Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging

Arroyo, Jaime Ortega; Cole, Daniel; Kukura, Philipp

doi:10.1038/nprot.2016.022

Cited by 138 publications

(171 citation statements)

References 35 publications

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“…We used a custom-built iSCAT microscope2324 to follow the colloidal aggregate dynamics and perovskite crystal growth. The microscope set-up is as follows.…”

Section: Methodsmentioning

confidence: 99%

Mechanism for rapid growth of organic–inorganic halide perovskite crystals

et al. 2016

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Optoelectronic devices based on hybrid halide perovskites have shown remarkable progress to high performance. However, despite their apparent success, there remain many open questions about their intrinsic properties. Single crystals are often seen as the ideal platform for understanding the limits of crystalline materials, and recent reports of rapid, high-temperature crystallization of single crystals should enable a variety of studies. Here we explore the mechanism of this crystallization and find that it is due to reversible changes in the solution where breaking up of colloids, and a change in the solvent strength, leads to supersaturation and subsequent crystallization. We use this knowledge to demonstrate a broader range of processing parameters and show that these can lead to improved crystal quality. Our findings are therefore of central importance to enable the continued advancement of perovskite optoelectronics and to the improved reproducibility through a better understanding of factors influencing and controlling crystallization.

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“…We used a custom-built iSCAT microscope2324 to follow the colloidal aggregate dynamics and perovskite crystal growth. The microscope set-up is as follows.…”

Section: Methodsmentioning

confidence: 99%

Mechanism for rapid growth of organic–inorganic halide perovskite crystals

et al. 2016

Self Cite

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“…These features are time invariant, do not change with lateral sample displacements, and can therefore be removed. We call the process of isolating and subsequently removing these signatures “flat-fielding” (Arroyo, Cole, & Kukura, 2016). First, the sample is translated laterally while recording a stack of 100 images.…”

Section: Methods and Protocolsmentioning

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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“…3a) and iSCAT imaging of unlabeled microtubules revealed that EB3 decorated growing plus and minus ends of wt microtubules in the expected comet-like manner ( Fig. 2a-b, Supplementary Video 4) 12,13,16 . In clear contrast, EB3 decorated the entire lattice of GTPasedeficient E254A mutant microtubules ( Fig.…”

mentioning

confidence: 92%

“…3i). Quantitative analysis of the intensity profiles allows to extract the characteristic comet lengths and, knowing the measured growth speeds, also the lifetime of the EB3 binding sites 12,13,17,26 . This revealed a four-fold increase in the lifetime of the EB3 binding sites at the ends of E254D microtubules compared to wt microtubules (Fig 3i).…”

mentioning

confidence: 99%

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

Roostalu

Thomas

Cade

et al. 2019

Preprint

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Microtubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1 . Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5 . The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6 . Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.2

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Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging

Cited by 138 publications

References 35 publications

Mechanism for rapid growth of organic–inorganic halide perovskite crystals

Mechanism for rapid growth of organic–inorganic halide perovskite crystals

Interferometric Scattering Microscopy for the Study of Molecular Motors

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

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