of membrane-associated protein molecules show that they behave as if caged in nanodomains. We show that the robustness and efficiency of our method provides a tool for the examination of single-molecule behaviour with unprecedented spatial detail and high acquisition rates.

scholarly journals Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2826 ◽  
Author(s):  
Shangguo Hou ◽  
Courtney Johnson ◽  
Kevin Welsher
Keyword(s):  
Fluorescence Spectroscopy ◽  
Real Time ◽  
Temporal Resolution ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Single Particle Tracking ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

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