Plasmid ID by comparing individual experimental barcodes to a theory database.

<p>(Left) Pearson correlation coefficients between the experimental barcodes (<i>pUUH</i>, <i>pEC005A</i> and <i>pEC005B</i>) and the plasmid theory database (as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref024" target="_blank">24</a>]) were calculated and turned into histograms. As input experimental barcode we used: unfiltered single-time frame (<i>ust</i>) barcodes, filtered (with Gaussian filter) single-time frame (<i>fst</i>) barcodes, and time-averaged (<i>ta</i>) barcodes. The vertical line gives the correlation coefficient for the correct plasmid. Notice that <i>ust</i> barcodes are not very good at plasmid ID (s-scores between 0.4-10%), but, <i>fsb</i> and <i>ta</i> barcodes are better (with s-scores less than 3%). Experiments were only matched to theory barcodes which had a length within ±3<i>σ</i>_length of the experimental barcode, with <i>σ</i>_length = {23, 7, 15} pixels for <i>pUUH, pEC005A and pEC005B</i>, respectively. The Gumbel fits to the histograms were done using the same method as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref024" target="_blank">24</a>]. (Right) The panels on the right show the change in between the experiment and the theoretical barcode (i.e., theory barcodes from <i>pUUH</i>, <i>pEC005A</i> or <i>pEC005B</i>, respectively) after filtering single time frame barcodes. On the top-right plot, in grey is shown the correlation coefficients for the case without filtering, and in blue/green the correlation after filtering. The four lines in the top plot are smoothed for visualization purposes and are moving averages of the result over the 300 nearest neighbor barcodes (each of the three plasmid types treated separately). The right-bottom panel shows the change for all 6400 single time-frames for the case of Gaussian filter. On average, the single frames match to theory improves by 0.11 ± 0.04 points after filtering (average over all three filters).</p

Kymographs from plasmids

<div>DNA barcodes used in 'Noise Reduction in Single Time Frame Optical DNA Maps' (2017), PLOS One. </div><div><br></div><div>DNA kymographs from plasmids obtained from nano-channel based competitive binding assays. The set consists of 32 kymograph from three types of plasmids: pUUH239.2 (8 kymographs), pEC005A (11 kymographs) and pEC005B (13 kymographs). Each kymograph corresponds to one molecule and contains 200 single time frames of 0.1s each (6400 single time frames in total).</div><div><br></div><div>The 32 raw kymographs are in named 'plasmidName_moleculeNumber'. </div><div><br></div

Increase in the Pearson correlation coefficient between filtered a single time frame and the aligned kymograph time average.

<p>The table shows the improvement, , in the Pearson correlation coefficient, (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.e011" target="_blank">Eq (2)</a>), between each single time frame barcode and its aligned kymograph time average after filtering the single frame. Correlation coefficients were averaged over all 6400 time-frame barcodes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.g003" target="_blank">Fig 3</a>) for each type of filter used (Gaussian, Moving Average and Windowed-Sinc filter). The improvement is defined as , where is the correlation coefficient between the filtered single time-frame (<i>fst</i>) barcode and the time averaged (<i>ta</i>) barcode, and is the correlation coefficient between the unfiltered (noisy) single time frame barcode and the kymograph time average. We see that all filters lead to a similar average improvement in the correlation, roughly 0.2 points. Results for by type of plasmid (<i>pUUH</i>, <i>pEC005A</i>, <i>pEC005B</i>) are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.s001" target="_blank">S1 Table</a> in Supplementary Information.</p

Examples of optical mapping kymographs (raw, aligned and time averaged) from a linearized plasmid DNA stretched in a nanochannel.

<p>(a) A raw kymograph (i.e. a stack of images of stretched and fluorescently labeled DNA) from a linearized plasmid obtained using the competitive binding assay described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref038" target="_blank">38</a>]. The horizontal direction corresponds to the nano-channel extension (i.e., the direction of the stretched DNA) and vertical axes are different time points (0.1 s between time frames). The kymograph consists of 200 single time frame images (d, e). (b) The raw kymograph is aligned (using <i>WPAlign</i> from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref041" target="_blank">41</a>]) and subsequently averaged over all 200 time frames in order to produce (c, f) noise-reduced, time averaged DNA barcodes. The noisy curves in (d, e) represent the intensities along two single time-frame (snap-shot) barcodes, see (a). For visualization purposes, the snap-shot barcodes were shifted globally to the position where they have the maximum correlation coefficient with the time-averaged barcode. The challenge addressed in this study is how to make the noisy single-time frame barcodes of the form illustrated above resemble the (more reproducible) time-averaged barcode to a higher degree by using low-pass filtering. The barcodes shown are from plasmid <i>pEC005A</i>, see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179041#pone.0179041.ref024" target="_blank">24</a>] for further information about the experiments.</p

Summary of three low-pass filters used in this study: Gaussian, Moving average and Window-Sinc filter.

<p>Summary of three low-pass filters used in this study: Gaussian, Moving average and Window-Sinc filter.</p

Example barcode filtered using our noise-reducing filtering method.

<p>(grey) A noisy single time-frame (snap-shot) barcode taken with 0.1s exposure time. (orange) The time average of the aligned kymograph. Such time-averages are used as reference (“true” barcode) throughout this study and used to judge the quality of the filtering process. (blue barcodes) From top to bottom Gaussian, Moving average and Sinc filter, respectively, is applied recursively to the single-time frame barcode (grey) until all peaks in the filtered barcode have a FWHM of at least that of the FWHM of the PSF of the system. Notice the visual similarity of the filtered barcodes to the time-averaged barcode. The Pearson correlation coefficient between the time average of the aligned kymograph and the barcode before and after the the filtering changes from 0.6 without the filtering, to 0.8 after filtering. The original raw kymograph consists of 200 single time-frame shots (exposure time 0.1s) from plasmid <i>pEC005B</i>.</p

Lipid-Based Passivation in Nanofluidics

Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA–DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein–DNA interactions with high spatial and temporal resolution

Binding of Thioflavin‑T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction

Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative <i>in vitro</i> studies

Self-Assembly and Near Perfect Macroscopic Alignment of Fluorescent Triangulenium Salt in Spin-Cast Thin Films on PTFE

Highly fluorescent, discotic trioxatriangulenium dyes were aligned by simple spin-casting on substrates with friction transferred PTFE layers. The fluorescent crystalline thin films show near perfect macroscopic alignment on centimeter large areas directly from spin-casting. Gracing Incidence X-ray Diffraction (GIXD) unambiguously allowed the determination of a long-range order unit cell as well as its orientation with respect to the PTFE fibers. Further analysis of the X-ray data, in conjunction with polarized absorption spectroscopy, suggest a lamellar packing model with alternating layers of alkyl chains and ionic dyes oriented parallel to the substrate. This structure results in a highly anisotropic electrostatic potential around the cationic chromophore, causing significant shifts in energy and orientation of the optical transitions. Thus, the optical properties of the material are, to a large extent, controlled by the position of the otherwise inert PF₆^– counterions. The bright fluorescence from the films is also polarized parallel to the PTFE alignment layer. Doping of the thin films with fluorescent energy acceptor traps shows that efficient exciton migration takes place in the thin films. The excellent exciton transfer capabilities, in conjunction with the perfect alignment, might be of interest in future applications in solar energy harvesting or as thin film sensors

Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers

Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59–184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δ<i>f</i>) and in dissipation (Δ<i>D</i>). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine