Methods and measures for investigating microscale motility

Motility is an essential factor for an organism's survival and diversification. With the advent of novel single-cell technologies, analytical frameworks and theoretical methods, we can begin to probe the complex lives of microscopic motile organisms and answer the intertwining biological and physical questions of how these diverse lifeforms navigate their surroundings. Herein, we give an overview of different experimental, analytical, and mathematical methods used to study a suite of microscale motility mechanisms across different scales encompassing molecular-, individual- to population-level. We identify transferable techniques, pressing challenges, and future directions in the field. This review can serve as a starting point for researchers who are interested in exploring and quantifying the movements of organisms in the microscale world.Comment: 24 pages, 2 figure

Microfluidic Devices for Analysis of Spatial Orientation Behaviors in Semi-Restrained Caenorhabditis elegans

This article describes the fabrication and use of microfluidic devices for investigating spatial orientation behaviors in nematode worms (Caenorhabditis elegans). Until now, spatial orientation has been studied in freely moving nematodes in which the frequency and nature of encounters with the gradient are uncontrolled experimental variables. In the new devices, the nematode is held in place by a restraint that aligns the longitudinal axis of the body with the border between two laminar fluid streams, leaving the animal's head and tail free to move. The content of the fluid streams can be manipulated to deliver step gradients in space or time. We demonstrate the utility of the device by identifying previously uncharacterized aspects of the behavioral mechanisms underlying chemotaxis, osmotic avoidance, and thermotaxis in this organism. The new devices are readily adaptable to behavioral and imaging studies involving fluid borne stimuli in a wide range of sensory modalities

Of Cell Shapes and Motion: The Physical Basis of Animal Cell Migration.

Motile cells have developed a variety of migration modes relying on diverse traction-force-generation mechanisms. Before the behavior of intracellular components could be easily imaged, cell movements were mostly classified by different types of cellular shape dynamics. Indeed, even though some types of cells move without any significant change in shape, most cell propulsion mechanisms rely on global or local deformations of the cell surface. In this review, focusing mostly on metazoan cells, we discuss how different types of local and global shape changes underlie distinct migration modes. We then discuss mechanical differences between force-generation mechanisms and finish by speculating on how they may have evolved

Model-based image analysis of a tethered Brownian fibre for shear stress sensing

The measurement of fluid dynamic shear stress acting on a biologically relevant surface is a challenging problem, particularly in the complex environment of, for example, the vasculature. While an experimental method for the direct detection of wall shear stress via the imaging of a synthetic biology nanorod has recently been developed, the data interpretation so far has been limited to phenomeno-logical random walk modelling, small-angle approximation, and image analysis techniques which do not take into account the production of an image from a three-dimensional subject. In this report, we develop a mathematical and statistical framework to estimate shear stress from rapid imaging sequences based firstly on stochastic modelling of the dynamics of a tethered Brownian fibre in shear flow, and secondly on a novel model-based image analysis, which reconstructs fibre positions by solving the inverse problem of image formation. This framework is tested on experimental data, providing the first mechanistically rational analysis of the novel assay. What follows further develops the established theory for an untethered particle in a semi-dilute suspension, which is of relevance to, for example, the study of Brownian nanowires without flow, and presents new ideas in the field of multi-disciplinary image analysis

Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species

Microswimmers and Microfluidics: Understanding and Manipulating the Locomotion of Undulatory Microswimmers

Undulatory microswimmers, such as nematodes, are of great importance to agriculture, animal and human health, and fundamental biological research. The nematode Caenorhabditis (C.) elegans is widely used as a model organism for medical studies. My work focuses on studying the locomotion of nematodes; their interactions with surfaces, fluid flow, and each other; and developing new tools to manipulate their motion for diverse applications. In the first half (chapters 2-4) of this dissertation, I investigate experimentally and theoretically the effects of flat solid surfaces, external channel flow, and other swimmers on the swimming dynamics of undulatory microswimmers. I discovered that 1) when swimming in close proximity, undulatory microswimmers synchronize their swimming gait. This synchronization is facilitated by direct collisions among the swimmers, rather than by long-range hydrodynamic interactions or deliberate actions of the swimmers; 2) undulatory micro-swimmers have a tendency to accumulate near and swim along surfaces. This behavior does not require touch sensation ability of the swimmers, and can be explained by a short-range hydrodynamic interaction between the swimmers and adjacent surface; 3) undulatory microswimmers exhibit positive rheotaxis (upstream swimming behavior) near solid surfaces. This behavior is induced by the combination of a hydrodynamic surface attraction effect and the velocity gradient of external flow near solid surfaces. These findings help explain certain intriguing behaviors of undulatory microswimmers, highlight the diverse roles of hydrodynamic forces in microswimmers\u27 life cycles, and lay the foundations for novel microswimmer manipulation methods for fundamental biological research and clinical applications. In the second half (chapters 5-7) of this dissertation, I present the design, fabrication, characterization, and applications of a few engineering devices/methods for dynamic trapping, motility measurement, motility-based sorting, and directing the motion of microswimmers. These new devices/methods enabled many studies that would be impossible or impractical with conventional methods

Control and navigation problems for model bio-inspired microswimmers

Navigation problems for a model bio-inspired micro-swimmer, consisting of a cargo head and propelled by multiple rotating flagella or propellers and swimming at low Reynolds numbers, are formulated and solved. We consider both the direct problem, namely, predicting velocity and trajectories of the swimmer as a consequence of prescribed rotation rates of the propellers, and inverse problems, namely, find the rotation rates to best approximate desired translational and rotational velocities and, ultimately, target trajectories. The equations of motion of the swimmer express the balance of the forces and torques acting on the swimmer, and relate translational and rotational velocities of the cargo head to rotation rates of the propellers. The coefficients of these equations, representing hydrodynamic resistance coefficients, are evaluated numerically through a custom-built finite-element code to simulate the (Stokes) fluid flows generated by the movement of the swimmer and of its parts. Several designs of the propulsive rotors are considered: from helical flagella with different chirality to marine propellers, and their relative performance is assessed

Bio-inspired Magnetic Systems: Controlled Swimming, Fluid Pumps, and Collective Behaviour

This thesis details the original experimental investigations of magnetically actuated and controlled microscopic systems enabling a range of actions at low Reynolds number. From millimetre-robots and self-propelled swimmers to microfluidic and lab-on-a-chip technology applications. The main theme throughout the thesis is that the systems reply on the interactions between magnetic and elastic components. Scientists often take inspiration from nature for many aspects of science. Millimetre to micrometre machines are no exception to this. Nature demonstrates how soft materials can be used to deform in a manner to create actuation at the microscale in biological environments. Nature also shows the effectiveness of using beating tails known as flagella and the apparent enhancements in flow speeds of collective motion. To begin with, a swimmer comprised of two ferromagnetic particles coupled together with an elastic link (the two-ferromagnetic particle swimmer), was fabricated. The system was created to mimic the swimming mechanism seen by eukaryotic cells, in which these cells rely on morphological changes which allows them to propel resulting in approximate speeds of up to 2 body lengths per second. The aim of this system was to create a net motion and control the direction of propagation by manipulating the external magnetic field parameters. It was shown that the direction of swimming has a dependence on both the frequency and amplitude of the applied external magnetic field. A key factor discovered was that the influence of a small bias field, in this case, the Earth’s magnetic field (100 orders of magnitude smaller than the external magnetic field) resulted in robust control over the speed (resulting in typical swimming speeds of 4 body lengths per second) and direction of propulsion. Following this work, swimmers with a hard ferromagnetic head attached to an elastic tail (the torque driven ferromagnetic swimmer) were investigated. These systems were created to be analogous to the beating flagella of many natural microscopic swimmers, two examples would be sperm cells and chlamydomonas cells. These biological cells have typical speeds of 10s of body lengths per second. The main focus of this investigation was to understand how the tail length affects the swimming performance. An important observation was that there is an obvious length tail (5.7 times the head length) at which the swimming speed is maximised (approximately 13 body lengths per second). The experimental results were compared to a theoretical model based on three beads, one of which having a fixed magnetic moment and the other two non-magnetic, connected via elastic filaments. The model shows sufficient complexity to break time symmetry and create a net motion, giving good agreement with experiment. Portable point-of-care systems have the potential to revolutionise medical diagnostics. Such systems require active pumps with low power (USB powered devices) external triggers. Due to the wireless and localisation of magnetic fields could possibly allow these portable point-of-care devices to come to life. The main focus of this investigation was to create fluid pump systems comprising from the previously investigated two-ferromagnetic particle swimmer and the torque driven ferromagnetic swimmer. Building on the fact that if a system can generate a net motion it would also be able to create a net flow. Utilising the geometry of the systems, it has been demonstrated that a swimmer-based system can become a fluid pump by restricting the translational motion. The flow structure generated by a pinned swimmer in different scenarios, such as unrestricted flow around it as well as flow generated in straight, cross-shaped, Y-shaped and circular channels were investigated. This investigation demonstrated the feasibility of incorporating the device into a channel and its capability of acting as a pump, valve and flow splitter. As well as a single pump system, networks of the previously mentioned pump systems were fabricated and experimentally investigated. The purpose of this investigation was to utilise the behaviour of the collective motion. Such networks could also be attached to the walls or top of the channel to create a less invasive system compared to pump based within the channel system. The final investigation involved creating collective motion systems which could mimic the beating of cilia - known as a metachronal wave. Two methods were used to create an analogous behaviour. The first was using arrays of identical magnetic rotors, which under the influence of an external magnetic field created two main rotational patterns. The rotational patterns were shown to be controllable producing useful flow fields at low Reynolds numbers. The second system relied on the magnetic components having different fixed magnetisation to create a phase lag between oscillations. The magnetic components were investigated within a channel and the separation between the components was shown to be a key parameter for controlling the induced flow. In both cases, a simple model was produced to help understand the behaviour. Finally, a selection of preliminary investigations into possible applications were conducted experimentally. These investigations included, measuring the effective surface viscosity of lipid monolayers, created cell growth microchannels, as well as systems which could be used for blood plasma separation. The properties of lipid monolayers vary with the surface density, resulting on distinct phase transitions. Slight differences in the molecular lattice are often accompanied by significant changes in the surface viscosity and elasticity. The idea was to use a swimmer as a reporter of the monolayer viscosity, resulting in a less invasive method compared to current techniques to monitor monolayer viscosity, for example torsion pendulums and channel viscometers. The reported effective surface viscosity closely matched the typical Langmuir trough measurements (with a systematic shift of approximately 17 Ų/molecule). The blood plasma separation preliminary work shows the previously investigated two-ferromagnetic particle swimmer mixing a typical volume (100 μm) blood sample with a buffer solution in 21 seconds. The system was also able to create locations with a high population of red blood cells. This resulted in a separation between the blood plasma and red blood cells. Two other preliminary results of future investigations were presented; the collective motion of free swimmers, and the fabrication of ribbon-like structures with fixed magnetic moment patterns.European CommissionEngineering and Physical Sciences Research Council (EPSRC