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This thesis presents a discussion of a novel combination of state-of-the-art experimental techniques into a single microscope setup and the studies this has enabled. Optical tweezers, nanopipettes, and fluorescence were all combined to study the effect of salt species on nanoscale voltage driven flows, the mechanical behaviour of cell membranes, and the electrical activity from various cells. Voltage driven, or electroosmotic, flow was quantified by measuring the piconewton forces of the flow field outside nanopipettes in diff erent salt conditions using optical tweezers. Changing salt conditions revealed new flow behaviour, with flow reversing in CsCl compared to other salts. The role of substrate sti ness in cell membrane tension was investigated using optical tweezers. Optical tweezers were used to trap colloidal particles and then adhere them to the membranes of xenopus laevis retinal ganglion cell axons and NIH 3T3 fibroblasts to pull lipid tethers. Pulling these tethers allowed the e ffective cell membrane tension of these samples to be investigated. These tethers were pulled from cells plated on glass and hydrogels with di fferent elastic moduli. The force response was the same across hydrogels but di fferent on glass, for both neurons and fibroblasts. Fluorescence microscopy was used in-situ to confirm the presence of lipid tethers. Work towards a nanopipette electrophysiology platform was performed, with successful recordings of spontaneous activity taken from rat and mice neuron and astrocyte cultures for longer than 1 hour achieved. Recordings were attempted in brain tissue slices and also performed simultaneously with micro-electrode array recordings for comparison with network activity. Electrophysiological recordings from unmodified escherichia coli bacteria are also demonstrated. The outcome of this work is the advancement of a combined approach using tools from nanoscience to advance the toolset of biophysics. Biophysics concerns itself with complex phenomena, and our findings and demonstrations of method advancement have implications and applications in the understanding of nanoscale transport, cell mechanics, and electrophysiology.

Conventional bulk rheology presents a well-established technique for the mechanical characterisation of soft materials, ranging from liquid suspensions and emulsions to tenuous or even stiff gels. Probing the internal microstructure and understanding the relaxation of such materials has always been of great interest to industries, where the flow behaviour and stress response are of paramount importance in quality control for achieving high product performance. However, although generally reliable, bulk rheology suffers from certain limitations related to the frequency range and measurement sensitivity, which do not allow probing fast processes occurring on the microscale in complex viscoelastic fluids. This has sparked the search for an alternative method of extracting the high-frequency response and has led to a significant rise in the use of microrheology, where tracer particles provide indirect information about the viscoelasticity of the fluid, in which they are embedded. Recent advances in the fields of optics and electronics paved the way for using laser trapping and light scattering beyond their standard applications. In this thesis, I combine mechanical and optical measurements applied on newly emerging materials in order to extract their viscoelasticity over a broad frequency range in an attempt to gain better understanding of their internal self-organisation. In particular, I concentrate on DNA-based and clay-based hydrogels, which exhibit strong degree of swelling when dispersed in an aqueous solvent. These soft transient networks represent intriguing thermoresponsive and time-dependent systems, where the flexibility in the design opens up new exciting avenues within the field of nanotechnology.

Optical tweezers represent a powerful tool for studying forces in biological samples. In this thesis, I explored applications of optical tweezers that pushed the technique beyond its limitations. In fact I worked on extending the range of forces applicable by the tweezers and on applying optical tweezers for the study of cell-cell junctions in tissues. Typical values for optical tweezers forces range between tenths to hundreds of pN, with only few examples of tweezers reaching nN forces. I synthesised photonically structured probes and obtained tweezers capable of nN forces. Furthermore, I optimised the probes to make them more suitable for biological experiments, I investigated alternative synthesis methods and conducted proof-of-concept studies for their application in cellular biology. Regarding the application of tweezers in tissues, I demonstrated their use for the study of tension in developing chick embryos. At about 6h from the egg's deposition, the chick embryo initiates gastrulation: the embryo rearranges from a single layered structure into a multi-layered one. The process is regulated by the large-scale highly coordinated flows of the cells in the epiblast. There are evidences that contraction and preferential cells intercalation in the posterior area of the embryo drive the process. The contraction in the posterior area seems to be correlated with the presence of myosin II cables. I optically manipulated cell-cell junctions in chick embryos while recording their deformation. I measured the difference response of junctions studied under different conditions. The results support the idea that junctions in the posterior area of embryos starting gastrulation (5h old) are under stronger tensions than junctions in younger embryo and junctions in the anterior area of the organism. Moreover, when embryos are treated with myosin I or myosin II inhibitors, tension in the junctions is reduced.

The rate of progress of Brownian processes is not easily quantifiable. An importantmeasure of the "speed" of Brownian motion is themean first-passage time (FPT) to a given distance. FPTs exist in various flavours including exit- and transition-path times, which, for instance, can be used to quantify the length of reaction paths in folding transitions inmolecules such as DNA. Due to their inherently stochastic nature, measurements of any FPTs require repeated experiments under controlled conditions. In my thesis, I systematically explore FPTs in various contexts using a custom-built automated holographic optical tweezers (HOT) setup. More precisely, I investigate transition- and exit-path-time symmetries in equilibrium systems and demonstrate the breakdown of the symmetry in out-of-equilibriumsystems. Experimental data from folding DNA-hairpins show that the principles established on the mesoscale extend well into the molecular regime. In Kramers escape problem, the reciprocal of the escape rate corresponds to the time of first-passage to leave the initial state. A lower bound for the achievable FPT, e.g. of the reaction coordinate of a folding molecule, therefore corresponds to a speed-limit of the ensemble reaction rate. Using my setup, I show that certain barrier shapes can substantially lower the escape time across the barrier without changing the overall energy balance. This result has deep implications for reaction kinetics, e.g. in protein folding. Furthermore, I investigate the role of entropic forces in Brownian transport, show that hydrodynamic drag plays a crucial role in Brownian motion in confined systems, and give an experimental realisation of Fick-Jacobs theory. The thermodynamic applications of HOTs considered here necessitate the creation of fine-tuned optical landscapes, which requires precise phase-retrieval to compute the necessary holograms. In order to address this problem, I explore novel algorithms based on deep conditional generative models and test whether such models can assist in finding holograms for a given desired light distribution. I compare several differentmodels, including conditional generative-adversarial networks and conditional variational autoencoders, which are trained on data sets sampled on the HOT setup. Furthermore, I propose a novel forward-loss-minimising architecture and demonstrate its excellent performance on both validation and artificially-created test data sets.

