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SummarySingle-molecule FRET (smFRET) is widely used to investigate dynamic (bio) molecular interactions taking place over distances of up to 10 nm. With the advent of recent super-resolution methods such as MINFLUX, MINSTED or RASTMIN, the spatiotemporal resolution of these techniques advanced towards the smFRET regime. While these methods do not suffer from the spatial restriction of FRET, they only visualize one emitter at a time, thus rendering fast dynamics of interactions out of reach. Here, we describe two approaches to overcome this limitation in pMINFLUX using its intrinsic fluorescence lifetime information. First, we combined pMINFLUX with smFRET. This enabled us to track a FRET donor fluorophore and simultaneously colocalize its FRET acceptor with nanometer precision. To extend co-localized tracking beyond the FRET range, we developed pMINFLUX lifetime multiplexing, a method to simultaneously track two fluorophores with similar spectral properties but distinct fluorescence lifetimes. We demonstrated its application on both static and dynamic DNA origami systems with a precision better than 2 nm. Within the FRET range, pMINFLUX lifetime multiplexing additionally uses a novel combined phasor-microtimegating approach. This paves the way for nanometer precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, closing the resolution gap between smFRET and co-tracking.Graphical Abstract

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.

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.

A single chromophore can only emit a maximum of one single photon per excitation cycle. This limitation results in a phenomenon commonly referred to as photon antibunching (pAB). When multiple chromophores contribute to the fluorescence measured, the degree of pAB has been used as a metric to "count" the number of chromophores. But the fact that chromophores can switch randomly between bright and dark states also impacts pAB and can lead to incorrect chromophore numbers being determined from pAB measurements. By both simulations and experiment, we demonstrate how pAB is affected by independent and collective chromophore blinking, enabling us to formulate universal guidelines for correct interpretation of pAB measurements. We use DNA-origami nanostructures to design multichromophoric model systems that exhibit either independent or collective chromophore blinking. Two approaches are presented that can distinguish experimentally between these two blinking mechanisms. The first one utilizes the different excitation intensity dependence on the blinking mechanisms. The second approach exploits the fact that collective blinking implies energy transfer to a quenching moiety, which is a time-dependent process. In pulsed-excitation experiments, the degree of collective blinking can therefore be altered by time gating the fluorescence photon stream, enabling us to extract the energy-transfer rate to a quencher. The ability to distinguish between different blinking mechanisms is valuable in materials science, such as for multichromophoric nanoparticles like conjugated-polymer chains as well as in biophysics, for example, for quantitative analysis of protein assemblies by counting chromophores.

Forschung im Ingenieurwesen (2021). doi:10.1007/s10010-021-00452-3, Published by Springer, Berlin ; Heidelberg

ZusammenfassungDie zunehmende Digitalisierung führt zu veränderten Arbeitsbedingungen in verschiedenen Bereichen der Logistik, wie der Intralogistik und den Post- und Zustelldiensten. Für beide Berufsgruppen gelten die Arbeitsbedingungen als äußerst belastend. Allerdings sind die Zusammenhänge mit der Arbeitsfähigkeit bislang nicht hinreichend untersucht. Ziel ist es deshalb, diese Zusammenhänge anhand empirischer Analysen und auf theoretischer Basis eines erweiterten Anforderungs-Ressourcen-Modells zu erklären und somit Ansatzpunkte für eine menschengerechte Arbeitsgestaltung aufzuzeigen. Datengrundlage ist die von der Bundesanstalt für Arbeitsschutz und Arbeitsmedizin durchgeführte Arbeitszeitbefragung. Anhand von Gruppenvergleichen und Regressionsmodellen werden beide Berufsgruppen miteinander verglichen. Ansätze für eine menschengerechte Gestaltung von Arbeitssystemen können für beide Berufsgruppen vor allem bei organisatorischen Arbeitsbedingungen wie der sozialen Unterstützung oder eines auf Anerkennung und Rollenklarheit basierenden Betriebsklimas ausgemacht werden.Praktische Relevanz Die vorliegenden Analysen können als Grundlage für eine menschengerechte Gestaltung von Arbeitssystemen je nach den spezifischen Bedingungen in der jeweiligen Berufsgruppe dienen. Sie verdeutlichen besonders das Bedingungsverhältnis von technischen und sozialen Aspekten und zeigen darüber hinaus für beide untersuchten Berufsgruppen sowohl bei der technischen Gestaltung der Arbeitssysteme als auch mit Blick auf organisationale Ressourcen Ansatzpunkte für Interventionen auf.

The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA–liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics., The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) through the ERC grant PhysProt (Agreement No. 337969). K.N.B., P.S.C., and T.P.J.K. are grateful for financial support from the Biotechnology and Biological Sciences Research Council (BBSRC), the Newman Foundation, the Wellcome Trust, and the Cambridge Centre for Misfolding Diseases. D.M. is supported by the Winton Programme for the Physics of Sustainability, as well as the Engineering and Physical Sciences Research Council (EPSRC). P.T. gratefully acknowledges funding by the Bavarian Ministry of Science and the Arts through the ONE MUNICH Project “Munich Multiscale Biofabrication” and by the LMU-Cambridge strategic partnership. S.H.A. acknowledges funding by the Gobierno de Aragón-FSE (Research Group E47_20R).

Tin-vacancy color centers in diamond are promising for quantum communication and information applications. Here, we determine the coherence time of orbital qubit states through coherent population trapping.

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.

Products derived from crude oil form the basis for large segments of energy and production systems. However, the availability of crude oil is limited and the combustion of fossil fuels contributes to global warming. Biorefineries can produce a product portfolio similar to that of crude-oil refineries from renewable resources and thus promote transformation towards a more sustainable bioeconomy. The actual product portfolio of a given biorefinery, however, depends on the choice and capacity of the production units used to upgrade raw materials to marketable products. Thus, to improve biorefinery product competitiveness, we apply an algorithm that combines an exact optimization algorithm nested in an Evolutionary Strategy with Geographic Information Systems to determine a wood-based biorefinery's optimal configuration, capacity, and location within the Cariboo District in Canada. The results indicate that there are numerous locations with similarly attractive economic potentials for biorefineries with 450,000 to 550,000 tons of biomass input capacity in the investigated area.

Drugs intoxications often lead to severe vasoplegia and cardiogenic shock, and VA-ECMO represents a viable therapy option. However, as cardiopulmonary support is not contributing to the removal of the causal agent from the blood, detoxification by a new hemoadsorption device (CytoSorb) could represent a potential therapeutic tool due to its highly efficient elimination capacity of endogenous but also exogenous hydrophobic substances for which otherwise no effective antidote exist. In this case series, four anecdotal cases of acute intoxications requiring VA-ECMO support used as extracorporeal cardiopulmonary resuscitation after intoxication-induced out-of-hospital cardiac arrest (OHCA) are presented, who were additionally treated with CytoSorb hemoadsorption in combination with renal replacement therapy. Combined treatment was associated with a considerable decrease in plasma levels of the overdosed drugs. Additionally, the combination of applied techniques was safe, practical, and technically feasible with no adverse or any device-related side effects documented during or after the treatment sessions. Based on the reported dramatic decline in drug levels during treatment, that fits in the device's characteristics, we strongly suggest to further investigate the potentially lifesaving role of CytoSorb therapy in patients with acute intoxications requiring multiple organ support techniques.

Forschung im Ingenieurwesen (2021). doi:10.1007/s10010-021-00462-1, Published by Springer, Berlin ; Heidelberg

Forschung im Ingenieurwesen = Engineering research (2021). doi:10.1007/s10010-021-00456-z, Published by Springer, Berlin ; Heidelberg

