Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics.

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

Quantitative Single-Molecule Measurements of Membrane Charges with DNA Origami Sensors.

Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1-10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.

Coherent Interactions between Silicon-Vacancy Centers in Diamond.

We report tunable excitation-induced dipole-dipole interactions between silicon-vacancy color centers in diamond at cryogenic temperatures. These interactions couple centers into collective states, and excitation-induced shifts tag the excitation level of these collective states against the background of excited single centers. By characterizing the phase and amplitude of the spectrally resolved interaction-induced signal, we observe oscillations in the interaction strength and population state of the collective states as a function of excitation pulse area. Our results demonstrate that excitation-induced dipole-dipole interactions between color centers provide a route to manipulating collective intercenter states in the context of a congested, inhomogeneous ensemble.

The object space task shows cumulative memory expression in both mice and rats.

Declarative memory encompasses representations of specific events as well as knowledge extracted by accumulation over multiple episodes. To investigate how these different sorts of memories are created, we developed a new behavioral task in rodents. The task consists of 3 distinct conditions (stable, overlapping, and random). Rodents are exposed to multiple sample trials, in which they explore objects in specific spatial arrangements, with object identity changing from trial to trial. In the stable condition, the locations are constant during all sample trials even though the objects themselves change; in the test trial, 1 object's location is changed. In the random condition, object locations are presented in the sample phase without a specific spatial pattern. In the overlapping condition, 1 location is shared (overlapping) between all trials, while the other location changes during sample trials. We show that in the overlapping condition, instead of only remembering the last sample trial, rodents form a cumulative memory of the sample trials. Here, we could show that both mice and rats can accumulate information across multiple trials and express a long-term abstracted memory.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Hidden Silicon-Vacancy Centers in Diamond.

We characterize a high-density sample of negatively charged silicon-vacancy (SiV^{-}) centers in diamond using collinear optical multidimensional coherent spectroscopy. By comparing the results of complementary signal detection schemes, we identify a hidden population of SiV^{-} centers that is not typically observed in photoluminescence and which exhibits significant spectral inhomogeneity and extended electronic T_{2} times. The phenomenon is likely caused by strain, indicating a potential mechanism for controlling electric coherence in color-center-based quantum devices.

Roadmap on quantum nanotechnologies.

Quantum phenomena are typically observable at length and time scales smaller than those of our everyday experience, often involving individual particles or excitations. The past few decades have seen a revolution in the ability to structure matter at the nanoscale, and experiments at the single particle level have become commonplace. This has opened wide new avenues for exploring and harnessing quantum mechanical effects in condensed matter. These quantum phenomena, in turn, have the potential to revolutionize the way we communicate, compute and probe the nanoscale world. Here, we review developments in key areas of quantum research in light of the nanotechnologies that enable them, with a view to what the future holds. Materials and devices with nanoscale features are used for quantum metrology and sensing, as building blocks for quantum computing, and as sources and detectors for quantum communication. They enable explorations of quantum behaviour and unconventional states in nano- and opto-mechanical systems, low-dimensional systems, molecular devices, nano-plasmonics, quantum electrodynamics, scanning tunnelling microscopy, and more. This rapidly expanding intersection of nanotechnology and quantum science/technology is mutually beneficial to both fields, laying claim to some of the most exciting scientific leaps of the last decade, with more on the horizon.

Interchromophoric Interactions Determine the Maximum Brightness Density in DNA Origami Structures.

An ideal point light source is as small and as bright as possible. For fluorescent point light sources, homogeneity of the light sources is important as well as that the fluorescent units inside the light source maintain their photophysical properties, which is compromised by dye aggregation. Here we propose DNA origami as a rigid scaffold to arrange dye molecules in a dense pixel array with high control of stoichiometry and dye-dye interactions. In order to find the highest labeling density in a DNA origami structure without influencing dye photophysics, we alter the distance of two ATTO647N dyes in single base pair steps and probe the dye-dye interactions on the single-molecule level. For small distances strong quenching in terms of intensity and fluorescence lifetime is observed. With increasing distance, we observe reduced quenching and molecular dynamics. However, energy transfer processes in the weak coupling regime still have a significant impact and can lead to quenching by singlet-dark-state-annihilation. Our study fills a gap of studying the interactions of dyes relevant for superresolution microscopy with dense labeling and for single-molecule biophysics. Incorporating these findings in a 3D DNA origami object will pave the way to bright and homogeneous DNA origami nanobeads.

