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.

The thermal velocity fluctuations of microscopic particles mediate the transition from microscopic statistical mechanics to macroscopic long-time diffusion. Prior to this work, detection methods lacked the sensitivity necessary to resolve motion at the length and time scales at which thermal velocity fluctuations occur. This dissertation details two experiments which resulted in velocity measurement of the thermal motion of dielectric microspheres suspended by an optical trap in gases and liquids. First, optical tweezers were used to trap glass microspheres in air over a wide range of pressures and a detection system was developed to track the trapped microspheres' trajectories with MHz bandwidth and <100 fm/rt(Hz) position sensitivity. Low-noise trajectory measurements allowed for observation of fluctuations in the instantaneous velocity of a trapped particle with a signal to noise ratio (SNR) of 26 dB, and provided direct verification of the equipartition theorem and of the Maxwell-Boltzmann velocity distribution for a single Brownian particle. Next, the detection technology was further optimized and used to track optically trapped silica and barium titanate glass microspheres in water and acetone with >50 MHz bandwidth and <3 fm/rt(Hz) sensitivity. Brownian motion in a liquid is influenced by hydrodynamic, time-retarded coupling between the particle and the fluid flow its motion generates. Our measurements allowed for instantaneous velocity measurement with an SNR of up to 16 dB and confirmed the Maxwell Boltzmann distribution for Brownian motion in a liquid. The measurements also revealed several unusual features predicted for Brownian motion in the regime of hydrodynamic coupling, including faster-than-exponential decay of the velocity autocorrelation function, correlation of the thermal force and non-zero cross-correlation between the particle's velocity and the thermal force preceding it. / text

Mechanics and biological function of nucleic acids are intimately coupled. The DNA double helix must be opened to allow base pairing of RNA during transcription; RNA must bend and fold in its many cellular functions. Presented in this dissertation are two investigations of mechanical deformations of nucleic acids, conducted with optical tweezers.In the introduction, the mechanical properties of DNA and RNA and their relevance to their cellular functions are introduced, to give the reader context for the results presented in the Chapters 2 and 3. This is followed by an introduction to the theory of semiflexible polymer elasticity. The optical tweezers instruments used in conducting these investigations are then presented, along with calibration procedures and a short introduction to optical trapping physics.Chapter 2 presents an investigation of the effect of downstream DNA tension on initiation by T7 RNA polymerase. A hidden Markov model is fit to force-dependent lifetimes obtained from optical tweezers experiments, allowing us to identify which steps in initiation are force-dependent and estimate rates and transition state distances. We find that 1-2 pN of tension is sufficient to turn o gene expression by causing transcription bubble collapse and destabilizing the bound state. Our force-dependence scheme and estimated transition distances provide independent supportfor the \scrunching" model of initiation.The effects of cation binding and screening on single-stranded helix formation in poly(A) RNA are presented in Chapter 3. Magnesium and calcium bind to poly(A), stabilize the helix, and change its mechanical properties. A new model of helix-coil transitions is presented and used to estimate energetics and mechanical properties.Chapter 4 presents the first fully objective algorithm for use in analyzing the noisy staircaselike data that is often produced by single-molecule fluorescence experiments. A test based on the SIC (BIC) statistic is used in conjunction with a progressive step-placement scheme to locate changepoints (steps) in noisy data. Its performance is compared to other step detection algorithms in use by biophysicists by repeating tests performed in a recent review.Experimental protocols and computer codes used in these investigations are presentedin detail in the appendices.

Single atoms are promising candidates for physically implementing quantum bits, the fundamental unit of quantum information. We have built an apparatus for cooling, trapping and imaging single rubidium atoms in microscopic optical tweezers. The traps are formed from a tightly focused off-resonant laser beam, which traps atoms using the optical dipole force. The traps have a diameter of ~1 &mu;m and a depth of ~1 mK. The novelty of our approach is the use a digital mirror device (DMD) to generate multiple independently movable tweezers from a single laser beam. The DMD consists of an array of micro-mirrors that can be switched on and off, thus acting as a binary amplitude modulator. We use the DMD to imprint a computer-generated hologram on the laser beam, which is converted in to the desired arrangement of traps in the focal plane of a lens. We have developed fast algorithms for calculating binary holograms suitable for the DMD. In addition, we use this method to measure and correct for errors in the phase of the wavefront caused by optical aberrations, which is necessary for producing diffraction-limited focal spots. Using this apparatus, we have trapped arrays of up to 20 atoms with arbitrary geometrical arrangements. We exploit light-assisted collisions between atoms to ensure there is at most one atom per trapping site. We measure the temperature of the atoms in the traps to be 12 &mu;K, and their lifetime to be 1.4 s. Finally, we demonstrate the ability to select individual atoms from an array and transport them over a distance of 14μm with laser cooling, and 5 &mu;m without.