The particle-like nature of light becomes evident in the photon statistics of fluorescence from single quantum systems as photon antibunching. In multichromophoric systems, exciton diffusion and subsequent annihilation occurs. These processes also yield photon antibunching but cannot be interpreted reliably. Here we develop picosecond time-resolved antibunching to identify and decode such processes. We use this method to measure the true number of chromophores on well-defined multichromophoric DNA-origami structures, and precisely determine the distance-dependent rates of annihilation between excitons. Further, this allows us to measure exciton diffusion in mesoscopic H- and J-type conjugated-polymer aggregates. We distinguish between one-dimensional intra-chain and three-dimensional inter-chain exciton diffusion at different times after excitation and determine the disorder-dependent diffusion lengths. Our method provides a powerful lens through which excitons can be studied at the single-particle level, enabling the rational design of improved excitonic probes such as ultra-bright fluorescent nanoparticles and materials for optoelectronic devices., Photon antibunching typically measures the time-averaged photophysics of multichromophoric nanoparticles. Here, the authors report on time-resolving photon antibunching, allowing the true number of chromophores and exciton diffusion to be measured in DNA origami and conjugated polymer aggregates.

We use two coherent spectroscopy techniques to measure the strain tensor local to an ensemble of silicon-vacancy centers in diamond. Our results provide a possible pathway for using diamond as a tensorial strain gauge.

The Science of Making Torque from Wind, TORQUE 2020, , 28 Sep 2020 - 2 Oct 2020; Journal of physics / Conference series 1618: The Science of Making Torque from Wind (TORQUE 2020) 28 September – 2 October 2020, The Netherlands (online)(5) 052002 (2020). doi:10.1088/1742-6596/1618/5/052002 special issue: "Turbine Technology", Published by IOP Publ., Bristol

We characterize a high-density sample of negatively charged silicon-vacancy (${\mathrm{SiV}}^{\ensuremath{-}}$) centers in diamond using collinear optical multidimensional coherent spectroscopy. By comparing the results of complementary signal detection schemes, we identify a hidden population of ${\mathrm{SiV}}^{\ensuremath{-}}$ 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.

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.

A miniaturized implantable multi-sensor system for cardiovascular monitoring of physiological parameters is presented. High accuracy pressure measurements within the vessel can be performed by a capacitive pressure sensor. Additional information about the patient, e. g., sudden movement, inclination or increased temperature can be obtained by additional sensor components such as an acceleration sensor and a temperature sensor unit. This information facilitates compensation of interferences for more accurate pressure measurements. A multi-functional ASIC enables, amongst others, sensor signal processing, power management and telemetric communication with extracorporeal electronics. Sensor chips, the multi-functional ASIC and passive components are assembled on a LTCC circuit board in which an antenna coil is integrated for telemetric energy and data transmission at a frequency of 13.56 MHz. In order to support further miniaturization, the implant shall be encapsulated with a stack of very thin and hermetic ceramics applied by ALD instead of using bulky metal housings. Further encapsulation with polymers, which can be functionalised with appropriate biomolecules, is necessary for a proper shape, a biocompatible interface to the surrounding tissue and, thereby, reduction of thrombogenicity.

Biorefineries can provide a product portfolio from renewable biomass similar to that of crude oil refineries. To operate biorefineries of any kind, however, the availability of biomass inputs is crucial and must be considered during planning. Here, we develop a planning approach that uses Geographic Information Systems (GIS) to account for spatially scattered biomass when optimizing a biorefinery’s location, capacity, and configuration. To deal with the challenges of a non-smooth objective function arising from the geographic data, higher dimensionality, and strict constraints, the planning problem is repeatedly decomposed by nesting an exact nonlinear program (NLP) inside an evolutionary strategy (ES) heuristic, which handles the spatial data from the GIS. We demonstrate the functionality of the algorithm and show how including spatial data improves the planning process by optimizing a synthesis gas biorefinery using this new planning approach.

Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of Forster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with

An ensemble of silicon vacancy centers in diamond (\ce{SiV-}) is probed using two coherent spectroscopy techniques. Two main distinct families of \ce{SiV-} centers are identified using multidimensional coherent spectroscopy, and these families are paired with two orientation groups by comparing spectra from different linear polarizations of the incident laser. By tracking the peak centers in the measured spectra, the full diamond strain tensor is calculated local to the laser spot. Such measurements are made at multiple points on the sample surface and variations in the strain tensor are observed.

