The Control of Tetrahymena pyriformis Using Behavioral Responses to Various Stimuli as a Biological Actuator

There is great interest in developing viable robotic swimmers at the microscale for applications such as microassembly, micromanipulation, and drug efficacy testing and other biomedical tasks. One of the greatest obstacles for developing microrobots is the often expensive and complicated fabrication techniques. Microorganisms are continuously targeted for microrobotics research because they possess many of the required components needed for robotic systems, including power systems, sensing abilities, and swimming mechanisms. They are also relatively inexpensive to produce. We investigated the single cell microorganism Tetrahymena pyriformis as a candidate for a microrobot. This ciliated protozoan responds to a variety of stimuli. Here, we demonstrated its control through a variety of control modalities, including electric and magnetic fields. Turning behavior and response to electric fields were quantitatively characterized. We also investigated its swimming capabilities by stripping the cell of its motile organelles and observe their regeneration and recovery. While this cell does not naturally respond to magnetic fields, they were modified through the uptake iron oxide particles and then imparted with a magnetic dipole using a permanent magnet. Magnetic control was used only to steer the cell with negligible translational force. Each cell possessed a magnetic dipole after magnetization, whose strength is a function of the strength the a permanent magnet used to magnetize the cells as well as function of the amount of ingested iron oxide. By studying the effects of rotational fields and each cell’s unique response to the rotational frequency, discrete multi-cell control becomes possible. This swarm control was validated in theory, simulation, and experiments. The development and control of this organism as a microrobot will give us valuable insight to harness and develop versatile biologically inspired robotic systems in the microscale.Ph.D., Mechanical Engineering -- Drexel University, 201

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell\u27s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliat

Artificially generated turbulence: A review of phycological nanocosm, microcosm, and mesocosm experiments

Building on a summary of how turbulence influences biological systems, we reviewed key phytoplankton-turbulence laboratory experiments (after Peters and Redondo in Scientia Marina: Lectures on plankton and turbulence, International Centre for Coastal Resources, Barcelona, 1997) and Peters and Marrase (Marine Ecology Progress Series 205:291-306, 2000) to provide a current overview of artificial turbulence generation methods and quantification techniques. This review found that most phytoplankton studies using artificial turbulence feature some form of quantification of turbulence; it is recommended to use turbulent dissipation rates (epsilon) for consistency with physical oceanographic and limnological observations. Grid-generated turbulence is the dominant method used to generate artificial turbulence with most experiments providing quantified epsilon values. Couette cylinders are also commonly used due to the ease of quantification, albeit as shear rates not epsilon. Dinoflagellates were the primary phytoplanktonic group studied due to their propensity for forming harmful algal blooms (HAB) as well as their apparent sensitivity to turbulence. This study found that a majority of experimental setups are made from acrylate plastics that could emit toxins as these materials degrade under UV light. Furthermore, most cosm systems studied were not sufficiently large to accommodate the full range of turbulent length scales, omitting larger vertical overturns. Recognising that phytoplankton-turbulence interactions are extremely complex, the continued promotion of more interdisciplinary studies is recommended

MicroBioRobots for Single Cell Manipulation

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable micro actuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a bio-integrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 μm coated with a monolayer of the swarming Serratia marcescens. The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments, which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment

The Screening, Fabrication and Production of Microalgae Biocomposites for Carbon Capture and Utilisation

PhD ThesisThe use of microalgae for carbon dioxide sequestration and as a feedstock for biodiesel production has been a topic of active research since the late 1950s. It has not been adopted as a technology due to the difficulties in growing the microalgae, harvesting it and the excessive cost of the fuel produced via this route. This research work focuses on a novel idea of attached microalgae growth method to cultivate different species of freshwater and marine microalgae on a solid substrates to sequester carbon dioxide and use the biomass to produce biofuel. Initially, we undertake a study to prove the concept of nourishing microalgae cells attached to filter paper via capillary. The initial results indicate a good survivability of the immobilised cells with limited nutrients for 8 weeks. The average cumulative CO2 fixation of C. vulgaris cells (3.33 mmol g-1 day-1 ) attached to the paper was more than twice the suspended culture cultivation (0.924 mmol g-1 day-1 ) using 5% CO2/air mixture. The next stage in this research work investigated the use of binders for cell immobilisation on a biodegradable substrate. A binder screening protocol which took into account toxicity and adhesion strength was developed to produce a biocomposite using the best combinations of microalgae species and binders upon different substrates. We subsequently developed an experimental system to continuously sequester carbon dioxide for 6 weeks using biodegradable microalgae bio composites made from loofa sponge and latex binders. D. salina cumulative CO2 fixation of 5.96 mmol/g day-1 when immobilised with latex binder Baymedix CH-120 polyurethane resin dispersion was 15 times higher than the 0.40 mmol/g day-1 recorded for suspended culture. This also translate to reduction in land and water usage when compared to open pond algae cultivation or photobioreactor. The immobilised cells lipid content production improved for two of the algae species (C. vulgaris and D. salina) that were tested. The lipid content was 69.38% and 66.22% biomass dry weight for C. vulgaris and D. salina respectively. This novel research work has the potential to substantially reduce the cost associated with biological carbon capture and biofuel production using microalgae when compared with the open ponds and photobioreactors.Petroleum Technology Development Fund, Nigerian Arm

Actuation and control of microfabricated structures using flagellated bacteria

In this work methods of actuation and control of microfabricated structures are investigated using bacteria as configurable, scalable actuators. Bacteria offer many benefits as microfluidic actuators. They draw chemical energy directly from their environment, they can be operated in a wide range of temperature and pH, and literally billions of bacteria may be cultured within hours. Additionally, the well-documented responses of individual motile bacterial cells may be expected to scale up to arrays of cells. On this population scale, the cellular responses can be employed en masse creating controlled forces that actuate inorganic microfabricated elements. For these investigations the bacterium Serratia marcescens has been chosen. S. marcescens has properties that are particularly appropriate for engineering applications. When cultured on soft agar, the bacteria demonstrate a form of surface motility known as swarming. These investigations start with an experimental analysis of the swarming cell motility using a non-labeled cell tracking technique. The results of these studies reveal that the most energetic bacteria populate the progressing edge of the swarm. A technique of biocompatible microfabrication and chemical release of bacteria-driven microstructures is also presented. This method is used to pattern structure surfaces with the rigorous swarming cells by direct blotting. The self-coordinated motion of the cells is investigated for use as arrays of actuators. Control mechanisms are investigated to adjust rotational and translational motion using optical and electrical stimuli, respectively. The fundamentals of the electrokinetics are also investigated and integrated into a system demonstrating controlled manipulation of target objects and phenotypic chemical sensing.Ph.D., Mechanical Engineering -- Drexel University, 200

Long-Term Quantitative Microscopy: From Microbial Population Dynamics to Growth of Plant Roots

Quantitative optical measurements at the micron scale have been crucial to the study of multiple biological processes, including bacterial chemotaxis, eukaryotic gene expression and y development. Extending measurements to long time scales allows complete observation of processes that are otherwise studied piecemeal, such as development and evolution. This thesis describes the development of two types of microscope for making long term, quantitative measurements, and the tools for image analysis. The rst device is a digital holographic microscope for measuring microbial population dynamics. It allows three dimensional localization of hundreds of cells within a mm3 sized volume, at micron resolution and an acquisition period of minutes. The technique is simple and inexpensive, which enabled us to construct ten replicate devices for parallel measurements. Each device incorporates precise and programmable control of light and temperature for the microbial ecosystem. Experiments were performed with the green algae Chlamydomonas reinhardtii and the ciliate Tetrahymena reinhardtii, both together and in isolation, and continued for as long as 90 days. The population dynamics exhibited a striking degree of repeatability, despite the presence of added noise in the illumination, spatial gradients of cell density, convection currents and phenotypic changes of both species. The second device is a thin light sheet fluorescence microscope for tracking nuclei in growing roots of the flowering plant Arabidopsis thaliana. The device incorporates a chamber designed to maintain optical quality while providing conditions for root growth. Optical feedback to a translation stage is used to maintain the root tip in the fi eld of view as the root grows by centimeters over several days. Data from a three day experiment is presented to demonstrate the technique. Over 1,000 nuclei were tracked simultaneously, and hundreds of cell divisions were automatically identif ed. The device was also used to image the regeneration of a root tip after surgical excision. The data corroborate earlier investigations at a more detailed level than was previously possible