Tandem-repeat proteins differ from globular proteins, both in their biophysical characteristics and in how they interact with their respective partners, yet they comprise nearly one third of the human proteome and are central to many cellular processes and disease phenotypes. Repeat proteins have been shown to behave like nano-sized biological springs: they are flexible, dynamic and elastic. Using coarse-grained models, I discuss how intrinsic flexibility may arise in repeat proteins and how it could be crucial for the biological function of two systems: PR65, the scaffold protein of the protein phosphatase 2A, and Rap proteins, which are involved in quorum sensing. To interrogate α-solenoids at physiologically relevant forces, I performed force spectroscopy experiments using a dumbbell optical tweezers set up for which it is necessary to attach the relevant protein to DNA. As PR65 is not amenable to current DNA-protein attachment methods, I developed a protocol that allows the cross-linking of DNA oligos to proteins using bio-orthogonal chemistry. I then explored the mechanics of the natural repeat protein, PR65, and a series of designed TPR proteins. I find that these proteins respond to forces in a novel manner which is significantly different to what has been previously reported. TPRs unfold and refold in quasi-equilibrium at constant force without energy loss. In contrast, PR65 unfolds in separate domains and refolds along an entirely different pathway. In conclusion, my doctoral studies explore the physical characteristics of repeat proteins in more detail. Using both experimental and computational techniques, I provide unique perspectives on different aspects of their mechanical and dynamic capabilities. This work provides the basis for future investigations of how such interesting mechanical behaviour relates to biological function. Are repeat proteins simply a molecular recognition platforms for their multitude of binding partners, or do their mechanics matter in a biological context?

Dinoflagellates hold vast diversities and are major contributors to overall marine primary production. Close relatives to the apicomplexan parasites and ciliates, the dinoflagellates, however, have a shockingly different nuclear biology. The dinoflagellates have massively inflated genomes (2-245 Gbps, up to 80X that of humans), permanently condensed liquid crystalline chromosomes throughout the life cycle, minimal histone proteins expression, and loss of nucleosome-mediated chromosome organisation. These changes co-occur with adoption of a novel nuclear protein termed DVNP (Dinoflagellate/Viral NucleoProtein). DVNP is highly positively charged, nucleus-located, and is found in only dinoflagellates and numerous marine large DNA viruses. I will address two issues in this thesis, 1) what are the properties of the protein DVNP, and can we infer if and how DVNP manages the permanently condensed chromosome state; and 2) how do other proteins accommodate this drastically different organisation of genetic material, more specifically how does transcription work in a cell with permanently condensed chromosomes? To elucidate the location of transcription, specific probe were designed and synthesised to locate RNA polymerase and newly synthesised nascent RNA in relation to the compacted chromosomes, and result showed that both are distributed at the periphery but not the inside of the chromosomes. On the other end, DVNPs are expressed and purified to perform biophysical and biochemical characterisation. In addition, using single-molecule optical tweezers microscopy, DVNP was observed to compact and change the mechanical properties of DNA. Most interestingly, the DVNP/DNA aggregates showed a propensity to travel along the DNA strand en masse. I then endeavoured to solve the NMR structure of DVNP. The results suggest that DVNP plays a central role in chromatin packaging in dinoflagellates. Finally, to marry the results obtained from both experiments, at the end of the thesis I propose a model of a novel nucleosome-independent chromatin management in dinoflagellates and how its transcription could proceed.

The mammalian hippo signalling pathway controls cell proliferation and apoptosis via transcriptional co-activators YAP and TAZ, and as such is a key regulator of organ and tissue growth. Multiple cellular components converge in this pathway, including the actin cytoskeleton, which is required for YAP/TAZ activity. The precise mechanism by which the mechanical actomyosin network regulates Hippo signalling, however, is unknown. Optical methods provide a non-invasive way to image and study the biomechanics of cells. In the past two decades, super-resolution fluorescence microscopy techniques that break the diffraction limit of light have come to the fore, enabling visualisation of intracellular detail at the nanoscale level. Optical trapping, on the other hand, allows precise control of micron-sized objects such as cells. Here, super resolution structured illumination microscopy (SIM) and elastic resonator interference stress microscopy (ERISM) were used to investigate a potential role for the FERM-domain protein FRMD6, or Willin, in the mechanical control of the Hippo pathway in a neuronal cell model. A double optical trap was also integrated with the Nikon-SIM with the aim of cell stretching. Willin expression was shown to modify the morphology, neuronal differentiation, actin cytoskeleton and forces of SH-SY5Y cells. Optical trapping from above the SIM objective, however, was demonstrated to be ineffective for manipulation of adherent cells. The results presented here indicate a function for Willin in the assembly of actin stress fibres that may be the result of an interaction with the Hippo pathway regulator AMOT. Further investigation, for example by direct cell stretching, is required to elucidate the exact role of Willin in the mechanical control of YAP/TAZ.