Nanostructures can be used for boosting the light outcoupling of color centers in diamond; however, the fiber coupling performance of these nanostructures is rarely investigated. Here, we use a finite element method for computing the emission from color centers in inverted nanocones and the overlap of this emission with the propagation mode in a single-mode fiber. Using different figures of merit, the inverted nanocone parameters are optimized to obtain maximal fiber coupling efficiency, free-space collection efficiency, or rate enhancement. The optimized inverted nanocone designs show promising results with 66% fiber coupling or 83% free-space coupling efficiency at the tin-vacancy center zero-phonon line wavelength of 619 nm. Moreover, when evaluated for broadband performance, the optimized designs show 55% and 76% for fiber coupling and free-space efficiencies respectively, for collecting the full tin-vacancy emission spectrum at room temperature. An analysis of fabrication insensitivity indicates that these nanostructures are robust against imperfections. For maximum emission rate into a fiber mode, a design with a Purcell factor of 2.34 is identified. Finally, possible improvements offered by a hybrid inverted nanocone, formed by patterning into two different materials, are investigated, and increases the achievable fiber coupling efficiency to 71%., The following article has been accepted by Applied Physics Letters. After it is published, it will be found at https://doi.org/10.1063/5.0050338

We report the direct integration and efficient coupling of nitrogen vacancy (NV) color centers in diamond nanophotonic structures into a fiber-based photonic architecture at cryogenic temperatures. NV centers are embedded in diamond micro-waveguides (μWGs), which are coupled to fiber tapers. Fiber tapers have low-loss connection to single-mode optical fibers and hence enable efficient integration of NV centers into optical fiber networks. We numerically optimize the parameters of the μWG-fiber-taper devices designed particularly for use in cryogenic experiments, resulting in 35.6% coupling efficiency, and experimentally demonstrate cooling of these devices to the liquid helium temperature of 4.2 K without loss of the fiber transmission. We observe sharp zero-phonon lines in the fluorescence of NV centers through the pigtailed fibers at 100 K. The optimized devices with high photon coupling efficiency and the demonstration of cooling to cryogenic temperatures are an important step to realize fiber-based quantum nanophotonic interfaces using diamond spin defect centers.

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor compound the fundamental core of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional intiation factor Bdp1, which is unique to the RNA polymerase (RNAP) III sytem, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain, that arises from DNA compaction and transcriptional activity, on the efficiency of initiation complex formation. We made use of a new nanotechnological tool – a DNA origami-based force clamp - to follow the assembly of human initiation complexes in the Pol II and Pol III system at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is necessary and sufficient to stabilise TBP on a strained RNAP II promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of Pol III genes for the first time.

We present a training procedure and maze equipped with sensors and automated feeders for training spatial behavioral tasks in rodents. The maze can be transformed from an enclosed box to a maze of variable dimensions. The modularity of the protocol and setup makes it highly flexible and suitable for training a wide variety of spatial tasks, and facilitates incremental training stages of increasing maze size for more efficient learning. The apparatus, in its software and hardware, is able to adapt to animal performance, adjusting task challenges and difficulty. Two different methods of automatic behavioral scoring are evaluated against manual methods. Sensors embedded in the maze provide information regarding the order of reward locations visited and the time between the activation of the cue via the nose-poke and the activation of the reward location sensors. The distributions of these reaction times differ between correct and incorrect trials, providing an index of behavior and motivation. The automated maze system allows the trainer to operate and monitor the task away from the experimental set-up, minimizing human interference and improving the reproducibility of the experiment. We show that our method succeeds in training a binary forced-choice task in rats.

t a novel miniaturized multi sensor implant for monitoring hemodynamic parameters in cardiovascular regions. Pressure measurements are performed with a highly accurate capacitive pressure sensor. An additional acceleration and temperature sensor allows compensating the impact of patient's inclination and temperature variations on the pressure measurement, respectively. A multi-functional transponder application-specific integrated circuit (ASIC) manages sensor signal processing, storage of ID, sensor calibration data, telemetric energy, and data transmission with an extracorporeal reading unit. Each component of the implant is assembled on a low temperature co-fired ceramics (LTCC) circuit board with an integrated antenna coil enabling an inductive near-field coupling at a frequency of 13.56 MHz. For a streamlined shape and reduction of thrombogenicity, the implant is encapsulated by biocompatible polymers.