Efficient Extraction of Light from a Nitrogen-Vacancy Center in a Diamond Parabolic Reflector.

Quantum emitters in solids are being developed for a range of quantum technologies, including quantum networks, computing, and sensing. However, a remaining challenge is the poor photon collection due to the high refractive index of most host materials. Here we overcome this limitation by introducing monolithic parabolic reflectors as an efficient geometry for broadband photon extraction from quantum emitter and experimentally demonstrate this device for the nitrogen-vacancy (NV) center in diamond. Simulations indicate a photon collection efficiency exceeding 75% across the visible spectrum and experimental devices, fabricated using a high-throughput gray scale lithography process, demonstrating a photon extraction efficiency of (41 ± 5)%. This device enables a raw experimental detection efficiency of (12 ± 1)% with fluorescence detection rates as high as (4.114 ± 0.003) × 106 counts per second (cps) from a single NV center. Enabled by our deterministic emitter localization and fabrication process, we find a high number of exceptional devices with an average count rate of (3.1 ± 0.9) × 106 cps.

A DNA Walker as a Fluorescence Signal Amplifier.

Sensing nucleic acids typically involves the recognition of a specific sequence and reporting by, for example, a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a "DNA walker") can release the fluorescence of many molecules by acting as the catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with single-nucleotide sensitivity. Using Monte Carlo simulations, we rationalize that the random self-avoiding walk is mainly terminated when steps to nearest neighbors are exhausted. Finally, we demonstrate that the DNA walker is also active in a plasmonic hotspot for fluorescence enhancement, indicating the potential of combining different amplification mechanisms enabled by the modularity of DNA nanotechnology.

Isoflurane Sedation on the ICU in Cardiac Arrest Patients Treated With Targeted Temperature Management: An Observational Propensity-Matched Study.

OBJECTIVE: Targeted temperature management after cardiac arrest requires deep sedation to prevent shivering and discomfort. Compared to IV sedation, volatile sedation has a shorter half-life and thus may allow more rapid extubation and neurologic assessment. DESIGN: Observational analysis of clinical data. SETTING: University hospital, medical ICU. PATIENTS: Four hundred thirty-two cardiac arrest survivors underwent targeted temperature management; of those, 110 were treated with volatile sedation using an anesthetic conserving device and isoflurane, and 322 received standard IV sedation. INTERVENTION: No intervention. MEASUREMENT AND MAIN RESULTS: A matched pairs analysis revealed that time on ventilator (difference of median, 98.5 hr; p = 0.003) and length of ICU stay (difference of median, 4.5 d; p = 0.006) were significantly shorter in patients sedated with isoflurane when compared with IV sedation although no differences in neurologic outcome (45% of patients with cerebral performance category 1-2 in both groups) were observed. Significant hypercapnia occurred more frequently during anesthetic conserving device use (6.4% vs 0%; p = 0.021). CONCLUSIONS: Volatile sedation is feasible in cardiac arrest survivors. Prospective controlled studies are necessary to confirm the beneficial effects on duration of ventilation and length of ICU stay observed in our study. Our data argue against a major effect on neurologic outcome. Close monitoring of PaCO2 is necessary during sedation via anesthetic conserving device.

Efficient photon collection from a nitrogen vacancy center in a circular bullseye grating.

Efficient collection of the broadband fluorescence from the diamond nitrogen vacancy (NV) center is essential for a range of applications in sensing, on-demand single photon generation, and quantum information processing. Here, we introduce a circular "bullseye" diamond grating which enables a collected photon rate of (2.7 ± 0.09) × 10(6) counts per second from a single NV with a spin coherence time of 1.7 ± 0.1 ms. Back-focal-plane studies indicate efficient redistribution of the NV photoluminescence into low-NA modes by the bullseye grating.

Generation of ensembles of individually resolvable nitrogen vacancies using nanometer-scale apertures in ultrahigh-aspect ratio planar implantation masks.

A central challenge in developing magnetically coupled quantum registers in diamond is the fabrication of nitrogen vacancy (NV) centers with localization below ∼20 nm to enable fast dipolar interaction compared to the NV decoherence rate. Here, we demonstrate the targeted, high throughput formation of NV centers using masks with a thickness of 270 nm and feature sizes down to ∼1 nm. Super-resolution imaging resolves NVs with a full-width maximum distribution of 26 ± 7 nm and a distribution of NV-NV separations of 16 ± 5 nm.

