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This thesis explores the structures and dynamics of optically confined matter, ranging from single particle traps to complex optically bound colloidal arrays, investigating and quantifying the behaviour of each system. It begins with an introduction to optical manipulation techniques and a discussion of the development of the single beam gradient force trap, more commonly referred to as optical tweezers. Following this, the building of a single beam optical trap will be presented alongside a discussion of some of the key components in such a setup, before it is calibrated, allowing a demonstration of some of the techniques which are utilised later in the thesis. The optical trapping of aerosol droplets is an area of key importance in atmospheric chemistry, as optical tweezers provide a valuable and versatile tool for droplet manipulation and characterisation. Trapping single aerosol droplets is facilitated by using annular rather than conventional Gaussian beams, as will be demonstrated, with significant advantages in increasing the size range of trappable droplets, and improving their axial localisation. These improvements will be demonstrated experimentally with an in-depth comparison of Gaussian and annular beam trapping. These enhancements are also verified theoretically using a model developed by Burnham and McGloin, showing excellent agreement with experimental results. Ionic liquids, defined as organic salts with melting points below room temperature, are another area of great contemporary interest. They are highly tunable and so have been referred to as "designer solvents", and also have important applications as "green" solvents in organic chemistry. Trapping particles within these novel liquids allows a micro-rheological investigation of their properties to be conducted. This is demonstrated by determining the temperature dependent viscosity changes of these media, showing excellent agreement with previous macro-rheological studies. In addition, hydrodynamic effects such as Faxen's correction to viscous drag in proximity to a surface, and hydrodynamic coupling between pairs of colloids trapped in ionic liquids are demonstrated. Following these single and dual particle studies, this thesis continues with an investigation of the structures and dynamics of optically bound matter formed of larger numbers of particles. The behaviour of these optically bound structures is particularly sensitive to the number of particles involved, and so a counter-propagating evanescent field trap in conjunction with an inverted optical tweezers setup is utilised in order to controllably assemble these structures and study the factors affecting their behaviour. Initially one-dimensional chains of optically bound 3.5 um diameter silica particles are studied, allowing an implementation of Generalized Lorentz-Mie Theory (GLMT) to be developed through collaboration with Dr. Jonathan Taylor of The University of Glasgow. Experimental and theoretical insights allow further understanding of the processes involved in the formation of these structures. Having studied the behaviour of 3.5 um diameter silica particles in a counter-propagating evanescent wave trap, the effects of changing particle size and refractive index are presented by using smaller silica and melamine particles. These results are explained in terms of the increased importance of interference fringes in determining the arrangement of the optically bound structures of smaller particles, and due to the increased interaction of the melamine particles with the evanescent field as a result of the larger refractive index contrast between them and the trapping medium. The thesis then concludes with a study of the dynamics of the previously presented optically bound chains. Initially the diffusion of single particles in the evanescent field is compared to their freely-diffusing behaviour, quantifying the confining effect of the field. The addition of particles to the field then allows the diffusive behaviour to be studied as a function of particle number, and understood in terms of on-axis confinement by adjacent particles. The tilting of these optically bound chains relative to the inter-beam axis is also explored as a function of particle number, as is the rigidity of these chains. Finally a more complex, dynamic effect is presented, dubbed "Newton's Cradle", in which particles are ejected from the ends of the chains before returning and repeating this process. This behaviour is understood by utilising the previously developed GLMT simulations.

It has been proposed that coated gas microbubbles, currently used as ultrasound contrast agents could also be used as microscale sensors due to the sensitivity of their acoustic response to changes in their environment. However, their behaviour is not fully understood and there remains considerable scope for improving their characterisation. The aim of this thesis is to improve the theoretical description of microbubble dynamics under ultrasound excitation with the ultimate aim of assessing the regimes in which they could be exploited most effectively as sensors. Previous theoretical and experimental work relating to the confinement and acoustic excitation of microbubbles is reviewed. Specifically, optical trapping as a method for the isolation and manipulation of individual bubbles is studied for use in developing a sensor. An assessment of the existing models’ validity is undertaken. This is followed by the development of models for optical trapping of single microbubbles, and the coupled radial and translational motion of a microbubble under ultrasound excitation, which includes time dependent phenomena. The latter model is used to perform a sensitivity analysis to determine the uncertainty associated with using microbubbles as sensors. The potential for uniquely characterising the shell of the microbubble from experimental data is also assessed. Subsequent chapters present the results from a combination of computer simulations and experimental data, used to develop and assess the validity of the new models for describing microbubble behaviour. Particularly, the model is used to simulate the response of a dilute suspension of microbubbles undergoing large amplitude oscillations and single microbubbles undergoing lipid shedding. The optimal regimes in which microbubbles may be utilised as sensors for liquid physical properties and local pressure variations are then assessed. Finally, a summary of the conclusions and areas for further work is presented.