Atmospheric aerosols affect global temperatures by scattering or absorbing solar radiation. Aerosols are often coated in a film of organic or inorganic material. To correctly understand atmospheric aerosols and their influence on climate, it is paramount that atmospheric aerosols are correctly understood. In the thesis, optical trapping techniques were applied alongside neutron and x-ray reflectivity methods to study atmospheric aerosol and atmospheric aerosol thin films. Optical trapping applied simultaneously with Mie spectroscopy determined the refractive index of an aerosol. Optical trapping was applied to extracts sourced from atmospheric aerosol collected at urban, remote and wood burning sources: the refractive index of the extracts ranged from 1.450 to 1.588 at 589 nm. Optical trapping techniques were also successfully applied to changing systems: the refractive index of squalene upon exposure to ozone or oxygen changed by 0.0169±0.0026 or 0.0173±0.0026 respectively. All three experimental techniques were applied to study thin films. Optical trapping demonstrated the consequence of film formation upon aerosol light scattering. When a 0.190 µm thick film of sulfuric acid formed on a silica particle with a diameter of 1.930 µm, the refractive index resembled sulfuric acid. Application of neutron and x-ray experiments extended thin film studies by allowing the oxidation of thin films to be followed. The scattering length density and film thickness of aerosol extract films composed of urban, remote and wood burning aerosol extracts at the air water interface were determined. Subsequently, continuous collection of reflectivity data during exposure to gas-phase OH radicals allowed the oxidation kinetics to be studied and film lifetime to be estimated. Film lifetime was on the timescale of minutes for all samples. Additionally, neutron reflectivity was applied to determine the oxidation kinetics of an aerosol proxy film at the silica-water interface. The film demonstrated degradation decay upon exposure to aqueousphase OH radicals.

There are a growing variety of research fields requiring non-contact micro- manipulation. An increasing number of these fields are turning to optical tweezers as a solution, owing to their high spatial and temporal resolution. Optical tweezers have the ability to quantitively exert and measure forces on the piconewton scale, a convenient force scale for soft biological materials, and are hugely versatile due to the wide assortment of beam shaping techniques that can be employed. The work in this thesis can be broadly divided into two main themes: that quantifying the optical trapping forces in shaped beams; and bringing control and simplification of complex systems to non-expert users who may utilise optical tweezers as part of interdisciplinary collaborations. Static beam shaping is used to generate a conically refracted optical trap and the trapping properties are characterised. It is shown that trapping in the lower Raman spot gives full, 3D gradient trapping, while the upper Raman spot allows for particle guiding due to its levitation properties. Particles in the Lloyd/Poggendorff rings experience a lower trap stiffness than particles in the lower Raman spot but benefit from rotational control. Dynamic beam shaping techniques are exploited for the simplification of complex systems through the development and testing of the HoloHands program. This software allows a holographic optical tweezers experiment to be controlled by gestures that are detected by a Microsoft Kinect. Multiple particle manipulation is demonstrated, as well as a calibration of the tweezers system. Application of trapping forces is demonstrated through an examination of integrin – ligand bond strength. Both wild type effector T cells and those with a kindlin-3 binding site mutation similar to that found in neutrophils from Leukocyte Adhesion Deficiency sufferers are investigated. Through the use of back focal plane interferometry, a bond rupture force of (17.9 ± 0.6) pN at a force loading rate of (30 ± 4) pN/s, was measured for single integrins expressed on wild type cells. As expected, a significant drop in rupture force of bonds was found for mutated cells, with a measured rupture force of (10.1 ± 0.9) pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects bond strength of single integrin-ligand bonds. An experimental system for studying these cells under more physiologically relevant conditions is also presented. Additionally, a low-cost optical micromanipulation system that makes use of simple microfabricated components coupled to a smartphone camera for imaging is proposed and demonstrated. Through the layering of hanging droplets of polydimethylsiloxane (PDMS) on microscope coverslips, lenses capable of optical trapping are created. Combination of PDMS with Sudan II dye led to the fabrication of long pass filters. An extension of this low-cost system into the life sciences is proposed through the adaptive use of bubble wrap, which allows for the culturing of cells in a chamber compatible with optical trapping.

Optical tweezers have allowed us to harness the momentum of light to trap, move, and manipulate microscopic particles with Angstrom-level precision. Position and force feedback systems grant us the ability to feel the microscopic world. As a tool, optical tweezers have allowed us to study a variety of biological systems, from the mechanical properties of red blood cells to the quantised motion of motor-molecules such as kinesin. They have been applied, with similar impact, to the manipulation of gases, atoms, and Bose-Einstein condensates. There are, however, limits to their applicability. Historically speaking, optical tweezers have only been used to trap relatively simple structures such as spheres or cylinders. This thesis is concerned with the development of a fabricational and optical manipulation protocol that allows holographical optical tweezers to trap photonic membranes. Photonic membranes are thin, flexible membranes, that are capable of supporting nanoplasmonic features. These features can be patterned to function as metamaterials, granting the photonic membrane the ability to function as almost any optical device. It is highly desirable to take advantage of these tools in a microfluidic environment, however, their extreme aspect ratios mean that they are not traditionally compatible with the primary technology of microfluidic manipulation: optical tweezers. In line with recent developments in optical manipulation, an holistic approach to optical trapping is used to overcome these limitations. Full six-degree-of-freedom control over a photonic membrane is demonstrated through the use of holographical optical tweezers. Furthermore, a photonic membrane (PM)-based surface-enhanced Raman spectroscopy sensor is presented which is capable of detecting rhodamine dye from a topologically undulating sample. This work moves towards marrying these technologies such that photonic membranes, designed for bespoke applications, can be readily deployed into a microfluidic environment. Extending the range of tools available in the microfluidic setting helps pave the way toward the next set of advances in the field of optical manipulation.

