iSMS: single-molecule FRET microscopy software

Preus, Søren; Noer, Sofie L.; Hildebrandt, Lasse L.; Gudnason, Daniel; Birkedal, Victoria

doi:10.1038/nmeth.3435

Cited by 111 publications

(129 citation statements)

References 5 publications

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“…Fluorescence from the donor and acceptor fluorophores was spatially separated onto the EMCCD camera (Andor, iXON 3) by a wedge mirror. Movies were recorded with a 200 ms integration time per frame with a total length of 600 s (10 min) and were analyzed using the home-built iSMS software (34). Only the trajectories with single-step donor and/or acceptor photobleaching, or showing clear anti-correlated donor–acceptor dynamics, were selected for further analysis.…”

Section: Methodsmentioning

confidence: 99%

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

et al. 2016

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G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.

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Section: Methodsmentioning

confidence: 99%

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

et al. 2016

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“…The videos were analyzed employing the iSMS software (Preus et al 2015) using the program's default settings. Molecule spots were detected using a threshold of 200 for Alexa647 spots.…”

Section: Single-molecule Coimmunoprecipitation Experimentsmentioning

confidence: 99%

Structural and functional insights into the fly microRNA biogenesis factor Loquacious

Jakob¹,

Treiber²,

Treiber³

et al. 2016

RNA

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In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.

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“…1 b, inset) (5,(12)(13)(14)(15)20). The advantage of the local aperture mean is that it is simple, fast, and robust when the peak of interest in the image frame is well isolated from neighboring peaks (17). In this work, the use of background estimators based on local apertures was much faster computationally (by orders of magnitude) than Gaussian profile fitting.…”

Section: Performance Of Traditional Background Estimatorsmentioning

confidence: 91%

“…Data analysis was performed using the smFRET microscopy software package iSMS (http://isms.au.dk) (17). Colocalized donor/acceptor fluorescence spots were identified.…”

Section: Methodsmentioning

confidence: 99%

Optimal Background Estimators in Single-Molecule FRET Microscopy

Preus

Hildebrandt

Birkedal

2016

Biophysical Journal

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Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Fö rster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

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iSMS: single-molecule FRET microscopy software

Cited by 111 publications

References 5 publications

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

Structural and functional insights into the fly microRNA biogenesis factor Loquacious

Optimal Background Estimators in Single-Molecule FRET Microscopy

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