Technological limitations have prevented interrogation and manipulation of many signaling pathways in model and living systems required for the development of diagnostic and therapeutic modalities in diseases, such as cancer. Liposome-supported plasmon resonant gold nanoshells are biologically inspired composite structures, in which the liposome allows for the encapsulation of substances, and the plasmon resonant structure facilitates rapid release of encapsulated contents upon laser light illumination. As shown in this work, we overcome current limitations in cellular manipulation using plasmon resonant gold-coated liposomes in conjunction with light-activated release to achieve accurate probing of complex cellular responses. Development toward this goal was demonstrated with four specific aims. The first specific aim was to develop a computational model of heat diffusion to investigate the light-induced heating of gold-coated liposomes. This model was used to optimize the photothermal process for release of an encapsulated payload. The second aim was to demonstrate encapsulation and on-demand release of molecules in a spectrally-controlled manner, where plasmon resonant nanoparticles only release content upon illumination with a wavelength of light matching their plasmon resonance band. The third specific aim was to demonstrate that this release mechanism can be used in a biological setting to deliver a peptide and extracellularly activate surface membrane receptors with single-cell spatial and high temporal resolution. The fourth specific aim further refined the level of spatial and temporal control of payload release using gold-coated liposomes with optical trapping to demonstrate mirco-manipulation of liposome movement and rapid content release to enable accurate perturbation of cellular functions in response to released compounds. Through this work, we have developed an experimental system with the potential for the delivery and localized release of an encapsulated agent with high spatial and temporal resolution. This on-demand release system is compatible with a broad range of molecules and uses biologically safe near-infrared light. In combination with the spectral tunability of these plasmon resonant nanoshells and spectrally-selective release, this technology may allow for interrogation of complex and diverse signaling pathways in living tissues or their models with unprecedented spatial and temporal control.

The delivery of biomolecules into living cells is an important methodology in cell and molecular biology. Optical methods using lasers are attractive tools for such application. However, the interaction of the laser with the cell depends on the laser type and the parameters used. Hence, in this thesis, optical transfection and injection of both mammalian and embryonic cells is demonstrated using a variety of laser sources. Furthermore, some key issues are addressed by demonstrating alternative configurations of optoinjection and transfection systems to develop a robust, user-friendly device with potential for commercialisation. Most optical methods for the delivery of molecules rely on complex and expensive laser systems that occupy a large footprint. In order for the system to be accessible to end-users, transient transfection of plasmid DNA into mammalian cells using an inexpensive continuous wave 405 nm diode laser is demonstrated. In this work, the laser parameters are varied in order to optimise the transfection efficiency. By calculating the temperature change upon irradiation of the focused violet light, the mechanism of violet diode laser transfection is elucidated. Furthermore, the system is used to deliver small interfering RNA molecules to specifically knock down a particular protein within the cell. This work is a major step towards an inexpensive and portable optical transfection system. The critical issue of accurate targeting of the cell membrane is also addressed in conventional near-infrared femtosecond optical transfection systems. A near-infrared femtosecond holographic system is built utilising a spatial light modulator in order to provide fast three dimensional beam translation. Computer control of dosage and targeting allows us to explore the potential of different targeting modalities. An enhanced optoinjection and transfection on mammalian cells is demonstrated. Furthermore, the system is applied to optically manipulate a developing Pomatoceros lamarckii embryo. The holographic system can be employed to optoinject a variety of macromolecules into the embryo, as well as orient and position the embryo by switching to the continuous wave mode of the laser. Such development of optical techniques to deliver biomolecules and orient embryos will benefit the field of developmental biology. Lastly, to achieve controlled cavitation, limiting the mechanical effects of a nanosecond laser source, an optically trapped microsphere undergoes laser induced breakdown in the presence of a cell monolayer. Laser induced breakdown of a trapped microsphere allows control over several parameters, such as the microsphere material, position of the breakdown from the monolayer and the size of the microsphere. Optimising these parameters provide limited mechanical effects, particularly suited for cell transfection. This technique is an excellent tool for plasmid-DNA transfection of multiples of cells with both reduced energy requirements and cell lysis compared to previously reported approaches. Demonstrating optimised and successful delivery of macromolecules with the variety of laser sources used in this thesis will advance the applicability of optical injection and transfection and allow more potential users to access the technique. This thesis advances optical injection and transfection for optimised delivery of macromolecules to both mammalian cells and a developing embryo.

