High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy

Cheng, Ching-Ya; Liao, Yi‐Hung; Hsieh, Chia-Lung

doi:10.1039/c8nr06789a

Cited by 59 publications

(100 citation statements)

References 56 publications

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“…28 Recent advancements in interferometric microscopy techniques improved system sensitivity with pupil engineering and led to single protein detection in sparse samples, yet they do not address the inadequate lateral resolution. 20,21,24 Here, we present a computational imaging approach that extends single-particle interferometric reflectance (SPIR) microscopy's capability from direct detection to detailed morphological characterization of sub-diffraction-limited particles. Recently, several computational coherent imaging techniques have demonstrated great advancements in improving spatial resolution.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

et al. 2020

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Section: Introductionmentioning

confidence: 99%

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

et al. 2020

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“…[26][27] By using interferometric scattering (iSCAT) microscopy, 70 very small nanoparticles (as small as 10 nm gold nanoparticle) can be visualized and tracked at a high speed over a long observation time. 71 The higher spatiotemporal resolution can potentially reveal nanoscopic molecular dynamics in the polymer-cushioned plasma membrane bilayers.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

Wong¹,

Juo²,

Liao³

et al. 2019

Preprint

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Native cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics.Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm 2 /s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

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“…21 Interferometric scattering microscopy (iSCAT) has enabled the tracking of nanoparticles as small as 2 nm at high spatial and temporal resolution. 22,23 It has been used to observe the motion of motor proteins, 24,25 the interactions of membrane proteins, 26 protein diffusion in lipid membranes and live cells, 27,28 label-free detection of single proteins, 29,30 and protein conformational changes. 31 Here, we explore fine details of the interaction of the Ase1 protein with the MT lattice by employing fast 3D tracking of single proteins with nanometer precision and 20-µs temporal resolution.…”

Section: Introductionmentioning

confidence: 99%

Fast leaps between millisecond confinements govern Ase1 diffusion along microtubules

Bujak

Holanová

Marín

et al. 2021

Preprint

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Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, we used interferometric scattering microscopy to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. We resolved millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments, revealing Ase1's mode of diffusive translocation along the microtubule's periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. We anticipate that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, will provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

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High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy

Abstract: Direct visualization of single 10 nm nanoparticles at 1000 frames per second is achieved by using coherent brightfield (COBRI) microscopy.

Cited by 59 publications

References 56 publications

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

Fast leaps between millisecond confinements govern Ase1 diffusion along microtubules

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