Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds

Lin, Ying-Hsiu; Chang, Wei-Lin Melody; Hsieh, Chia-Lung

doi:10.1364/oe.22.009159

Cited by 82 publications

(90 citation statements)

References 34 publications

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“…When controlled appropriately, iSCAT has been shown to detect and image single, unlabeled proteins (Ortega Arroyo et al, 2014; Piliarik & Sandoghdar, 2014) and to track 20 nm gold labels at up to 500 kHz frame rates with nm precision (Andrecka et al, 2015; Lin, Chang, & Hsieh, 2014; Mickolajczyk et al, 2015). Interferometric detection leads to a lesser drop in scattering contrast with decreasing particle size compared to dark-field microscopy, simplifying the detection of very weak scatterers.…”

Section: Interferometric Scattering Microscopymentioning

confidence: 99%

Interferometric Scattering Microscopy for the Study of Molecular Motors

Andrecka

Takagi

Mickolajczyk

et al. 2016

Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

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Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20 nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

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Section: Interferometric Scattering Microscopymentioning

confidence: 99%

Interferometric Scattering Microscopy for the Study of Molecular Motors

Andrecka

Takagi

Mickolajczyk

et al. 2016

Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

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“…Using 40 nm gold nanoparticles (GNPs) as labels with bright-field detection, single lipids and membrane proteins were tracked in the plasma membrane with 17 nm spatial precision at 40 kHz from which membrane compartmentalization of hundreds of nanometers was discovered17. Recently, a novel concept of interferometric detection of scattering signal (iSCAT) has been introduced that shows promise of further improving the sensitivity and spatiotemporal resolution of scattering-based SPT1819202122232425. The interferometric detection makes it possible to circumvent the limitation of signal-to-noise ratio (SNR) set by the electronic noise of high-speed detectors, reaching the optimal sensitivity required for super localization of very small particle via intrinsic scattering24.…”

mentioning

confidence: 99%

“…Recently, a novel concept of interferometric detection of scattering signal (iSCAT) has been introduced that shows promise of further improving the sensitivity and spatiotemporal resolution of scattering-based SPT1819202122232425. The interferometric detection makes it possible to circumvent the limitation of signal-to-noise ratio (SNR) set by the electronic noise of high-speed detectors, reaching the optimal sensitivity required for super localization of very small particle via intrinsic scattering24. By iSCAT microscopy, detection and localization of single nanoparticles, ranging from metallic particles to virus particles have been demonstrated202122232425.…”

mentioning

confidence: 99%

“…The interferometric detection makes it possible to circumvent the limitation of signal-to-noise ratio (SNR) set by the electronic noise of high-speed detectors, reaching the optimal sensitivity required for super localization of very small particle via intrinsic scattering24. By iSCAT microscopy, detection and localization of single nanoparticles, ranging from metallic particles to virus particles have been demonstrated202122232425. The stable and unsaturated scattering signal allows for long-term detection with unlimited observation time.…”

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confidence: 99%

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Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

Lin

Yen

et al. 2016

Sci Rep

Self Cite

102

126

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Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

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“…New technologies are capable of extending the trajectory length whilst retaining high sampling rates and high spatial resolution. For example interferometric scattering microscopy (iSCAT) can generate very long (50,000 step) trajectories, with high spatial (< 2 nm) and temporal (up to 500 kHz) resolution (3)(4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

A Hidden Markov Model for Detecting Confinement in Single Particle Tracking Trajectories

Slator

Burroughs

2018

Preprint

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State-of-the-art single particle tracking (SPT) techniques can generate long trajectories with high temporal and spatial resolution. This offers the possibility of mechanistically interpreting particle movements and behaviour in membranes. To this end, a number of statistical techniques have been developed that partition SPT trajectories into states with distinct diffusion signatures, allowing a statistical analysis of diffusion state dynamics and switching behaviour. Here we develop a confinement model, within a hidden Markov framework, that switches between phases of free diffusion, and confinement in a harmonic potential well. By using a Markov chain Monte Carlo (MCMC) algorithm to fit this model, automated partitioning of individual SPT trajectories into these two phases is achieved, which allows us to analyse confinement events. We demonstrate the utility of this algorithm on a previously published dataset, where gold nanoparticle (AuNP) tagged GM1 lipids were tracked in model membranes. We performed a comprehensive analysis of confinement events, demonstrating that there is heterogeneity in the lifetime, shape, and size of events, with confinement size and shape being highly conserved within trajectories. Our observations suggest that heterogeneity in confinement events is caused by both individual nanoparticle characteristics and the binding site environment. The individual nanoparticle heterogeneity ultimately limits the ability of iSCAT to resolve molecular dynamics to the order of the tag size; homogeneous tags could potentially allow the resolution to be taken below this limit by deconvolution methods. In a wider context, the presented harmonic potential well confinement model has the potential to detect and characterise a wide variety of biological phenomena, such as hop diffusion, receptor clustering, and lipid rafts.

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Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds

Cited by 82 publications

References 34 publications

Interferometric Scattering Microscopy for the Study of Molecular Motors

Interferometric Scattering Microscopy for the Study of Molecular Motors

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

A Hidden Markov Model for Detecting Confinement in Single Particle Tracking Trajectories

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