Optical manipulation covers a wide range of techniques to guide and trap cells using only the forces exerted by light. Another optical tool is photoporation, the technique of injecting membrane-impermeable molecules using light, which has become an important alternative to other injection techniques. Together they provided sterile tools for manipulation and molecule delivery at the single-cell level. In this thesis, the properties of low Reynolds fluid flows are exploited to guide cells though a femtosecond Bessel beam. This design allows for high-throughput optical injection of cells without the need to individually target cells. A method of 'off-chip' hydrodynamic focusing was evaluated and was found to confine 95.6% of the sample within a region which would receive a femtosecond dose compared to 20% without any hydrodynamic focusing. The system was tested using two cell lines to optically inject the membrane-impermeable dye, propidium iodide. This resulted in an increase of throughput by an order of magnitude compared to the previous microfluidic design (to up to 10 cells per second). Next optical trapping and photoporation were combined to create a multimodal workstation. The system provides 3D beam control using spatial light modulators integrated into a custom user interface. The efficiency of optical injection of adherent cells and trapping capabilities were tested. The development of the system provides the groundwork for exploration of the parameters required for photoporation of non-adherent cells. Finally optical trapping is combined with temporally focused multiphoton illumination for scanless imaging. The axial resolution of the system was measured using different microscope objectives before imaging cells stained with calcein. Both single and a pair of recently trypsinised cells were optically trapped and imaged. The position of the trapped cells was manipulated using a spatial light modulator in order to obtain a z-stack of images without adjusting the objective position.

Optical cell manipulation allows precise and non-invasive exploration of mammalian cell function and physiology for medical applications. Plants, however, represent a vital component of the Earth's ecosystem and the knowledge gained from using optical tools to study plant cells can help to understand and manipulate useful agricultural and ecological traits. This thesis explores the potential of several biophotonic techniques in plant cells and tissue. Laser-mediated introduction of nucleic acids and other membrane impermeable molecules into mammalian cells is an important biophotonic technique. Optical injection presents a tool to deliver dyes and drugs for diagnostics and therapy of single cells in a sterile and interactive manner. Using femtosecond laser pulses increases the tunability of multiphoton effects and confines the damage volume, providing sub-cellular precision and high viability. Extending current femtosecond photoporation knowledge to plant cells could have sociological and environmental benefits, but presents different challenges to mammalian cells. The effects of varying optical and biological parameters on optical injection of a model plant cell line were investigated. A reconfigurable optical system was designed to allow easy switching between different spatial modes and pulse durations. Varying the medium osmolarity and optoinjectant size and type affected optoinjection efficacy, allowing optimisation of optical delivery of relevant biomolecules into plant cells. Advanced optical microscopy techniques that allow imaging beyond the diffraction limit have transformed biological studies. An ultimate goal is to merge several biophotonic techniques, creating a plant cell workstation. A step towards this was demonstrated by incorporating a fibre-based optical trap into a commercial super-resolution microscope for manipulation of cells and organelles under super-resolution. As proof-of-concept, the system was used to optically induce and quantify an immunosynapse. The capacity of the super-resolution microscope to resolve structure in plant organelles in aberrating plant tissue was critically evaluated.