Scalable fabrication of high purity diamond nanocrystals with long-spin-coherence nitrogen vacancy centers.

The combination of long spin coherence time and nanoscale size has made nitrogen vacancy (NV) centers in nanodiamonds the subject of much interest for quantum information and sensing applications. However, currently available high-pressure high-temperature (HPHT) nanodiamonds have a high concentration of paramagnetic impurities that limit their spin coherence time to the order of microseconds, less than 1% of that observed in bulk diamond. In this work, we use a porous metal mask and a reactive ion etching process to fabricate nanocrystals from high-purity chemical vapor deposition (CVD) diamond. We show that NV centers in these CVD nanodiamonds exhibit record-long spin coherence times in excess of 200 µs, enabling magnetic field sensitivities of 290 nT Hz(-1/2) with the spatial resolution characteristic of a 50 nm diameter probe.

Improvement of cerebral oxygen saturation after successful electrical cardioversion of atrial fibrillation.

AIMS: Cerebral and microvascular perfusion is reduced in atrial fibrillation (AF). Maintenance of brain perfusion is important in acute disease and long-term course. Assessment of brain perfusion and oxygenation is difficult in clinical practice. Our study aimed to determine changes in cerebral tissue oxygen saturation (SctO2) with bedside near-infrared spectroscopy (NIRS). METHODS AND RESULTS: Twenty patients (mean age 67.7 ± 10.2 years, 50% men) in whom electrical cardioversion (CV) was successful were prospectively studied. Ten patients (mean age 64.2 ± 7.7 years, 80% men) in whom CV was not successful served as control group. Bilateral SctO2, mean arterial pressure (MAP), arterial oxygen saturation (SaO2), and heart rate were recorded and changes of all parameters before and after CV were compared between the groups. Our results show an increase in SctO2 after successful CV that was significantly higher compared with patients who remained in AF (right SctO2 3.25 ± 2.5 vs. -0.13 ± 0.52%, P = 0.001; left SctO2 4.27 ± 3.56 vs. -0.38 ± 2.4%, P < 0.001). Neither arterial blood pressure nor SaO2 changes differed significantly between the two groups. No correlation could be detected between the significant increase of SctO2 after successful CV and arterial blood pressure, SaO2, or heart rate. CONCLUSION: Cerebral tissue oxygen saturation increases significantly after restoration of sinus rhythm. Near-infrared spectroscopy monitoring can identify changes of SctO2 after successful CV of AF independent from standard monitoring parameters (MAP, SaO2). Near-infrared spectroscopy can be used to detect cerebral oxygen saturation deficits in AF patients or patients at high risk for AF. Clinical applications may include monitoring during ablation procedures and in critical care.

The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Comparative assessment of two-phase class II treatment with Activator or Bionator followed by fixed appliances: A retrospective controlled before-and-after study.

AIM: Two-phase treatment for children with Class II malocclusion with several functional appliances is still performed by many orthodontists, while the Activator and the Bionator appliances are two of the most popular ones. Aim of this study was to compare the skeletal and dentoalveolar effects of treatment with these two appliances. METHODS: Class II children treated with Activator or Bionator in the first phase, followed by a phase of fixed appliances were included. Skeletal and dentoalveolar parameters were assessed from lateral cephalograms and analysed with linear regressions at 5%. RESULTS: A total of 89 patients (mean age 10.0 years; 47% female) were included. During the first phase, Bionator increased less the SNB (difference in mean treatment-induced changes [MD] -0.7°; 95% confidence interval [CI] -1.3 to -0.2°; P=0.01) and decreased less the ANB angle (MD 0.6°; 95% CI 0 to 1.1°; P=0.03) compared to Activator. Activator slightly increased the facial axis and Bionator reduced it (MD -1.6°; 95% CI -2.3 to -0.8°; P<0.001). Compared to Activator, the Bionator retroclined more the upper incisors (MD -2.4°; 95% CI -4.6 to -0.2°; P=0.03) and increased more the interincisal angle (MD 2.9°; 95% CI 0.5 to 5.4°; P=0.02). After the second phase (6.2 years after baseline), the only differences were a reduced facial axis (MD -1.3°; 95% CI -2.2 to -0.3°; P=0.008) and an increased maxillary rotation (MD 0.9°; 95% CI 0 to 1.8°; P=0.04) with Bionator compared to Activator. CONCLUSION: Similar dentoalveolar effects were seen overall with two-phase treatment with either appliance, with Bionator being associated with more vertical increase compared to Activator.