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Malaria pathogenesis and parasite multiplication depend on the ability of Plasmodium merozoites to invade human erythrocytes. Invasion is a complex multi-step process involving multiple parasite proteins which can differ between species and has been most extensively studied in P. falciparum. However, dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed P. falciparum merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified strains including some generated specifically for this work, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment interactions. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. By comparing over 4000 individual merozoite-erythrocyte interactions in a range of conditions and strains, we establish that the PfEBA/PfRH families play a central role in P. falciparum merozoite attachment, not the major merozoite surface protein PfMSP1. Author summary: Malaria is a devastating disease caused by a parasitic infection. The deadliest species is Plasmodium falciparum, which causes more than 600,000 deaths annually. The Plasmodium life cycle is complex, but all the symptoms of malaria are caused when the parasites replicate in human red blood cells. Replication depends on the invasion of the red blood cells by the parasites, a process involving multiple molecular interactions and multiple steps. Invasion begins with the attachment of the parasite to the red blood cell, making this step of particular interest in the development of new therapeutics. We used an optical tweezer assay to directly measure the binding force between individual parasites and red blood cells, and combined this assay with a range of molecular and genetic tools that target specific interactions known to have a role in invasion. This approach showed that loss of a protein commonly thought to be critical to the early stages of invasion, PfMSP1, had no effect on attachment strength, whereas disruptions of several members from two families of proteins (the Erythrocyte Binding Like protein family and the Reticulocyte Binding-like protein family) did affect attachment strength. [ABSTRACT FROM AUTHOR]

Three-dimensional crystalline frameworks with nanoscale periodicity are valuable for many emerging technologies, from nanophotonics to nanomedicine. DNA nanotechnology has emerged as a prime route for constructing these materials, with most approaches taking advantage of the structural rigidity and bond directionality programmable for DNA building blocks. Recently, we have introduced an alternative strategy reliant on flexible, amphiphilic DNA junctions dubbed C-stars, whose ability to crystallize is modulated by design parameters, such as nanostructure topology, conformation, rigidity, and size. While C-stars have been shown to form ordered phases with controllable lattice parameter, response to stimuli, and embedded functionalities, much of their vast design space remains unexplored. Here, we investigate the effect of changing the chemical nature of the hydrophobic modifications and the structure of the DNA motifs in the vicinity of these moieties. While similar design variations should strongly alter key properties of the hydrophobic interactions between C-stars, such as strength and valency, only limited differences in self-assembly behavior are observed. This finding suggests that long-range order in C-star crystals is likely imposed by structural features of the building block itself rather than the specific characteristics of the hydrophobic tags. Nonetheless, we find that altering the hydrophobic regions influences the ability of C-star crystals to uptake hydrophobic molecular cargoes, which we exemplify by studying the encapsulation of antibiotic penicillin V. Besides advancing our understanding of the principles governing the self-assembly of amphiphilic DNA building blocks, our observations thus open up new routes to chemically program the materials without affecting their structure. [ABSTRACT FROM AUTHOR]

Synchronization of driven oscillators is a key aspect of flow generation in artificial and biological filaments such as cilia. Previous theoretical and numerical studies have considered the "rotor" model of a cilium in which the filament is coarse grained into a colloidal sphere driven with a given force law along a predefined trajectory to represent the oscillating motion of the cilium. These studies pointed to the importance of two factors in the emergence of synchronization: the modulation of the driving force around the orbit and the deformability of the trajectory. In this work it is shown via experiments, supported by numerical simulations and theory, that both of these factors are important and can be combined to produce strong synchronization (within a few cycles) even in the presence of thermal noise. [ABSTRACT FROM AUTHOR]

Motile cilia are highly conserved structures in the evolution of organisms, generating the transport of fluid by periodic beating, through remarkably organized behavior in space and time. It is not known how these spatiotemporal patterns emerge and what sets their properties. Individual cilia are nonequilibrium systems with many degrees of freedom. However, their description can be represented by simpler effective force laws that drive oscillations, and paralleled with nonlinear phase oscillators studied in physics. Here a synthetic model of two phase oscillators, where colloidal particles are driven by optical traps, proves the role of the average force profile in establishing the type and strength of synchronization. We find that highly curved potentials are required for synchronization in the presence of noise. The applicability of this approach to biological data is also illustrated by successfully mapping the behavior of cilia in the alga Chlamydomonas onto the coarse-grained model. [ABSTRACT FROM AUTHOR]

Optical tweezers are used to manipulate the shape of artificial dioleoyl-phosphatidylcholine (DOPC) phospholipid vesicles of around 30µm diameter. Using a time-shared trapping system, a complex of traps drives oscillations of the vesicle equator, with a sinusoidal time dependence and over a range of spatial and temporal frequencies. The mechanical response of the vesicle membrane as a function of the frequency and wavelength of the driving oscillation is monitored. A simple model of the vesicles as spherical elastic membranes immersed in a Newtonian fluid, driven by a harmonic trapping potential, describes the experimental data. The bending modulus of the membrane is recovered. The method has potential for future investigation of nonthermally driven systems, where comparison of active and passive rheology can help to distinguish nonthermal forces from equilibrium fluctuations. [ABSTRACT FROM AUTHOR]

Magneto-optical tweezers have been constructed and used for studying interactions between colloids in nematic liquid crystals. We used super-paramagnetic beads which induced planar anchoring at the surface and were suitable for optical trapping as well as manipulation by external magnetic field. Depending on the thickness of the sample, attractive or repulsive forces between two immersed spheres were observed: repulsive for sample thickness comparable to the bead size, and attractive in thick samples. This behaviour corresponds to quadrupolar interaction between two beads. [ABSTRACT FROM AUTHOR]

A stroboscope designed to observe pulsars in the optical spectrum is presented. The absolute phase of the stroboscope is synchronized to better than 2.5 μs with the known radio ephemerides for a given pulsar. The absolute timing is provided by the Global Positioning System clock. With such a device resolved photometry of pulsars can be performed. We demonstrated the instrument’s capabilities with the results of a set of observations of the Crab pulsar, the brightest of the known optical pulsars, with a visual magnitude of 16.5, and a rotational frequency of ∼29 Hz. © 2003 American Institute of Physics. [ABSTRACT FROM AUTHOR]

A number of colloidal systems, including polymers, proteins, micelles and hard spheres, have been studied in thermal gradients to observe and characterize their driven motion. Here we show experimentally the thermophoretic behaviour of unilamellar lipid vesicles, finding that mobility depends on the mean local temperature of the suspension and on the structure of the exposed polar lipid head groups. By tuning the temperature, vesicles can be directed towards hot or cold, forming a highly concentrated region. Binary mixtures of vesicles composed of different lipids can be segregated using thermophoresis, according to their head group. Our results demonstrate that thermophoresis enables robust and chemically specific directed motion of liposomes, which can be exploited in driven processes. [ABSTRACT FROM AUTHOR]

Using videomicroscopy imaging, we demonstrate the existence of a short-ranged equilibrium attraction between heavy silica colloids diffusing on soft surfaces of cross-linked polymer gels. The intercolloid potential can be tuned by changing the gel stiffness or by coating the colloids with a polymer layer. On sufficiently soft substrates, the interaction induced by the polymer matrix leads to large-scale colloidal aggregation. We correlate the in-plane interaction with a colloid-surface attraction. [ABSTRACT FROM AUTHOR]

There has been an increasing, and welcome, open hardware trend towards science teams building and sharing their designs for new instruments. These devices, often built upon low-cost microprocessors and microcontrollers, can be readily connected to enable complex, automated and smart experiments. When designed to use open communication web standards, devices from different laboratories and manufacturers can be controlled using a single protocol and even communicate with each other. However, science labs still have a majority of old, perfectly functional equipment which tends to use older, and sometimes proprietary, standards for communications. In order to encourage the continued and integrated use of this equipment in modern automated experiments, we develop and demonstrate LabThings Retro. This allows us to retrofit old instruments to use modern Web-of-Things standards, which we demonstrate with closed-loop feedback involving an optical microscope, digital imaging and fluid pumping., Competing Interests: At the time of writing, P.C. is a Board Member of Royal Society Open Science but had no involvement in the review or assessment of the paper., (© 2024 The Authors.)

We describe a phenotypic antibiotic susceptibility testing (AST) method that can provide an eightfold speed-up in turnaround time compared with the current clinical standard by leveraging advances in microscopy and single-cell imaging. A newly developed growth plate containing 96 agarose pads, termed the multipad agarose plate (MAP), can be assembled at low cost. Pads can be prepared with dilution series of antibiotics. Bacteria are seeded on the pads and automatically imaged using brightfield microscopy, with a fully automated segmentation pipeline quantifying microcolony formation and growth rate. Using a test set of nine antibiotics with very different targets, we demonstrate that accurate minimum inhibitory concentration (MIC) measurements can be performed based on the growth rate of microcolonies within 3 h of incubation with the antibiotic when started from exponential phase. Faster, reliable and high-throughput methods for AST, such as MAP, could improve patient care by expediting treatment initiation and alleviating the burden of antimicrobial resistance.

Cilia density, distribution and beating frequency are important properties of airway epithelial tissues. These parameters are critical in diagnosing primary ciliary dyskinesia and examining in vitro models, including those derived from induced pluripotent stem cells. Video microscopy can be used to characterize these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the ciliary beat frequency of very small areas, while requiring human intervention and training for their use. We propose a novel and open-source method to fully characterize cilia beating frequency and motile cilia coverage in an automated fashion without user intervention. We demonstrate the ability to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterize the cilia beating frequency of all moving areas. We also show that the method can be used to combine multiple fields of view to better describe a sample without relying on small pre-selected regions of interest. This is released with a simple graphical user interface for file handling, enabling a full analysis of individual fields of view in a few minutes on a typical personal computer., Competing Interests: At the time of writing, Prof Pietro Cicuta is a Board Member of Royal Society Open Science, but had no involvement in the review or assessment of the paper., (© 2023 The Authors.)

In many organs, thousands of microscopic 'motile cilia' beat in a coordinated fashion generating fluid flow. Physiologically, these flows are important in both development and homeostasis of ciliated tissues. Combining experiments and simulations, we studied how cilia from brain tissue align their beating direction. We subjected cilia to a broad range of shear stresses, similar to the fluid flow that cilia themselves generate, in a microfluidic setup. In contrast to previous studies, we found that cilia from mouse ependyma respond and align to these physiological shear stress at all maturation stages. Cilia align more easily earlier in maturation, and we correlated this property with the increase in multiciliated cell density during maturation. Our numerical simulations show that cilia in densely packed clusters are hydrodynamically screened from the external flow, in agreement with our experimental observation. Cilia carpets create a hydrodynamic screening that reduces the susceptibility of individual cilia to external flows., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The motility of microalgae has been studied extensively, particularly in model microorganisms such as Chlamydomonas reinhardtii. For this and other microalgal species, diurnal cycles are well known to control the metabolism, growth, and cell division. Diurnal variations, however, have been largely neglected in quantitative studies of motility. Here, we demonstrate using tracking microscopy how the motility statistics of C. reinhardtii are modulated by diurnal cycles. With nine independently inoculated cultures synchronized to the light-dark cycle at the exponential growth phase, we repeatedly observed that the mean swimming speed is greater during the dark period of a diurnal cycle. From this measurement, using a hydrodynamic power balance, we infer the mean flagellar beat frequency and conjecture that its diurnal variation reflects modulation of intracellular ATP. Our measurements also quantify the diurnal variations of the orientational and gravitactic transport of C. reinhardtii. We use this to explore the population-level consequences of diurnal variations of motility statistics by evaluating a prediction for how the gravitactic steady state changes with time during a diurnal cycle. Finally, we discuss the consequences of diurnal variations of microalgal motility in soil and pelagic environments., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as μNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

Eukaryotic cilia and flagella are chemo-mechanical oscillators capable of generating long-range coordinated motions known as metachronal waves. Pair synchronization is a fundamental requirement for these collective dynamics, but it is generally not sufficient for collective phase-locking, chiefly due to the effect of long-range interactions. Here we explore experimentally and numerically a minimal model for a ciliated surface: hydrodynamically coupled oscillators rotating above a no-slip plane. Increasing their distance from the wall profoundly affects the global dynamics, due to variations in hydrodynamic interaction range. The array undergoes a transition from a traveling wave to either a steady chevron pattern or one punctuated by periodic phase defects. Within the transition between these regimes the system displays behavior reminiscent of chimera states.

