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Tracking single particles for hours via continuous DNA-mediated fluorophore exchange
Get PDFMonitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales. The length of single-particle tracking experiments are limited due to photobleaching. Here the authors achieve long-term single-particle tracking with continuous fluorophore exchange in DNA-PAINT and use this to observe DNA origami on lipid bilayers for tens of minutes.
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Cell Tracking for Organoids: Lessons From Developmental Biology
Get PDFOrganoids have emerged as powerful model systems to study organ development and regeneration at the cellular level. Recently developed microscopy techniques that track individual cells through space and time hold great promise to elucidate the organizational principles of organs and organoids. Applied extensively in the past decade to embryo development and 2D cell cultures, cell tracking can reveal the cellular lineage trees, proliferation rates, and their spatial distributions, while fluorescent markers indicate differentiation events and other cellular processes. Here, we review a number of recent studies that exemplify the power of this approach, and illustrate its potential to organoid research. We will discuss promising future routes, and the key technical challenges that need to be overcome to apply cell tracking techniques to organoid biology
Intermittency of CsPbBr3 Perovskite Quantum Dots Analyzed by an Unbiased Statistical Analysis
Get PDFWe analyze intermittency in intensity and fluorescence lifetime of CsPbBr3 perovskite quantum dots by applying unbiased Bayesian inference analysis methods. We apply change-point analysis (CPA) and a Bayesian state clustering algorithm to determine the timing of switching events and the number of states between which switching occurs in a statistically unbiased manner, which we have benchmarked particularly to apply to highly multistate emitters. We conclude that perovskite quantum dots display a plethora of gray states in which brightness, broadly speaking, correlates inversely with decay rate, confirming the multiple recombination centers model. We leverage the CPA partitioning analysis to examine aging and memory effects. We find that dots tend to return to the bright state before jumping to a dim state and that when choosing a dim state, they tend to explore the entire set of states available
A plausible mechanism for longitudinal lock-in of the plant cortical microtubule array after light-induced reorientation
Get PDFThe light-induced reorientation of the cortical microtubule array in dark-grown Arabidopsis thaliana hypocotyl cells is a striking example of the dynamical plasticity of the microtubule cytoskeleton. A consensus model, based on katanin-mediated severing at microtubule crossovers, has been developed that successfully describes the onset of the observed switch between a transverse and longitudinal array orientation. However, we currently lack an understanding of why the newly populated longitudinal array direction remains stable for longer times and re-equilibration effects would tend to drive the system back to a mixed orientation state. Using both simulations and analytical calculations, we show that the assumption of a small orientation-dependent shift in microtubule dynamics is sufficient to explain the long-term lock-in of the longitudinal array orientation. Furthermore, we show that the natural alternative hypothesis that there is a selective advantage in severing longitudinal microtubules, is neither necessary nor sufficient to achieve cortical array reorientation, but is able to accelerate this process significantly
Particle diffusion in extracellular hydrogels
Get PDFHyaluronic acid is an abundant polyelectrolyte in the human body that forms extracellular hydrogels in connective tissues. It is essential for regulating tissue biomechanics and cell–cell communication, yet hyaluronan overexpression is associated with pathological situations such as cancer and multiple sclerosis. Due to its enormous molecular weight (in the range of millions of Daltons), accumulation of hyaluronan hinders transport of macromolecules including nutrients and growth factors through tissues and also hampers drug delivery. However, the exact contribution of hyaluronan to tissue penetrability is poorly understood due to the complex structure and molecular composition of tissues. Here we reconstitute biomimetic hyaluronan gels and systematically investigate the effects of gel composition and crosslinking on the diffusion of microscopic tracer particles. We combine ensemble-averaged measurements via differential dynamic microscopy with single-particle tracking. We show that the particle diffusivity depends on the particle size relative to the network pore size and also on the stress relaxation dynamics of the network. We furthermore show that addition of collagen, the other major biopolymer in tissues, causes the emergence of caged particle dynamics. Our findings are useful for understanding macromolecular transport in tissues and for designing biomimetic extracellular matrix hydrogels for drug delivery and tissue regeneration