On the dynamics of the flagellar beat under load

Eukaryotic flagella are lash-like cell appendages that can actively bend in order to serve different purposes, from cell propulsion to fluid transport. A remarkable phenomenon which can be observed for beating flagella is stable synchronisation, although a central internal clock seems to be missing. By exposing the biflagellate microswimmer Chlamydomonas reinhardtii, whose flagella are termed cis and trans depending on their proximity to the cell’s eyespot, to controlled fluid flow, we determine the phase-dependent load response of the flagellar beat which is thought to play an important role for synchronisation. Over a certain range, the beating frequency changes linearly with the applied load. If the external load exceeds a certain threshold, the flagellar beat comes to a halt. This threshold depends on the direction of the applied load and if the load is gradually increased from zero to maximum or vice versa, revealing a more or less pronounced hysteresis for cis- and trans-flagellum, individually. For intermediate load, we find two previously unknown, dynamic beating modes of C. reinhardtii’s flagella which occur only if the flow direction is opposite to the swimming direction with one of these new beating modes being almost exclusive to the cis-flagellum. In general, we observe a different behaviour of cis- and trans-flagellum under load. At last, we find that the capability for flagellar synchronisation depends on the strength and the direction of the applied load.Eukaryotische Flagellen sind fadenartige Zellausstülpungen, die sich aktiv verbiegen können und von der Fortbewegung von Zellen bis hin zum Flüssigkeitstransport unterschiedlichen Zwecken dienen. Ein erstaunliches Phänomen, das man bei schlagenden Flagellen beobachten kann, ist deren stabile Synchronisation, wenngleich ein zentraler Taktgeber zu fehlen scheint. Indem wir Chlamydomonas reinhardtii, einen Mikroschwimmer, dessen Flagellen abhängig von ihrer Nähe zum Augenfleck als cis- und trans-Flagellum bezeichnet werden, kontrollierten Flüssen aussetzen, können wir die Lastantwort des Flagellenschlags, welche eine wichtige Rolle für die Synchronisation spielt, bestimmen. In einem gewissen Bereich ändert sich die Frequenz des Flagellenschlags linear mit der angelegten Last. Überschreitet die Last einen bestimmten Schwellenwert, der sowohl von der Richtung der angelegten Last als auch davon abhängt, ob die Last schrittweise von Null auf das Maximum erhöht wird oder umgekehrt, so kommt der Flagellenschlag zum Erliegen. Ebenso zeigen cis- und trans-Flagellum eine mehr oder weniger stark ausgeprägte Hysterese. Im Bereich mittlerer Last finden wir zwei bisher unbekannte, dynamische Schlagmoden, sofern die Zelle entgegen der Flussrichtung schwimmt, wobei eine Schlagmode fast ausschließlich für das cis-Flagellum beobachtet werden konnte. Außerdem hängt die Fähigkeit zur Synchronisation des Flagellenschlags sowohl von der Richtung als auch von der Stärke der angelegten Last ab

The bank of swimming organisms at the micron scale (BOSO-Micro).

Unicellular microscopic organisms living in aqueous environments outnumber all other creatures on Earth. A large proportion of them are able to self-propel in fluids with a vast diversity of swimming gaits and motility patterns. In this paper we present a biophysical survey of the available experimental data produced to date on the characteristics of motile behaviour in unicellular microswimmers. We assemble from the available literature empirical data on the motility of four broad categories of organisms: bacteria (and archaea), flagellated eukaryotes, spermatozoa and ciliates. Whenever possible, we gather the following biological, morphological, kinematic and dynamical parameters: species, geometry and size of the organisms, swimming speeds, actuation frequencies, actuation amplitudes, number of flagella and properties of the surrounding fluid. We then organise the data using the established fluid mechanics principles for propulsion at low Reynolds number. Specifically, we use theoretical biophysical models for the locomotion of cells within the same taxonomic groups of organisms as a means of rationalising the raw material we have assembled, while demonstrating the variability for organisms of different species within the same group. The material gathered in our work is an attempt to summarise the available experimental data in the field, providing a convenient and practical reference point for future studies