Aerosols and their indirect contribution to the Earth's climate remains a poorly understood field. The indirect effect of aerosols on the climate refers to aerosol ability to form cloud condensation nuclei (CCN) and affect cloud properties. Naturally forming, thin organic films exist on the surface of atmospheric aerosols and have the ability to influence aerosol behaviour. Given the oxidative nature of the atmosphere, thin organic films on the surface of aerosols are readily oxidised which will impact on CCN formation and the properties of cloud droplets, affecting the Earth's climate indirectly. The purpose of this thesis was to investigate the oxidation of atmospherically relevant thin films at the air water interface using the complementary techniques of optical trapping and neutron and X-ray reflectometry, to determine whether such reactions are important in the atmosphere. An optical trapping technique was developed to coat individual solid silica beads with a thin film of oleic acid and characterise both the size and wavelength dependent refractive index of the core silica aerosol and the oleic acid coating using white light Mie scattering. The size of the core aerosol and coating thickness were determined to a precision of ± 0.5 nm and 1 nm respectively. The oxidation of monolayer organic films of atmospheric proxies at the air-water interface was studied using neutron reflectometry and the common atmospheric oxidants, O3, •OH, NO3•, SO4•−. These studies provided insight into the oxidation mechanism of the film as well as film persistence and thickness. Furthermore, the oxidation of monolayer films was also found to be independent of the viscosity and ionic strength of the solution that the film was present on. X-ray reflectivity studies were carried out on the oxidation of monolayer organic films extracted from real atmospheric aerosol and sea-water samples at the air-water interface. The films were found to be inert to gaseous ozone indicating a lack of unsaturated material in atmospheric samples and that oxidation by gaseous ozone may not be important in the atmosphere.

Nanopores are apertures of nanometric dimensions in an insulating matrix. They are routinely used to sense and measure properties of single molecules such as DNA. This sensing technique relies on the process of translocation, whereby a molecule in aqueous solution moves through the pore under an applied electric field. The presence of the molecule modulates the ionic current through the pore, from which information can be obtained regarding the molecule's properties. Whereas the electrical properties of the nanopore are relatively well known, much less work has been done regarding their fluidic properties. In this thesis I investigate the effects of fluid flow within the nanopore system. In particular, the charged nature of the DNA and pore walls results in electrically-driven flows called electroosmosis. Using a setup which combines the nanopore with an optical trap to measure forces with piconewton sensitivity, we elucidate the electroosmotic coupling between multiple DNA molecules inside the confined environment of the pore. Outside the pore, these flows produce a nanofluidic jet, since the pore behaves like a small electroosmotic pump. We show that this jet is well-described by the low Reynolds number limit of the classical Landau-Squire solution of the Navier-Stokes equations. The properties of this jet vary in a complex way with changing conditions: the jet reverses direction as a function of salt concentration, and exhibits asymmetry with respect to voltage reversal. Using a combination of simulations and analytic modelling, we are able to account for all of these effects. The result of this work is a more complete understanding of the fluidic properties of the nanopore. These effects govern the translocation process, and thus have consequences for better control of single molecule sensing. Additionally, the phenomena we have uncovered could potentially be harnessed in novel microfluidic applications, whose technological implications range from lab-on-a-chip devices to personalised medicine.

To respond to rapidly-changing stresses in their environment, bacterial cells must be able to sense a variety of chemical cues and respond to them by activating the relevant genes. The lysine riboswitch is a short RNA motif, located just upstream of a gene encoding a lysine biosynthesis protein, that suppresses the expression of that gene when sufficient lysine is present in the cell. It acts by binding a lysine monomer in a region called the aptamer, which in turn rearranges an adjacent domain called the expression platform, sequestering the ‘start' sequence of the gene and preventing it from being transcribed. In this thesis, the lysine riboswitch's ligand-binding transition is studied using single-molecule fluorescence microscopy, optical tweezers, and a hybrid optical force/fluorescence technique. Förster Resonance Energy Transfer (FRET) is used with a fluorescently-labeled aptamer to show that it has a previously-undescribed, partially-folded structural state with enhanced ligand affinity compared to the unfolded structure. The Mg²⁺ dependence of the transition between these states is shown to resolve existing debates in the literature about the sensitivity of the riboswitch. The kinetics of the folding transition are explored using FRET, optical force, and hybrid ‘Fleezers' to map the free energy landscape of ligand binding and show that the ligand itself promotes transitions into the aptamer's folded state, a so-called ‘induced fit' mechanism rare among riboswitches. Finally, high-resolution optical tweezers are used to explore the link between the aptamer's secondary structure (the sequence of paired nucleotides) and its tertiary structure (three-dimensional folding) to illuminate the role of ligand binding in gene regulation, which depends on the equilibrium between competing secondary structures. Hybrid biophysical techniques like optical force/fluorescence microscopy are shown to be indispensable for addressing all the states in the reaction pathways of complex biomolecules like riboswitches and for discriminating between multiple levels of structure formation and interaction with the environment. Not only do the results presented here shed light on the RNA folding problem, particularly the role of tertiary structure in determining the minimum-energy configuration of an RNA sequence, but they could have implications for biomedical research, as the lysine riboswitch has already been shown to be a potential target for next-generation antibiotics.

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.

