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34 results for “FRET”
Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy
<p>FRET-based methods are a special tool for detecting interactions between (bio)molecules and their immediate environment. The spatial distribution of molecular interactions and functional states can be seen using FLIM (Fluorescence Lifetime IMaging) and FRET imaging. The spatial information, accuracy, and dynamic range of the observed signals are, however, constrained by the fact that conventional FLIM and FRET imaging only provides average information over an ensemble of molecules within a diffraction-limited volume. On the other hand, conventional Single Molecule Localization Microscopy (SMLM) relies on highly sensitive multi-pixel detectors (e.g. sCMOS or EM-CCD) whose time resolution is not suitable for fluorescence lifetime measurements.</p> <p>Here, we demonstrate a method for obtaining super-resolved FRET imaging using confocal fluorescence-lifetime single-molecule localization microscopy. The proof of concept was carried out using a DNA origami sample for performing DNA-PAINT measurements in combination with fluorogenic probes for reducing background signal. With this method, We show that FRET events separated by sub-diffraction distances can be distinguished based on lifetime modifications.</p>
Neutron spin echo and intramolecular FRET and DEER-EPR measurements on hGBP1 (human guanylate binding protein 1)
<p>Neutron spin echo (NSE), double electron–electron resonance (<em>DEER</em>) <em>EPR</em>, ensemble time-correlated single photon counting (eTCSPC) fluorescence, and single-molecule detection (SMD) fluorescence spectroscopy data of the human guanylate binding protein 1 (hGBP1).</p> <p>CSH prepared samples for smFRET and performed protein activity assays. TV prepared sampled for EPR measurements. TOP, CSH, and AV performed the smFRET measurements under the supervision of CAMS. TOP analyzed the smFRET measurements. JPK performed and analyzed the EPR measurements.</p>
Dynamic FRET example videos related to "Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages"
<p>Videos of dynamic switching between iso-I and iso-II conformations of a holiday junction at 50 mM Magnesium resulting in high and low FRET from Cy3 and Alexa647 labels positioned on the arms. Holiday junctions are surface immobilized through a biotin attachment and imaged using TIRF microscopy. The camera sensor is split using a dual view so that the acceptor emission is on the top and the donor emission is on the bottom. Videos from each position are provided as compressed zip files containing a sequence of tif files and associated metadata text file. Image sequences were collected using Micro-Manager 2.0 using ALEX or alternating laser excitation with alternating 637 and 532 pulses separated as two different channels. Beam profile images are provided for 637 and 532 excitation allowing for correction of the non-uniform beam profiles. The following 2D affine transformation matrix can be used to transform from the top acceptor emission region to the bottom donor emission region during processing.</p> <p>Affine 2D transformation from top to bottom: (m00, m01, m02, m10, m11, m12), (1.00276, 0.000208, 1.01236, 0.000267, 1.00312, 507.21025)</p> <p>A detailed image processing workflow for this dataset using Mars can be found under the example section at <a href="https://duderstadt-lab.github.io/mars-docs/">https://duderstadt-lab.github.io/mars-docs/</a> or directly at <a href="https://duderstadt-lab.github.io/mars-docs/examples/FRET_dynamic/">https://duderstadt-lab.github.io/mars-docs/examples/FRET_dynamic/</a></p>
Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α-Fig. 2def
<p>smTIRF-FRET Data for Fig 2, for "Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α"</p>
Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α-Fig. 7cde
<p>smTIRF-FRET Data for Fig 7, for "Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α"</p>
Microsecond ALEX FRET analysis notebook using FRETbursts - corrections, FRET burst analysis of recurring molecules, FCS, 2CDE & BVA
