Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy

Spindler, Susann; Ehrig, Jens; König, Katharina; Nowak, Tristan; Piliarik, Marek; Stein, Hannah; Taylor, Richard W.; Garanger, Élisabeth; Lecommandoux, Sébastien; Alves, Isabel D.; Sandoghdar, Vahid

doi:10.1088/0022-3727/49/27/274002

Cited by 71 publications

(118 citation statements)

References 30 publications

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“…To study loosely and tightly docked intermediates, a GUV containing SNARE acceptor complexes was immobilized with a micropipette (see Figure 1a) and brought in close proximity to the cover glass. Next, we added large unilamellar vesicles (LUVs, 80-100 nm in diameter) containing synaptobrevin (syb) and used iSCAT microscopy to track LUVs on the lower hemisphere of individual GUVs from a single focus position (22). The LUVs readily bound to the GUV and either fused with it (as for WT syb, ref.…”

Section: Resultsmentioning

confidence: 99%

“…We employed interferometric scattering (iSCAT) microscopy to record three-dimensional (3D) trajectories of particle diffusion on the surface of GUVs. This technique has been shown to reach microsecond temporal resolution and nm precision in detecting and tracking individual nanoparticles via the interference of their scattered light with a reference beam (22,23, and see Methods). Using iSCAT, we could investigate the diffusion of unlabeled vesicles arrested at a loosely or tightly docked intermediate state using synaptobrevin point mutants as previously described (17).…”

Section: Introductionmentioning

confidence: 99%

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Differential diffusional properties in loose and tight docking prior to membrane fusion

Witkowska

Spindler

Mahmoodabadi

et al. 2020

Preprint

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Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study due to their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion. membrane fusion intermediates | iSCAT microscopy | SNARE proteins | particle tracking | tethered diffusion | GUV Correspondence: rjahn@gwdg.de (R.J.), vahid.sandoghdar@mpl.mpg.de (V.S.), and agata.witkowska@wp.eu (A.W.)

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differential diffusional properties in loose and tight docking prior to membrane fusion

Witkowska

Spindler

Mahmoodabadi

et al. 2020

Preprint

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“…In the following years, iSCAT was extended in our laboratory to different illumination and detection conditions [114,115] and used to detect single unlabeled viruses [116,117], semiconductor quantum dots [115], lipid vesicles [118,119] and unlabeled proteins [120]. In more recent years, several other groups have also successfully applied different illumination/detection variants of iS-CAT to detect single proteins [109,121,122], single viruses [83,107], lipids [123,124] and other nanoparticles [82], and even charge carriers [108].…”

Section: Foundationsmentioning

confidence: 99%

“…A more fundamental limit is introduced when concerned over photodamage of the sample when using very strong illuminations since every realistic substance also absorbs light at every wavelength, even if very weakly. When performing iSCAT on biological samples, illumination intensities on the order of 0.001-0.1 mWµm −2 have been reported for membranes [119,139], with powers as high as 5 mWµm −2 [119] for the fastest MHz imaging rates.…”

Section: Fast Measurements: No Saturationmentioning

confidence: 99%

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Interferometric Scattering (iSCAT) Microscopy and Related Techniques

Taylor

Sandoghdar

2019

Biological and Medical Physics, Biomedical Engineering

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Interferometric scattering (iSCAT) microscopy is a powerful tool for label-free sensitive detection and imaging of nanoparticles to high spatio-temporal resolution. As it was born out of detection principles central to conventional microscopy, we begin by surveying the historical development of the microscope to examine how the exciting possibility for interferometric scattering microscopy with sensitivities sufficient to observe single molecules has become a reality. We discuss the theory of interferometric detection and also issues relevant to achieving a high detection sensitivity and speed. A showcase of numerous applications and avenues of novel research across various disciplines that iSCAT microscopy has opened up is also presented.1

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Nanoparticle Transport Through Polymers and Along Interfaces

Boyle

Maguire

Rose

et al. 2022

Macromolecular Engineering

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Transport of nanoparticles has received increasing attention for both fundamental interest to test prevailing models as well as practical interest in separations and self‐healing applications. Methods for measuring particle dynamics are briefly reviewed to orient the reader to different methodologies. The transport of nanoparticles through polymer melts, solutions, and cross‐linked networks is described with emphasis on systems where the nanoparticle is on the length scale of the defining structure including the tube size, correlation length, and mesh size, respectively. Because nanoparticles can interact with the different mediums, the interactions between nanoparticles and polymer and the subsequent impacts on dynamics are described. The transport of particles at liquid–solid (polymer brush) interfaces is addressed due to growing interest in applications ranging from understanding dynamics in porous media to the translocation of DNA. Both hard particles and polymer grafted particles (soft particles) are described. The objective of this article is to provide the reader with fundamental descriptions of phenomena while reviewing the current state of understanding. Further directions will leverage advances in inorganic chemistry to prepare monodisperse, functional nanoparticles as well as polymer chemistry to create novel media, such as networks with monodisperse mesh size.

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Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy

Cited by 71 publications

References 30 publications

Differential diffusional properties in loose and tight docking prior to membrane fusion

Differential diffusional properties in loose and tight docking prior to membrane fusion

Interferometric Scattering (iSCAT) Microscopy and Related Techniques

Nanoparticle Transport Through Polymers and Along Interfaces

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