The use of optical fibre as a mechanism for the delivery of the trapping laser beam to the sample chamber significantly reduces both the size and the build costs of “Optical Tweezers”. Furthermore, the use of fibre facilitates the decoupling of the optical trapping beam from the microscope optics, which provides further scope for the development of a portable optical trapping system, and the potential for uncomplicated integration with other advanced microscopy systems such as an atomic force microscope (AFM) for example. For use with an AFM, the optical fibre must be inserted at an angle of 10° with respect to the sample chamber floor. However, previous literature suggests that 3D optical trapping with a single fibre inserted at an angle ≤20° is not feasible. This thesis presents the design, development, build and test of a single beam optical fibre based gradient force optical tweezers system and its associated software. An investigation is conducted to ascertain why optical trapping, using single fibre systems, cannot be achieved at sub 20° insertion angles, the result of which formed the basis of a hypothesis that explains this limitation. This finding led to the development of tapered optical fibre tips that are cable of 3D optical trapping at an insertion angle of ≤10°. The optimised optical fibre tapers are presented and their ability to trap both organic and inanimate material in 3D at an insertion angle of 10° is demonstrated. The near-horizontal insertion angle introduced a maximum trapping range (MTR). The MTR of the tips is determined empirically, evaluated against simulated data, and found to be tuneable through taper optimisation. Optical trap characterisation has been undertaken in terms of the optical trapping forces acting on the trapping subjects. Finally, the fibre tapering devices ability to reproduce identical tapers, or not, using the same device parameters, was investigated and the results in terms of geometric profile and optical performance are presented.

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 μm 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 μK, 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 μm without.

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.

This thesis presents an investigation of the dynamic properties of wide range of interfacial systems, from colloidal particles in solution, through the realm of aerosols and onto studies of molecular adsorption at an interface. The primary experimental technique utilized is optical tweezers. An exploration of the history of the use of radiation pressure to manipulate matter is presented, followed by an introduction to how optical tweezers work. Some of the more advanced methods of tweezing are discussed, with an emphasis on the use of spatial light modulators (SLMs) to realise dynamic holographic optical tweezers (DHOTs), an example of which has been constructed within our laboratory using off-the-shelf optical components, and combined with a spectrometer to facilitate high resolution spectroscopic studies of microscopic systems. The spectroscopic analysis of microparticles is greatly enhanced by optical feedback generated when the wavelength of light utilized is an integer number of wavelengths around the circumference of the microsphere. Enhanced signal occurs at these wavelengths, termed whispering gallery modes (WGMs). The absolute position of these resonances depends strongly upon the shape, size and refractive index of the particle, and is predicted by Mie theory. A discussion of the concepts behind Mie theory, as well as how to use an experimental WGM spectrum to deduce the size and composition of a microparticle, is provided. This technique is then put to use in a detailed study on the properties of single aerosols, comprised of sodium chloride solution, and generated using a handheld medical nebulizer. Studies have been carried out on both evaporating and growing droplets trapped with a Gaussian beam; in the latter case, periods of size stability are observed, owing to resonant absorption of radiation at the trapping laser wavelength. The SLM can be used to change the trapping laser to a Laguerre-Gaussian (LG) mode, and an investigation of how this affects the dynamics of the droplet is presented. It is found that the use of LG modes with $ellgeq10$ produced Raman spectra with significantly more intense WGMs, and also suppressed droplet evaporation. Through observations made with fluorescent polystyrene microspheres, it is argued that the LG modes are more efficient at coupling into WGMs of the droplets. Leading on from these experiments on salt water droplets, experiments have been conducted using ionic liquids (ILs). These fluids have many fascinating properties and potential applications. The optical trapping of droplets comprised of aqueous solutions of the ionic liquid ethylammonium nitrate (EAN) and water has been demonstrated for the first time. These droplets are analysed spectroscopically by illuminating them with the output from a broadband LED; WGMs that are observed in the backscattered light are used to determine their size and composition. The response of the droplets to conditions of varying relative humidity has also been investigated. In order to characterise the relative humidity experienced by both the salt water and IL droplets, the concentration of water vapour within the trapping cells has been measured using diode laser absorption spectroscopy. The spatially modulated laser beam is then utilized in a different fashion; instead of optically tweezing a sample, a low numerical aperture objective lens is utilized to focus the laser onto the surface of a gold coated microscope slide. When a colloidal sample is placed on this surface, the thermal gradients cause the particles to form two dimensional crystals. The SLM is utilized to form multiple nucleation sites, and the dynamics of the crystals are directly observed in real time using video microscopy. It is found that grain rotation-induced grain coalescence (GRIGC) occurs, with the rotation of both crystals before coalescence. Control over the grain size is achieved by altering the separation of the laser spots, and shows that the time scale for grain boundary annealing in our system is in good agreement with theoretical expressions formulated for nanocrystal growth. Finally, as a complimentary technique to the microparticle spectroscopy previously discussed, a bulk interface is probed by using evanescent wave broadband cavity enhanced absorption spectroscopy (EW-BBCEAS) specifically to study the adsorption of cytochrome c (cyt c) to a fused silica surface. Visible radiation from a supercontinuum source is coupled into an optical cavity consisting of a pair of broadband high reflectivity mirrors, and a total internal reflection (TIR) event at the prism/water interface. Aqueous solutions of cyt c are placed onto the TIR footprint on the prism surface and the subsequent protein adsorption is probed by the resulting evanescent wave. The time integrated cavity output is directed into a spectrometer, where it is dispersed and analysed. The broadband nature of the source allows observation of a wide spectral range (ca 250 nm in the visible). The system is calibrated by measuring the absorption spectra of dyes of a known absorbance. Absorption spectra of cyt c are obtained for both S and P polarized radiation, allowing information about the orientation of the adsorbed protein to be extracted.