Freezing of Aqueous Carboxylic Acid Solutions on Ice
Get PDFWe study the properties of acetic acid and propionic acid solutions at the surface of monocrystalline ice with surface-specific vibrational sum-frequency generation (VSFG) and heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). When we decrease the temperature towards the eutectic point of the acid solutions, we observe formation of a freeze concentrated solution (FCS) of the carboxylic acids that is brought about by a freeze induced phase separation (FIPS). The freeze concentrated solution freezes on top of the ice surface as we cool the system below the eutectic point. We find that for freeze concentrated acetic acid solutions the freezing causes a strong decrease of the VSFG signal, while for propionic acid an increase and a blue shift are observed. This different behavior points at a distinct difference in molecular-scale behavior when cooling below the eutectic point. We find that cooling of the propionic acid solution below the eutectic point leads to the formation of hydrogen-bonded dimers with an opposite alignment of the carboxylic acid O-H groups
Differences in plasticity between hard and soft spheres
Get PDFContact changes in packings of sheared hard spheres invariably trigger instabilities and irreversible rearrangements, providing an archetypal scenario for plasticity of disordered media. Here we show that the plasticity of jammed soft spheres at any finite pressure follows a different scenario, with only 14% of contact changes leading to irreversible rearrangements, irrespective of pressure, size, dimension or interaction potential. Moreover, we find that for sheared soft spheres, the nonlinear quantities associated with either contact changes or irreversible events exhibit the same finite-size scaling with pressure and system size as linear response quantities such as the shear modulus, suggesting an unexpected connection between curvature and saddle points in the potential energy landscape. Together our results indicate that soft spheres at finite pressure are not a smooth perturbation away from hard spheres, and that the nonlinear response of soft spheres is singular at zero pressure
Non-contact surface grating coupling for arrays of atom–photonic devices
Get PDFFor future atom-photonic devices, the incoupling method is expected to become the main bottleneck in the scal ing of the number of devices, as current devices use side-coupling, which is restricted to the sample edges. This paper describes how light from above is coupled directly from free space into a freestanding membrane of 220 nm thin silicon nitrite (Si3N4) with surface grating couplers. The design for these couplers is modeled, captured in an elegant design formula, and experimentally tested. A coupling efficiency of 12% makes experimental coupling to an array of atom-photonic devices possible. The elegant design formula makes grating design straightforward for all-dielectric materials and wavelengths. For this formula, determination of the correct effective index of t he specific waveguide mode inside the grating layer is essential
Quantifying Strain and Dislocation Density at Nanocube Interfaces after Assembly and Epitaxy
Get PDFNanoparticle self-assembly and epitaxy are utilized extensively to make 1D and 2D structures with complex shapes. High-resolution transmission electron microscopy (HRTEM) has shown that single-crystalline interfaces can form, but little is known about the strain and dislocations at these interfaces. Such information is critically important for applications: drastically reducing dislocation density was the key breakthrough enabling widespread implementation of light-emitting diodes, while strain engineering has been fundamental to modern high-performance transistors, solar cells, and thermoelectrics. In this work, the interfacial defect and strain formation after self-assembly and room temperature epitaxy of 7 nm Pd nanocubes capped with polyvinylpyrrolidone (PVP) is examined. It is observed that, during ligand removal, the cubes move over large distances on the substrate, leading to both spontaneous self-assembly and epitaxy to form single crystals. Subsequently, atomically resolved images are used to quantify the strain and dislocation density at the epitaxial interfaces between cubes with different lateral and angular misorientations. It is shown that dislocation- and strain-free interfaces form when the nanocubes align parallel to each other. Angular misalignment between adjacent cubes does not necessarily lead to grain boundaries but does cause dislocations, with higher densities associated with larger rotations
Effects of Diabetes Mellitus on Fibrin Clot Structure and Mechanics in a Model of Acute Neutrophil Extracellular Traps (NETs) Formation
Get PDFSubjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by S. aureus bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots