Nanometrology and super-resolution imaging with DNA

Peer reviewe

Quantifying absolute addressability in DNA origami with molecular resolution

Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines

Quantitative analysis of single particle trajectories: mean maximal excursion method

An increasing number of experimental studies employ single particle tracking to probe the physical environment in complex systems. We here propose and discuss new methods to analyze the time series of the particle traces, in particular, for subdiffusion phenomena. We discuss the statistical properties of mean maximal excursions, i.e., the maximal distance covered by a test particle up to time t. Compared to traditional methods focusing on the mean squared displacement we show that the mean maximal excursion analysis performs better in the determination of the anomalous diffusion exponent. We also demonstrate that combination of regular moments with moments of the mean maximal excursion method provides additional criteria to determine the exact physical nature of the underlying stochastic subdiffusion processes. We put the methods to test using experimental data as well as simulated time series from different models for normal and anomalous dynamics, such as diffusion on fractals, continuous time random walks, and fractional Brownian motion.Comment: 10 pages, 7 figures, 2 tables. NB: Supplementary material may be found in the downloadable source file

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05420j Click here for additional data file.

Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site

The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions

Bayesian Multiple Emitter Fitting using Reversible Jump Markov Chain Monte Carlo

In single molecule localization-based super-resolution imaging, high labeling density or the desire for greater data collection speed can lead to clusters of overlapping emitter images in the raw super-resolution image data. We describe a Bayesian inference approach to multiple-emitter fitting that uses Reversible Jump Markov Chain Monte Carlo to identify and localize the emitters in dense regions of data. This formalism can take advantage of any prior information, such as emitter intensity and density. The output is both a posterior probability distribution of emitter locations that includes uncertainty in the number of emitters and the background structure, and a set of coordinates and uncertainties from the most probable model

A mock circulation loop to test extracorporeal CO2 elimination setups

Background: Extracorporeal carbon dioxide removal (ECCO2R) is a promising yet limited researched therapy for hypercapnic respiratory failure in acute respiratory distress syndrome and exacerbated chronic obstructive pulmonary disease. Herein, we describe a new mock circuit that enables experimental ECCO2R research without animal models. In a second step, we use this model to investigate three experimental scenarios of ECCO2R: (I) the influence of hemoglobin concentration on CO2 removal. (II) a potentially portable ECCO2R that uses air instead of oxygen, (III) a low-flow ECCO2R that achieves effective CO2 clearance by recirculation and acidification of the limited blood volume of a small dual lumen cannula (such as a dialysis catheter). Results: With the presented ECCO2R mock, CO2 removal rates comparable to previous studies were obtained. The mock works with either fresh porcine blood or diluted expired human packed red blood cells. However, fresh porcine blood was preferred because of better handling and availability. In the second step of this work, hemoglobin concentration was identified as an important factor for CO2 removal. In the second scenario, an air-driven ECCO2R setup showed only a slightly lower CO2 wash-out than the same setup with pure oxygen as sweep gas. In the last scenario, the low-flow ECCO2R, the blood flow at the test membrane lung was successfully raised with a recirculation channel without the need to increase cannula flow. Low recirculation ratios resulted in increased efficiency, while high recirculation ratios caused slightly reduced CO2 removal rates. Acidification of the CO2 depleted blood in the recirculation channel caused an increase in CO2 removal rate. Conclusions: We demonstrate a simple and cost effective, yet powerful, “in-vitro” ECCO2R model that can be used as an alternative to animal experiments for many research scenarios. Moreover, in our approach parameters such as hemoglobin level can be modified more easily than in animal models

Comparison of Serial and Parallel Connections of Membrane Lungs against Refractory Hypoxemia in a Mock Circuit

Extracorporeal membrane oxygenation (ECMO) is an important rescue therapy method for the treatment of severe hypoxic lung injury. In some cases, oxygen saturation and oxygen partial pressure in the arterial blood are low despite ECMO therapy. There are case reports in which patients with such instances of refractory hypoxemia received a second membrane lung, either in series or in parallel, to overcome the hypoxemia. It remains unclear whether the parallel or serial connection is more effective. Therefore, we used an improved version of our full-flow ECMO mock circuit to test this. The measurements were performed under conditions in which the membrane lungs were unable to completely oxygenate the blood. As a result, only the photometric pre- and post-oxygenator saturations, blood flow and hemoglobin concentration were required for the calculation of oxygen transfer rates. The results showed that for a pre-oxygenator saturation of 45% and a total blood flow of 10 L/min, the serial connection of two identical 5 L rated oxygenators is 17% more effective in terms of oxygen transfer than the parallel connection. Although the idea of using a second membrane lung if refractory hypoxia occurs is intriguing from a physiological point of view, due to the invasiveness of the solution, further investigations are needed before this should be used in a wider clinical setting

Comparison of Circular and Parallel-Plated Membrane Lungs for Extracorporeal Carbon Dioxide Elimination

Extracorporeal carbon dioxide removal (ECCO2R) is an important technique to treat critical lung diseases such as exacerbated chronic obstructive pulmonary disease (COPD) and mild or moderate acute respiratory distress syndrome (ARDS). This study applies our previously presented ECCO2R mock circuit to compare the CO2 removal capacity of circular versus parallel-plated membrane lungs at different sweep gas flow rates (0.5, 2, 4, 6 L/min) and blood flow rates (0.3 L/min, 0.9 L/min). For both designs, two low-flow polypropylene membrane lungs (Medos Hilte 1000, Quadrox-i Neonatal) and two mid-flow polymethylpentene membrane lungs (Novalung Minilung, Quadrox-iD Pediatric) were compared. While the parallel-plated Quadrox-iD Pediatric achieved the overall highest CO2 removal rates under medium and high sweep gas flow rates, the two circular membrane lungs performed relatively better at the lowest gas flow rate of 0.5 L/min. The low-flow Hilite 1000, although overall better than the Quadrox i-Neonatal, had the most significant advantage at a gas flow of 0.5 L/min. Moreover, the circular Minilung, despite being significantly less efficient than the Quadrox-iD Pediatric at medium and high sweep gas flow rates, did not show a significantly worse CO2 removal rate at a gas flow of 0.5 L/min but rather a slight advantage. We suggest that circular membrane lungs have an advantage at low sweep gas flow rates due to reduced shunting as a result of their fiber orientation. Efficiency for such low gas flow scenarios might be relevant for possible future portable ECCO2R devices