The behaviour of colloidal particles in two-dimensional (2D) systems is addressed in real space and time using magnetic fields, optical tweezers and optical video microscopy. First, the fluctuations of a grain boundary in a 2D colloidal crystal are analysed. A real space analogue of the capillary fluctuation method is derived and successfully employed to extract the key parameters that characterise the grain boundary. Good agreement is also found with a fluctuation-dissipation based method recently suggested in simulation. Following on from analysis of the interface fluctuations, the properties of the individual grain boundary particles are analysed to investigate the long standing hypothesis that suggests that grain boundary particle dynamics are similar to those in supercooled liquids. The grain boundary particle dynamics display cage breaking at long times, highly heterogeneous particle dynamics and the formation of cooperatively moving regions along the interface, all typical behaviour of a supercooled liquid. Next, the frustration induced by confining colloidal particles inside a pentagonal environment is investigated. The state of the system is adjusted via two separate control parameters: the inter-particle interaction potential and the number density. A gradual crystalline to confined liquid-like transition is observed as the repulsive inter-particle interaction potential is decreased. In contrast, re-entrant orientational ordering and dynamical effects result as the number density of the confined colloidal particles is increased. Finally, the dynamics of colloidal particles distributed amongst a random array of fixed obstacle particles is probed as a function of both the mobile particle and fixed obstacle particle number densities. Increasing the mobile and the obstacle particle number density drives the system towards a glass transition. The dynamics of the free particles are shown to behave in a similar way to the normal glass transition at low obstacle density and more analogous to a localisation glass transition at high obstacle density.

The material that is covered in this thesis concerns the calibration and application of a set of optical tweezers to be used for one- and two-point micro-rheology experiments on hard sphere colloidal suspensions. The colloidal suspensions that were used in this study were all quasi-monodisperse density- and refractive index-matched PMMA particles that had a radii, a = 0:90 ± 0:05μm or a = 0:86 ± 0:07 for one-point microrheology experiments and radii a = 0:90 ± 0:05μm or a = 0:133 ± 0:010μm for the two-point micro-rheology experiments. By collecting the forward scattered light from a single optically trapped particle the particle's displacements in time were used to determine passive microviscosity, η(Passive) μ , for colloidal suspension in the range of 0:10 < Ø < 0:57 and comparison with literature data has been made and agreement found. Actively dragging an optically trapped particle through suspensions with volume fractions of the same range has yielded the active microviscosities, η(Active) μ , for both high and low shear regimes, displaying shear thinning behaviour. Comparison to literature data has been made and agreement found as well. Collecting the forward scattered light from two optically trapped particles has been used to determine the cross-correlated motion of the two particles in bare solvent and in suspensions with volume fraction Ø = 0:02. The friction coefficients ξ1;1 and ξ1;2 were extracted from the cross-correlated motion of the particles and agreement was found with theoretical predictions for bare solvent only. The suspensions with volume fraction Ø = 0:02 were found to have a friction coefficient ξ1;1 that was greater than what theory predicted with the suspension with bath particles a = 0:90 ± 0:05μm had the greater magnitude. The magnitude ξ1;2 was found to decrease for the suspension with bath particles of radius a = 0:133 ± 0:010μm and to increase for the suspension with bath particles a = 0:90 ± 0:05μm.

The two main themes in this thesis are the implementation of interference methods with optically trapped particles for measurements of position and optical phase (optical interferometric metrology) and the optical manipulation of nanoparticles for studies in the assembly of nanostructures, nanoscale heating and nonlinear optics (nanophotonics). The first part of the thesis (chapter 1, 2) provides an introductory overview to optical trapping and describes the basic experimental instrument used in the thesis respectively. The second part of the thesis (chapters 3 to 5) investigates the use of optical interferometric patterns of the diffracting light fields from optically trapped microparticles for three types of measurements: calibrating particle positions in an optical trap, determining the stiffness of an optical trap and measuring the change in phase or coherence of a given light field. The third part of the thesis (chapters 6 to 8) studies the interactions between optical traps and nanoparticles in three separate experiments: the optical manipulation of dielectric enhanced semiconductor nanoparticles, heating of optically trapped gold nanoparticles and collective optical response from an ensemble of optically trapped dielectric nanoparticles.

Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

Over the last few decades, the use of light to control and manipulate microscopic particles has become widespread. These methods are enabling new areas of research to flourish across the physical and biological sciences. This thesis describes investigations into both optical trapping and the closely related field of optical sorting. It documents the development of a variety of new techniques. The thesis begins with a short review of optical trapping and existing methods for sorting mixtures of microscopic particles. The first half of this chapter highlights some of the reasons behind optical trapping's rapid growth in popularity. By reviewing an array of methods for sorting particles and discussing the relative merits of each, the case for optical sorting is established. The second chapter describes research into using a spatial light modulator to create three-dimensional optically trapped colloidal structures using the time-sharing technique. Limiting factors inherent in the technology are discussed in detail. The third chapter reviews a sophisticated particle-tracking software package that has proved to be a considerable success. It was developed explicitly with colloidal microscopy in mind and experimental plots produced by the software are used throughout the thesis. Experimental studies have been performed into the behaviour of microscopic particles moving under the influence of two classes of propagation-invariant beams: Mathieu beams and Bessel beams. The Bessel beam studies have been complimented by a theoretical model and have led ultimately to a new method for the static optical sorting of both solid particles and biological cells, with particular emphasis on human blood. The fifth and final chapter describes how re-configurable optical devices can be implemented to spatially separate different colloidal species. A new method for creating arbitrary optical landscapes using an acousto-optic modulator is reported. This new technique is then used to optically sort four particle species simultaneously - the first experimental demonstration of polydisperse optical fractionation. Additionally, experiments are reported that demonstrate controlled, static optical sorting using a spatial light modulator.

This thesis describes the experimental and theoretical work that investigated the transfer of orbital angular momentum from light to matter. This was achieved by combining two established areas of laser physics which were "optical tweezers" and Laguerre-Gaussian laser modes. The optical tweezers are essentially a tightly focussed laser beam from a high numerical aperture microscope objective lens, which traps particles in three dimensions just below the beam focus. By incorporating a Laguerre- Gaussian laser mode into the tweezers system, the trapping efficiency was doubled. These improved optical tweezers have been successfully demonstrated both theoretically and experimentally. In addition to the spin angular momentum which is associated with the polarisation state, the Laguerre-Gaussian laser modes also possess orbital angular momentum. The "optical spanners" utilised this property by transferring orbital angular momentum from the laser beam to the trapped particle, causing it to rotate whilst being held in the optical trap. This effect was theoretically modelled and experimentally observed. Using the optical spanners, the spin angular momentum of the laser was used to directly cancel the orbital angular momentum in the beam, which was observed as a cessation in rotation of the trapped particle. This demonstrated the mechanical equivalence of the spin and orbital components of angular momentum in a light beam, and gave experimental evidence for the well defined nature of the orbital angular momentum present in Laguerre-Gaussian laser modes.

