Continuous and discrete phasor analysis of binned or time-gated periodic decays

Time-resolved analysis of periodically excited luminescence decays by the phasor method in the presence of time-gating or binning is revisited. Analytical expressions for discrete configurations of square gates are derived and the locus of the phasors of such modified periodic single-exponential decays is compared to the canonical uni-versal semicircle. The effects of IRF offset, decay truncation and gate shape are also discussed. Finally, modified expressions for the phase and modulus lifetimes are pro-vided for some simple cases. A discussion of a modified phasor calibration approach is presented, and illustration of the new concepts with examples from the literature conclude this work

Nanometer Distance Measurements between Multicolor Quantum Dots

Quantum dot dimers made of short double-stranded DNA molecules labeled with different color quantum dots at each end were imaged using multicolor stage-scanning confocal microscopy. This approach eliminates chromatic aberration and color registration issues usually encountered in other multicolor imaging techniques. We demonstrate nanometer accuracy in individual distance measurement by suppression of quantum dot blinking and thoroughly characterize the contribution of different effects to the variability observed between measurements. Our analysis opens the way to accurate structural studies of biomolecules and biomolecular complexes using multicolor quantum labeling

Background rates as a function of time.

<p>Estimated background rate as a function of time for two μs-ALEX measurements. Different colors represent different photon streams. (<i>Panel a</i>) A measurement performed with a sealed sample chamber exhibiting constant a background as a function of time. (<i>Panel b</i>) A measurement performed on an unsealed sample exhibiting significant background variations due to sample evaporation and/or photobleaching (likely impurities on the cover-glass). These plots are produced by the command dplot(d, timetrace_bg) after estimation of background. Each data point in these figures is computed for a 30 s time window.</p

E-S histogram after filtering out D-only and A-only populations.

<p>2-D ALEX histogram after selection of FRET population using the composition of two burst selection filters: (1) selection of bursts with counts in D_ex stream larger than 15; (2) selection of bursts with counts in A_exA_em stream larger than 15. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160716#pone.0160716.g004" target="_blank">Fig 4</a> where all burst populations (FRET, D-only and A-only) are reported.</p

BVA distribution for a hairpin sample undergoing dynamics.

<p>The left panel shows the E-S histogram for a single stranded DNA sample (<i>A</i>₃₁-TA, see text), designed to form a transient hairpin in 400mM NaCl. The right panel shows the corresponding BVA plot. Since the transition between hairpin and open structure causes a significant change in FRET efficiency, <i>s</i>_<i>E</i> lies largely above the static standard deviation curve (<i>red curve</i>).</p

Photon selection syntax (non-ALEX).

<p>Photon selection syntax (non-ALEX).</p

E-S histogram showing FRET, D-only and A-only populations.

<p>A 2-D ALEX histogram and marginal E and S histograms for a 40-bp dsDNA with D-A distance of 17 bases (Donor dye: ATTO550, Acceptor dye: ATTO647N). Bursts are selected with a size-threshold of 30 photons, including A_ex photons. The plot is obtained with alex_jointplot(ds). The 2D E-S distribution plot (join plot) is an histogram with hexagonal bins, which reduce the binning artifacts (compared to square bins) and naturally resembles a scatter-plot when the burst density is low (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160716#pone.0160716.s004" target="_blank">S4 Appendix</a>). Three populations are visible: FRET population (middle), D-only population (top left) and A-only population (bottom, <i>S</i> < 0.2). Compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160716#pone.0160716.g005" target="_blank">Fig 5</a> where the FRET population has been isolated.</p

FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET

<div><p>Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.</p></div

Inter-photon delays fitted with and exponential function.

<p>Experimental distributions of inter-photon delays (<i>dots</i>) and corresponding fits of the exponential tail (<i>solid lines</i>). (<i>Panel a</i>) An example of inter-photon delays distribution (<i>red dots</i>) and an exponential fit of the tail of the distribution (<i>black line</i>). (<i>Panel b</i>) Inter-photon delays distribution and exponential fit for different photon streams as obtained with dplot(d, hist_bg). The <i>dots</i> represent the experimental histogram for the different photon streams. The <i>solid lines</i> represent the corresponding exponential fit of the tail of the distributions. The legend shows abbreviations of the photon streams and the fitted background rates.</p

Photon selection syntax (ALEX).

<p>Photon selection syntax (ALEX).</p