<p>The herein Python notebook uses FRETbursts (download from here: https://github.com/tritemio/FRETBursts) to show how to analyze microsecond alternating laser excitation (usALEX) confocal-based FRET measurements of freely diffusing single molecules. It includes a step-by-step calculation and implementation of correction factors, donor fluorescence leakage to the acceptor detection channel (Lk), acceptor fluorescence caused by acceptor excitation by the laser intended for donor excitation (Dir), the imbalance in acceptor/donor fluorecence quantum yields and detection efficiencies (Gamma) and the imbalance in donor/acceptor excitation yields (Beta). The notebook implments a global Gamma correction, assuming the Gamma correction factor is constant for all FRET populations, based on the procedure from Lee et al. 2005. Burst search for showing the FRET population is a dual-channel burst search. After correction, the corrected FRET histogram is presented (after burst selection takes into account Beta & Gamma corrected burst sizes). We also present analysis of bursts from recurring molecules, as well as the FCS (a bit irrelevant here, due to the lasr alternation in microeconds), 2CDE & BVA plots, helping in identifying whether a FRET population is a time average of FRET states, occurring faster then molecular diffusion time, or wheather the FRET population is static and represents a single conformational state. The sample data is a result of measurement of 50 pM of hairpin 3 presented in Tsukanov et al. 2013, labeled with ATTO dyes (ATTO 550 & ATTO 647N as donor and acceptor dyes, respectively) - 532 & 640 nm cw excitation, with an alternation period of 50 microseconds. </p>
Screening routine for integrative dynamic structural biology using SAXS and intramolecular FRET and DEER-EPR on hGBP1 (human guanalyte binding protein 1)
<p>Initial and selected ensemble for major and minor species of the human guanalyte binding protein 1 with scripts for the reading routine to combine and analyse jointly SAXS, EPR and FRET data.</p>
Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α-Fig. 1df
<p>smTIRF-FRET Data for Fig 1, for "Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α"</p>
A FRET based biosensor for measuring Gα13 activation in single cells
<p>The figures and raw data that are presented in the paper "A FRET based biosensor for measuring Gα13 activation in single cells"</p>
Datasets for "Precision and accuracy of single-molecule FRET measurements – a multi-laboratory benchmark study"
<p>Supplementary material (raw data) for Fig. 2 in "<strong>Precision and accuracy of single-molecule FRET measurements – a multi-laboratory benchmark study</strong>" to be published with Nature Methods</p> <p>The confocal data is given in ht3 and hdf5 format.</p> <p>For the TIRF data the original TIFF-stacks are uploaded including the calibration files.</p>
FRET efficiency of fluorescent protein pairs with mTurquoise2 as donor
<p>This dataset is was produced in the study of mTurquoise2 paired with a number of spectrally diverse fluorescent proteins as acceptors. For details and see the original paper: <a href="http://www.nature.com/articles/s41598-017-12212-x">https://www.nature.com/articles/s41598-017-12212-x</a></p> <p>These data are reported in table 3 of that paper.</p>
Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation
<p>This includes</p> <p>1- the data set collected at 100mW = 0.04 kW/cm^2 for the Cy3-Cy5 dye pair that is reported in the paper in Figure 2c and SI Figure 14ab (Supplementary Additional Data 1)</p> <p>2- Matlab script (zetacorrect.m) to do the simplified zeta correction and a Mathematica notebook (TestingSimplifiedZeta.nb) to show the derivation of simplified zeta correction</p> <p>3- the raw data corresponding to Figure 2c (Supplementary Additional Data 2)</p> <p>4- Main Text and Supplementary Figures Source Data</p> <p>----<br>## Citations</p> <p>If you use any of these shared materials, please use the relevant citatations:</p> <p> ```<br> * Avik K. Pati^{*}, Zeliha Kilic^{*}, Maxwell I. Martin^{#}, Daniel S. Terry^{#}, Alessandro Borgia, Sukanta Bar,<br> Steffen Jockusch, Roman Kiselev, Roger B. Altman, Scott C. Blanchard,<br> Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation,2024,Nature Methods<br> ```</p> <p>----<br>## Relevant Publications</p> <p>In our smFRET data analysis, we used SPARTAN<br>* Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond scale:<br> [paper](https://www.nature.com/articles/nmeth.3769)</p>
Can DyeCycling break the photobleaching limit in single-molecule FRET?