The selectivity of Watson-Crick base pairing has allowed the design of DNA-based functional materials bearing an unprecedented level of accuracy. Examples include DNA origami, made of tiles assembling into arbitrarily complex shapes, and DNA coated particles featuring rich phase behaviors. Frequently, the realization of conceptual DNA-nanotechnology designs has been hampered by the lack of strategies for effectively controlling relaxations. In this article, we address the problem of kinetic control on DNA-mediated interactions between Brownian objects. We design a kinetic pathway based on toehold-exchange mechanisms that enables rearrangement of DNA bonds without the need for thermal denaturation, and test it on suspensions of DNA-functionalized liposomes, demonstrating tunability of aggregation rates over more than 1 order of magnitude. While the possibility to design complex phase behaviors using DNA as a glue is already well recognized, our results demonstrate control also over the kinetics of such systems.

Live optical microscopy has become an essential tool for studying the dynamical behaviors and variability of single cells, and cell-cell interactions. However, experiments and data analysis in this area are often extremely labor intensive, and it has often not been achievable or practical to perform properly standardized experiments on a statistically viable scale. We have addressed this challenge by developing automated live imaging platforms, to help standardize experiments, increasing throughput, and unlocking previously impossible ones. Our real-time cell tracking programs communicate in feedback with microscope and camera control software, and they are highly customizable, flexible, and efficient. As examples of our current research which utilize these automated platforms, we describe two quite different applications: egress-invasion interactions of malaria parasites and red blood cells, and imaging of immune cells which possess high motility and internal dynamics. The automated imaging platforms are able to track a large number of motile cells simultaneously, over hours or even days at a time, greatly increasing data throughput and opening up new experimental possibilities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite-red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A two-state oscillator in a viscous liquid is composed of a micron-scale particle whose intrinsic dynamics is defined by linear potentials that undergo configuration-coupled transitions and is externally driven by a piecewise constant periodic force of varying amplitude and frequency. This elementary example of "active matter" has the minimal elements that allow us to study synchronization in the presence of thermal fluctuations. Experiments reveal the presence of synchronized states (and Arnol'd tongues), which we explain using analytical and numerical calculations. The system maintains synchronization by adjusting the phase between the bead and the clock. We discuss the relevance of this model to synchronization in real-world systems, including the role of thermal noise., (© 2011 American Physical Society)

The dynamics of disclination defect lines in a nematic liquid crystal are measured experimentally and considered theoretically. An optical trap is used to deform the line, enabling the previously unexplored regime of large deformation to be accessed. The relaxation follows a linear decay at large amplitude, crossing over into the well understood exponential decay at small amplitude. Both regimes can be described by simple theoretical arguments. The crossover point is well described by the theory, but the experiments show a faster than expected dynamics, indicating that the effective viscosity in the models is overestimated.

Colloidal particles are trapped harmonically on the vertices of planar regular polygons, using optical tweezers. The particles interact with each other via hydrodynamic coupling, which can be described adequately by Oseen's tensor. Because of the interaction, the dynamics of any individual sphere is complex. Thermal motion results in a spectrum of relaxation times. The configuration of a system of N particles can be decomposed into 2N normal modes. In this work it is shown how to calculate these modes and their relaxation time scale analytically. The mathematical structure of the matrix of interaction leads to general properties for the symmetry of the normal modes and their dynamics, differing between the cases of even and odd N. The theory is compared to experiments performed on a range of rings with 3 ≤ N ≤ 10, varying also the trap stiffness and the distance between particles.

We studied the confinement effect on the interaction force in nematic liquid crystal colloids with spherical particles inducing planar anchoring. Using magneto-optical tweezers, we measured the spatial dependence of the quadrupolar structural interparticle force over 4 orders of magnitude. For small separations, the interparticle potential follows the power law, whereas for separations larger than the sample thickness, it decreases exponentially with the decay length proportional to the sample thickness. Experimental results are reproduced by using the Landau-de Gennes free-energy minimization approach.

Magneto-optic tweezers were used for measurements of liquid-crystal-mediated forces between spherical beads with tangential anchoring in thin nematic samples. Repulsive force, which results from the quadrupolar symmetry of defects around the immersed beads, decreases proportionally to 1/x6, with x being the bead separation. The velocity with which the particles are pushed apart also follows the same separation dependence. We thus find the effective drag coefficient gamma(eff) independent of x for surface-to-surface distances as small as 10% of the bead diameter.