This thesis presents experimental work on using cavity optomechanics techniques to control the motion of levitated nanoparticles in high vacuum. The motivation behind this work was to develop quantum mechanics experiments at room temperature with increasingly large masses. Research on levitated nanoparticles started with the development of optical tweezers by Arthur Ashkin in the 1970s. Ashkin himself highlighted the outstanding mechanical isolation of levitated nanoparticles in vacuum. Cavity optomechanics, on the other hand, concerns itself with the coupling of an optical resonator (a cavity), and mechanical motion. Among the applications of cavity optomechanics are the use light to measure and control mechanical motion. In the experiments presented here, we couple the motion of a nanoparticle to the field of a deeply resolved-sideband cavity (Ω ≈ 10 · κ) using optomechanical interaction, by either actively driving the cavity or using the coherent scattering configuration. In a first experiment, we show that laser phase noise heating poses an important technical obstacle in cooling the motion of levitated nanoparticles through optomechanical interaction if the cavity field is driven through one of the cavity mirrors. With the demonstration of the coherent scattering technique, and its more favorable performance to phase noise, we performed experiments using this technique and show optomechanical strong coupling of the motion of levitated nanoparticles. Finally, we discuss the use of narrow linewidth cavities for similar experiments and find that they hinder both cooling, by means of strong coupling, and detection, by means of optical filtering, of the mechanical motion.

DNA replication inevitably encounters topological impasses created from the double helical structure of double stranded DNA. Incomplete resolution of the supercoils generated during fork progression leads to intertwining, or catenation, of the two sister chromatids which in turn leads to deleterious downstream effects such as aneuploidy. While biochemical aspects of regulation of catenation prevention and topoisomerase II, the enzyme primarily responsible for resolving supercoils during replication, activity on naked DNA have been deeply explored, the role of mechanical properties of chromatin and topoisomerase II activity on eukaryotic chromatin in its highly compacted form has been largely overlooked. Here I present single-molecule studies that provide insight into the effect of chromatin compaction via formation of nucleosomes in eukaryotic DNA replication and topoisomerase activity. First is the characterization of torsional mechanics of eukaryotic chromatin and its consequences followed up by observation of topoisomerase II activity on nucleosomal DNA. Secondly, I suggest a methodology to dissect the kinetic details of topoisomerase II activity and use it to observe response of the enzyme in physiologically relevant magnesium concentrations.

Optically levitated nanoparticles exhibit a measurable response to extremely minute forces. Examples include fluctuations introduced by thermal baths, and the stochastic recoil imparted by scattered photons. This high force sensitivity, together with the ability to measure the position with a precision close to the Heisenberg limit, makes this technology an outstanding platform for studying statistical mechanics, and for testing macroscopic quantum mechanics. In the first half of this Thesis, we engineer the stochastic properties of the thermal bath surrounding the particle. In a first experiment, these engineered fluctuations mimic a propulsive force with randomly precessing orientation. We then trap the nanoparticle in a bistable potential, and study the impact of this propulsive force on the transition rate. In a second experiment, we introduce a time dependence in the strength of the fluctuations. This technique mimics sudden changes in the temperature of the surrounding gas, and we use it to demonstrate nontrivial equilibration paths in the potential energy of the position. In the second half of this Thesis, we turn our attention to the role played by quantum fluctuations in our system. In a first experiment, we perform quantum state tomography of a particular optical mode into which the particle scatters. We demonstrate squeezing of this mode below the vacuum level, a consequence of the optomechanical interaction between light and particle. In a second experiment, we implement sudden jumps in the power of the trapping laser. We use these sudden jumps to show preliminary results towards coherent expansion of the motional wavefunction. This expansion represents a step closer towards generating a macroscopic quantum state of the motion of the particle.

Levitated micro- and nanoparticles have taken the stage as rich platforms for developing force sensors, and to study the classical to quantum transition of macroscopic objects. Optimal particle position detection has lead to exceptional control over its translational motion. More recently, researchers have extended the platform of levitated particles by addressing the rotational degrees of freedom. Manipulating rotational motion ranges from rotating a nanoparticle at ultrahigh speeds to feedback cooling its rotation from room temperature down to a few Kelvin. Both extremes show great potential for studying fundamental physics or enabling the development of highly sensitive sensors. In this thesis, we describe the interaction of light with an optically levitated nanodumbbell, formed by two nanospheres. The orientation of nanodumbbell couples to the polarization of the light beam, allowing for optical detection and control, while also interacting with a thermal bath of gas molecules. We initially demonstrate that the translational motion destabilizes the rotational motion, which we negate by feedback cooling the center-of-mass motion. In a circularly polarized beam, the nanodumbbell becomes a free rotor and is driven to high rotation frequencies. We study the fluctuations of the rotation frequency and demonstrate that those fluctuations are dominated by the coupling to the thermal bath. Besides optically driving the nanodumbbell, we also study and manipulate the nanodumbbell whilst constricting its orientation, creating so-called libration motion. More specifically, we feedback cool a libration mode to record-low temperatures and show evidence of its heating rate being dominated by radiation torque shot noise. Finally, we use theoretical insights to improve the optical detection scheme for the particle orientation, leading to a remarkable increase in signal-to-noise ratio.