In the atmosphere, cloud droplets can remain in a supercooled liquid phase at temperatures as low as -40 °C. Above this temperature, cloud droplets freeze via heterogeneous ice nucleation whereby a rare and poorly understood subset of atmospheric particles catalyze the ice phase transition. As the phase state of clouds is critical in determining their radiative properties and lifetime, deficiencies in our understanding of heterogeneous ice nucleation poses a large uncertainty on our efforts to predict human induced global climate change. Experimental challenges in properly simulating particle-induced freezing processes under atmospherically relevant conditions have largely contributed to the absence of a well-established model and parameterizations that accurately predict heterogeneous ice nucleation. Conversely, the sparsity of reliable measurement techniques available struggle to be interpreted by a single consistent theoretical or empirical framework, which results in layers of uncertainty when attempting to extrapolate useful information regarding ice nucleation for use in atmospheric cloud models. In this dissertation a new framework for describing heterogeneous ice nucleation is developed. Starting from classical nucleation theory, the surface of an ice nucleating particle is treated as a continuum of heterogeneous ice nucleating activity and a particle specific distribution of this activity g is derived. It is hypothesized that an individual particle species exhibits a critical surface area. Above this critical area the ice nucleating activity of a particle species can be described by one g distribution, 𝑔, while below it 𝑔 expresses itself expresses externally resulting in particle to particle variability in ice nucleating activity. The framework is supported by cold plate droplet freezing measurements for dust and biological particles in which the total surface area of particle material available is varied. Freezing spectra above a certain surface area are shown to be successfully fitted with 𝑔 while a process of random sampling from 𝑔 can predict the freezing behavior below the identified critical surface area threshold. The framework is then extended to account for droplets composed of multiple particle species and successfully applied to predict the freezing spectra of a mixed proxy for an atmospheric dust-biological particle system. The contact freezing mode of ice nucleation, whereby a particle induces freezing upon collision with a droplet, is thought to be more efficient than particle initiated immersion freezing from within the droplet bulk. However, it has been a decades’ long challenge to accurately measure this ice nucleation mode, since it necessitates reliably measuring the rate at which particles hit a droplet surface combined with direct determination of freezing onset. In an effort to remedy this longstanding deficiency a temperature controlled chilled aerosol optical tweezers capable of stably isolating water droplets in air at subzero temperatures has been designed and implemented. The new temperature controlled system retains the powerful capabilities of traditional aerosol optical tweezers: retrieval of a cavity enhanced Raman spectrum which could be used to accurately determine the size and refractive index of a trapped droplet. With these capabilities, it is estimated that the design can achieve ice supersaturation conditions at the droplet surface. It was also found that a KCl aqueous droplet simultaneously cooling and evaporating exhibited a significantly higher measured refractive index at its surface than when it was held at a steady state temperature. This implies the potential of a “salting out” process. Sensitivity of the cavity enhanced Raman spectrum as well as the visual image of a trapped droplet to dust particle collisions is shown, an important step in measuring collision frequencies of dust particles with a trapped droplet. These results may pave the way for future experiments of the exceptionally poorly understood contact freezing mode of ice nucleation.

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.

There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.

Les techniques de micromanipulation de molécules d’ADN uniques offrent des perspectives nouvelles pour lire et exploiter l’information contenue dans les génomes. Cela inclut le séquençage, la cartographie, le dénombrement de molécules et l'identification de modifications chimiques de l'ADN. Dans ce contexte, l'Équipe ABCDLab de l'ENS a développé une méthode utilisant l’ouverture et la fermeture mécanique répétée d’une molécule d’ADN en épingle à cheveux par pince magnétique. Cet outil permet de déterminer la position d'hybridation de petits oligonucléotides ainsi que celle d'anticorps révélant la position de marques épigénétiques. Un avantage de cette approche est de pouvoir travailler sur la même molécule pour d’une part identifier les marques épigénétiques et d'autre part réaliser une cartographie de sa position dans le génome. Mon travail de thèse consiste à développer un ensemble de méthodes bio-informatique visant à réaliser cette étape de cartographie. Le signal expérimental consiste en lecture des positions d’hybridation d’un, ou de plusieurs petits oligonucléotides sur la molécule étudiée. Cette mesure permet de construire une signature spécifique de la molécule que l’on peut rechercher dans le génome d’origine. Dans ce travail de thèse, j'ai réalisé des expériences avec sur pinces magnétiques pour acquérir des signatures moléculaires sur des molécules sélectionnées en aveugle dans E. coli. J'ai développé un logiciel capable de faire la recherche de ces signatures dans un génome et ensuite effectué l’ensemble du traitement des données pour tester le logiciel. Après plusieurs étapes d’optimisation, j’ai pu retrouver la position génomique des molécules étudiées, établissant ainsi une preuve de concept de cette stratégie de cartographie. Le travail a concerné l'ensemble de la chaîne de mesure : (1) le choix des sondes utilisées pour constituer la signature d’une molécule observée en optimisant un ensemble de critères liés aux conditions expérimentales et à la combinatoire des motifs de séquence. (2) la mise au point d’algorithmes de cartographie adaptés aux caractéristiques expérimentales des mesures. Enfin, j'ai testé ces algorithmes, à la fois sur des données simulées in silico et in vitro sur de l'ADN d'origine bactérienne. Je discuterais en quoi les performances des solutions de cartographie développées ici sont influencées par, d’une part les limites du montage expérimental actuel, et d’autre part les limites des approches bioinformatiques. Je présenterais les voies d’amélioration possibles de ces dernières. Mes travaux établissent qu'identifier des molécules d’ADN uniques par pinces magnétiques est possible dans le contexte d’application épigénétique et en génomique. / Single molecule micromanipulations technic offer new perspectives to read and unravel genome information. This includes sequencing, mapping, molecule counting and identification of DNA modifications. In this respect, ABCDLab team has developed a cutting edge method using repeated mechanical opening and closing of a DNA molecule with a hairpin shape using magnetic tweezers. This tool allows measuring along the DNA molecule the hybridization positions of oligonucleotides a few bases long and also to locate specific antibodies transiently bound to epigenetic markers. With this approach we can identify with the single molecule level epigenetics markers and localized them on the genome. My PhD work consisted of developing a set of bioinformatics methods to perform DNA mapping using magnetics tweezer signal consisting of hybridization positions along the studied molecule. This measurement may be viewed as a fingerprint of the molecule which can be searched on the reference genome. During my thesis, I have realized an experimental test using magnetic tweezers to acquire a set fingerprint data on a set of blinded selected molecules in the E. coli genome. I have developed a software performing a rapid search of these fingerprints inside the genome. Then I have performed the whole data treatment to check the software on the selected molecules. After several rounds of optimization, I have recovered the genetic position of the studied molecules, establishing a proof of concept of this cartography strategy. The work has addressed the whole measuring chain; (1) by choosing the oligonucleotides best adapted to obtain the molecular signature by optimizing the set of experimental constrains and combinatorial motifs of the sequence. (2) by tuning the cartography algorithm to adapt to the experimental measurement constrains. Finally, I have tested these algorithms, both on simulated data in silico and on experimental fingerprint in vitro. I shall discuss how the performances of these cartography solutions that have been developed here are impacted by the experimental limitations of the present technique, and by the bioinformatics limits. I shall present possible improvements to these methods. My studies constitute a proof of concept for genomic and epigenetic applications.

The process of a single cell evolving into a complex organism results from a series of coordinated movements of cells and tissues, especially during early embryo development. Although a wealth of morphological data characterises the shapes and movements of cells in embryos, how these movements are driven, patterned and controlled, and how this patterning is related to the mechanical properties of tissues remains unknown. Four-pole electromagnetic tweezers have been developed to probe the mechanical properties of living embryonic tissues that are undergoing active morphogenetic development. The device is capable of generating magnetic forces in the order of nano-Newtons on a grafted magnetic bead. The local passive mechanical properties of the tissues can be characterised by measuring the three-dimensional bead movement and analysing cell shape changes and cell rearrangement in response to this externally applied force. The magnetic device is used to probe the rheology in early zebrafish embryos between high stage (3.3 hpf) and the onset of gastrulation (5.3 hpf) when rapid cell cycles give way to a hollow sphere of cells. The tissue response to the applied force is modelled as linear visco- elastic. The embryo becomes stiffer and more viscous during this period of development, showing that a loose collection of cells becomes cohesive tissues. A computational model is used to explore how cells respond to local or global mechanical perturbations in two systems. First, the model simulates the movement of the bead within an embryo, and the results illustrate the generation, patterning and relaxation of the local cell stress around the bead. Second, the model reproduces the autonomous changes in mitotic cells within a stretched monolayer, and the results show that propensity of cells to divide along their long axis facilitates stress relaxation and contributes to tissue homoeostasis.

The need for non-contact micromanipulation methods is apparent for a number of different applications. Optical tweezers, a technique which uses highly focused laser beams to trap and move microscopic objects, has become an important tool for many applications owing to its incredible precision and dexterity. Optical trapping is, however, limited in several ways. It often struggles with particles larger than 10 micrometers, agglomerates and large numbers of particles. Complimentary technologies such as acoustic trapping, aim to overcome some of these limitations. This technique, also termed as Sonotweezers, uses ultrasonic fields to manipulate particles and can manipulate large particles with ease and manipulate large numbers of polydisperse particles and agglomerations, although they currently lack the dexterity of optical tweezers. Combining these two trapping modalities overcomes the some of the limitations of both of them and opens up a new range of useful applications. Three main types of hybrid optical and acoustic traps have been devised and are presented here. The first is an acoustic Bessel beam trap which is used to arrange a large number of polydisperse particles into concentric rings whereupon the smaller particles can then be further manipulated using a single beam optical tweezer. A rudimentary optical sorting system, which pushes particles in a flow laterally using an optical trap, has been combined with an acoustic levitator, which moves all particles away from the edges of the microfluidic channel reducing on sticking and other negative effects. A novel optically transparent ultrasonic device has been developed for easier integration into optical traps without the need for modication. This transparent trap has also been used in combination with a multibeam interference optical sorter to improve the separation between 5 and 10 micron particles.