<p><strong>Abstract. </strong>Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce <em>‘DyeCycling’</em>, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.</p>
Dataset: Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single molecule three-color FRET
<p>Dataset: Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single molecule three-color FRET</p>
Data deposition for "Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins"
<p>The deposited data for the publication "Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins".</p> <p>Data contains folder and sub-folders for the raw data, Main excel sheet named as "MasterTable_FRET-Challenge-Protein-Dynamics_Nat_Meth_Agam et al" has most of the data used in the publication. Another excel sheets "Data List for FIgures for Agam et al_revised" and "Data List for Supplmentary FIgures for Agam et al_revised" have the information regarding the Figure-wise data description and where the respective data locates.</p>
Data for: Optimal inference of molecular interaction dynamics in FRET microscopy
<p>Intensity-based time-lapse fluorescence resonance energy transfer (FRET) microscopy has been a major tool for investigating cellular processes, converting otherwise unobservable molecular interactions into fluorescence time series. However, inferring the molecular interaction dynamics from the observables remains a challenging inverse problem, particularly when measurement noise and photobleaching are nonnegligible—a common situation in single-cell analysis. The conventional approach is to process the time-series data algebraically, but such methods inevitably accumulate the measurement noise and reduce the signal-to-noise ratio (SNR), limiting the scope of FRET microscopy. Here, we introduce an alternative probabilistic approach, B-FRET, generally applicable to standard 3-cube FRET-imaging data. Based on filtering theory, B-FRET implements a statistically optimal way to infer molecular interactions and thus drastically improves the SNR. We validate B-FRET using simulated data and then apply it to real data, including the notoriously noisy in vivo FRET time series from individual bacterial cells to reveal signaling dynamics otherwise hidden in the noise.</p>
Upconversion FRET quantitation: the role of donor photoexcitation mode and compositional architecture on the decay and intensity based responses
<p>Abstract</p> <p>Lanthanide-doped colloidal nanoparticles capable of photon upconversion (UC) offer long luminescence lifetimes, narrowband absorption and emission spectra, and efficient anti-Stokes emission. These features are highly advantageous for Förster Resonance Energy Transfer (FRET) based detection. Upconverting nanoparticles (UCNPs) as donors may solve the existing problems of molecular FRET systems, such as photobleaching and limitations in quantitative analysis, but these new labels also bring new challenges. Here we have studied the impact of the core-shell compositional architecture of upconverting nanoparticle donors and the mode of photoexcitation on the performance of UC-FRET from UCNPs to Rose Bengal (RB) molecular acceptor. We have quantitatively compared luminescence rise and decay kinetics of Er<sup>3+</sup> emission using core-only NaYF<sub>4</sub>: 20% Yb, 2% Er and core-shell NaYF<sub>4</sub>: 20% Yb @ NaYF<sub>4</sub>: 20% Yb, 5% Er donor UCNPs under three photoexcitation schemes: (1) direct short-pulse photoexcitation of Er<sup>3+</sup> at 520 nm; indirect photoexcitation of Er<sup>3+</sup> through Yb<sup>3+</sup> sensitizer with (2) 980 nm short (5–7 ns) or (3) 980 nm long (4 ms) laser pulses. The donor luminescence kinetics and steady-state emission spectra differed between the UCNP architectures and excitation schemes. Aiming for highly sensitive kinetic upconversion FRET-based biomolecular assays, the experimental results underline the complexity of the excitation and energy-migration mechanisms affecting the Er<sup>3+</sup> donor responses and suggest ways to optimize the photoexcitation scheme and the architecture of the UCNPs used as luminescent donors.</p>
Data for: Optimal inference of molecular interaction dynamics in FRET microscopy
Open the record for dataset details and reuse information.
Data from: Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems
Open the record for dataset details and reuse information.
Simultaneous readout of multiple FRET pairs using photochromism: cell traces and derived data
<p>All cell traces and derived datasets for our publication on 'Simultaneous readout of multiple FRET pairs using photochromism'.</p>
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