The development of ultrasound contrast agents, or microbubbles, over the past 40 years has increased the possibilities for diagnostic imaging, although, more recently they have been proposed as a new vehicle for delivery of drugs and genes. However, there yet remains a considerable lack of fundamental understanding of microbubble behaviour under ultrasound excitation which has restricted their translation to therapeutic use. This project focussed on three key areas relating to the generation, observation, and bioeffects of microbubbles and the ultrasound used in their excitation. The experimental endeavour involved first, a full characterisation of the performance of a rotating mirror high-speed camera (Cordin 550-62) that was previously used by our group [and others] to investigate microbubble dynamics. Specifically, the investigation begins with an assessment of the frame-rate reporting accuracy of the system, a key aspect to the robustness of quantitative measurements extracted from recorded image sequences. This is then followed by the demonstration of a novel method of analysis for examining the image formation process in this type of camera, which facilitates a sensor-by-sensor assessment of performance that was not previously realised. Consolidating with previous work from within the group, this new analysis method was used to clarify previous data, and in the process suggested the presence of a temporal anomaly embedded within recorded images. In addition, the analysis also revealed empirical evidence for the mechanisms leading to this anomaly. Following on, a holographic optical tweezer system was developed for the purpose of exercising precise spatial control over microbubbles within their experimental environment. By positioning microbubbles in specific arrangements, interesting behaviours that were not previously achieved experimentally in the context of shelled microbubbles, were observed. Furthermore, by careful positioning of microbubbles within the imaging plane, it was possible to exploit the temporal anomaly present in the camera to greatly improve the integrity of data recorded, and to also operate in an enhanced imaging mode. Group aspirations to accelerate the development of therapeutic microbubbles had previously generated some early work on the in-house generation of bespoke bubble populations using microfluidic lab-on-a-chip techniques. In order to facilitate further development in this area, a finite-element computational model was herein developed to aid next generation chip design. Finally, in a slightly different context, considering not only the mechanical effect a microbubble may effect in a therapeutic treatment, a single biological cell assay was developed in order to probe any mechanical effects that were induced by the excitation ultrasound itself. Capitalising on the precise force control possible with atomic force spectroscopy, the elastic moduli of cells pre- and post-ultrasound insonation (sans microbubbles) were recorded. These new developments have extended the group capability and expertise in the areas of high-speed imaging, experimental observations of microbubble dynamics and with microfluidic generation of microbubbles. Additionally, the insights garnered have both served to consolidate the group's previous and as yet unpublished data, opening the way for circulation with absolute confidence in the integrity of that data.

Le piégeage optique se présente maintenant, depuis quelques décennies, comme une thématique majeure à l'intersection de diverses disciplines. Depuis les résultats d'Ashkin, de nombreux travaux ont été effectués dans le piégeage et le guidage d'objets physiques (particules, molécules, bactéries, etc.) de toutes tailles. Ces derniers caractériseront alors, devant la longueur d'onde, le domaine optique dans lequel nous nous placerons (Rayleigh, Mie, Optique géométrique).Notre travail porte donc sur l'étude des propriétés de chaînes linéaires périodiques de gouttelettes (huile), placées dans l'eau, et soumises à deux faisceaux laser horizontaux contra-propageants de profil gaussien. Nous démontrons qu'il est possible d'établir un ordre spatial sur un ensemble de grosses gouttes (devant la longueur d'onde) suivant une structure périodique. L'originalité d'un tel système réside dans le fait que la lumière peut alors être refocalisée par l'ensemble des gouttes espacées périodiquement. Cette périodicité peut ainsi, dans certains cas, conférer au faisceau une refocalisation périodique au sein du réseau. Cette première étude, en limite statique, nous permet ainsi de mettre en évidence les conditions de couplage des modes liés aux chaînes de gouttes. En particulier, nous caractérisons la présence de modes de Bloch où le faisceau se propage avec une périodicité équivalente à celle du réseau. Cela nous amène à remarquer que ces conditions modales sont soumises au paramètre de phase gaussien "Thêta" (phase de Gouy). Ainsi, bien que structuré à une échelle largement supérieure, nous mettons en évidence théoriquement des propriétés analogues à celle des cristaux photoniques, conférées par la périodicité des chaînes de gouttes. Ce qui nous permet, en conséquence, de démontrer l'existence de bandes interdites, nous amenant à définir un ensemble de modes guidants/nonguidants de cette chaîne. Cette étude statique est, par la suite, étendue d'un point de vue dynamique en considérant l'effet des forces optiques sur les gouttes. Nous démontrons ainsi qu'il est possible de piéger optiquement de telles gouttes sur des états d'équilibres stables. Au-delà desquels nous mettons en évidence, à travers une étude paramétrique, l'existence de modes oscillants périodiques ou pseudo-périodiques. Enfin, nous prenons en compte les phénomènes de collisions par coalescence, entraînant une réorganisation des répartitions de champs optiques qui peuvent se traduire par de nouvelles configurations de piégeage / Optical trapping appears now, since a few decades, as a major theme at the intersection of variousdisciplines. Since the results of Ashkin, many works were made in the trapping and the guidance of physical objects (particles, molecules, bacteria, etc.) of any sizes. The latter will characterize then, in front of the wavelength, the optical domain in which we shall take place (Rayleigh, Mie, Geometrical Optics).Our work thus concerns the study of the properties of periodic linear chains of droplets (oil), placed in water, and submitted to two counter-propagating horizontal laser beams of gaussian profile. We show that it is possible to establish a spatial order of a set of large drops (in front of the wavelength) in a periodic structure. The originality of such a system lies in the fact that the light can then be refocused by the set of periodically spaced drops. This periodicity may thus, in some cases, confer on the beam a periodic refocusing within the network. This first study, in static limit, allows us to identify the conditions of coupling modes associated with drop channels. In particular, we characterize the presence of Bloch modes where the beam propagates with similar frequency to that of the network. This leads us to note that these modal conditions are submitt to the gaussian phase parameter "Thêta" (Gouy phase). Thus, although structured at a widely higher scale, we highlight theoretically similar properties to that of the photonic crystals, conferred by the periodicity of the chains of drops. This allows us, consequently, to demonstrate the existence of bandgaps, leading us to define a set of guiding/not-guiding modes of this chain. This static study, thereafter, is extended from a dynamic point of view by taking into account the effect of the optical forces on the drops. We show that it is possible to optically trap such drops on stable equilibrium states. Beyond of which we highlight, through a parametric study, the existence of periodic or pseudo-periodic oscillating modes.Finally, we take into account the phenomena of collisions by coalescence, involving a reorganization of the distributions of optical fields which can result in new configurations of trapping.

Optical tweezers have been widely used by biophysicists to measure forces in single molecular processes, such as the force of a motor molecule walking and the force of a DNA molecule winding and unwinding. In these and similar force measurements, the usual assumption is that the force applied to a particle inside the tweezers is proportional to the displacement of the particle away from the trap center like Hookean springs, which would imply that the force field is conservative. However, the Gaussian beam model has indicated that the force field generated by optical tweezers is actually nonconservative, yet no experiments have measured or accounted for this effect. We introduce an experimental method -- the local drift method -- that can measure the force field in optical tweezers with high precision without any assumptions about the functional form of the force field. The force field is determined by analyzing the Brownian motion of a trapped particle. We successfully applied this method to different sizes of particles and measured the three dimensional force field with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is indeed nonconservative. The nonconservative contribution increases radially away from the optical axis for both small and large particles. The curl vector field -- a measurement of the nonconservative force field -- reverses direction from counter-clockwise for small particles in the Rayleigh regime to clockwise for large particles in the ray optics regime, consistent with the different scattering force profiles in the two distinct scattering regimes. Together with the thermal fluctuations of the trapped particle, the nonconservative force can cause a complex flux of energy into the system. Optically-confined Brownian motion is further used to probe nanostructures such as a biopolymer network. This technique -- thermal noise imaging -- uses a Brownian particle as a "natural scanner" to explore a biopolymer network by moving the Brownian particle through the network with optical tweezers. The position fluctuations of the probe particle reflect the location of individual filaments as excluded volumes. The resolution of thermal noise imaging is directly coupled to the size of the probe particle. A smaller probe is capable of exploring smaller pore sizes formed by dense network. Previously, a 200 nm polystyrene particle had been used to probe an agar network. In this work, 100 nm gold probe particles are used to enhance the resolution. A 100 nm particle explore a network with mesh 2³ times smaller and therefore enhance the network resolution by 2³ times. A 100 nm particle also improves the imaging speed by a factor of 2 because of its faster diffusion. Three-dimensional thermal noise images of agarose filaments are obtained and a resolution of 10 nm for the position of the filaments is achieved. In addition, a gold particle is trapped with significantly less power than a polystyrene particle of the same size, indicating the possibility for using even smaller gold particles to further improve the resolution. / text