A typical channel within a micro-scale acoustofluidic (MSAF) device has a small cross-section where the height is usually less than 200 μm and the width less than 5 mm; the length can be up to several cm, however, often is not of big interest. Direct measurement of, e.g., the pressure produced by the acoustic excitation is impossible due to the smallness of the region of interest. Additionally, the acoustic driving frequencies are generally above 100kHz such that the time resolution of measurements must be even at least twice as high if one is also interested in the transient behaviour and build up of, e.g., the acoustic pressure field and not only the steady-state. At the moment, the most common and straightforward way to approxi- mate the acoustic pressure within the channel is to optically measure the velocity of several objects of known size and material properties and then calculate back which pressure would have led to this velocity. The va- lidity and correctness of the pressure approximation depends on several uncertainties. Besides the object dimensions and the material parameters of the object and the fluid, the biggest uncertainty is the validity of the underlying theory of the acoustic radiation force (ARF) that is used for the calculation. There exist many MSAF models for the calculation of the acoustic forces which differ mainly in the assumptions regarding the physical model for the fluid and the immersed object. Each theory has its parameter space where it is superior to the others because it includes, e.g., the effect of visco-elasticity of the fluid. Here, an optical trapping (OT) apparatus is utilized to investigate two phenomena where controversies exist in the MSAF community: 1) the transient build up of the ARF and the drag force from acoustic streaming (AS) for a continuous and pulsed acoustic excitation; 2) the quantification of the steady-state rotational velocity of a spherical particle driven by the acoustic viscous torque where the viscous boundary layer (VBL) thickness is comparable to the particle radius itself. So far, OTs have mainly been used as force sensors on single particles within MSAF devices. For the measurements of both phenomena we take advantage of the fine spatial and temporal resolution that the OT offers, as well as the OT property that single particle measurements are possible. In order to measure the build up of the ARF and AS, an acoustic exci- tation frequency and measurement location within the standing pressure wave was used where the two forces were orthogonal to each other. The orthogonality as well as the division into ARF and drag force from AS was measured and validated by force measurements with the OT throughout the fluid channel with differently sized particles. The results of a continuous excitation showed that the ARF starts to build up almost instantaneously after the acoustic excitation was switched on, whereas the AS takes significantly longer. Interestingly, the fast ARF build up was expected from theoretical considerations, but the slow AS build up was underestimated by a factor of about 4. The pulsed excita- tion experiments revealed that depending on the specific pulse parame- ters the build up of AS can be suppressed substantially while the ARF is not affected as much as AS. Therefore, smaller particles can still be mainly manipulated by the ARF because the relative importance of AS decreases for a pulsed excitation faster than for the ARF. Our measure- ments strengthen experimental findings for a pulsed excitation that could not yet be explained theoretically. For the steady-state rotational speed measurement, a high viscosity mix- ture of water with glycerol (7 to 3) was created such that the formed VBL around the particle was about the same as the particle radius. The phase difference between two acoustic excitation sources spatially orthogonal to each other led to a time-averaged acoustic streaming field in the VBL of the particle along its circumference. This streaming field creates a driving viscous torque that causes a rotation with the rotational velocity at which the driving viscous torque equals the counteracting viscous drag torque. A theoretical formula overestimates the steady-state rotational speed for the experimental parameters by more than one order of magnitude. This was expected because, up to now, there are no theories that are valid for the regime where the radius is the same size or smaller than the VBL. However, a numerical study investigated exactly this regime and proposed a calculation for the final rotational velocity including the effects of the VBL. The rotational velocities measured with the OT confirmed two points: 1) the expected invalidity of the simplified theory in the regime of high viscosity (VBL in the same order of magnitude as the particle dimension) and, hence, the necessity of its inclusion in the calculations; 2) the correctness of the numerical results.

After pioneering work on optically levitated particles in the 1970s by Ashkin, so called optical tweezers have not only become a popular tool in biology and medicine but also experienced a renaissance in vacuum trapping over the last ten years. While the broader field of optomechanics already gained popularity previously and investigated the classical-to-quantum transition in cryogenic systems at the beginning of the last decade, levitated particle optomechanics opened the door to room temperature quantum experiments. The versatile levitated particle system has been utilized in various applications, such as rotation experiments at world record speeds exceeding 6 GHz, highly sensitive force sensors or to study stochastic effects of thermodynamics. Most intriguing, however, was the transition into the quantum regime by ground-state cooling of the particle's center-of-mass motion in one dimension, setting the first milestone of genuine quantum experiments. Ten years after the first cooling attempt, a technique adopted from the atom and ion cooling community finally led to ground-state cooling of a levitated particle. This technique, called cooling by coherent scattering, is the central scheme of this thesis. In the first part of this thesis we describe the construction of a double vacuum chamber system to efficiently trap and transfer a levitated particle into an optical cavity. In contrast to previous systems, no particle transfer to a second tweezer is necessary, minimizing the risk of particle loss and enabling experiments within minutes after particle loading. In the second part, we present the working principle of our coherent scattering setup. It was the first pure optical trapping configuration to transition a levitated particle into high vacuum, while being stabilized exclusively through cavity cooling. At the time, we achieved record low cavity cooling center-of-mass energies reflected by an effective temperature of a few millikelvin and demonstrated genuine 3D cooling of the motional degrees of freedom. The central feat of the coherent scattering setup is the ability to cool the particle motion in a cavity field node, reducing the impact of laser phase noise compared to the dispersive regime. In the last part of the thesis, we find that the mechanical instability of the particle position limits the suppression of phase noise, leading to particle cooling to about 10 phonons.

The nitrogen-vacancy (NV) in diamond has proved, in recent years, to be an invaluable tool across physics and biology. This naturally occurring colour centre exhibits an incredible diversity of properties and uses. It can act as bright and photostable fluorescent marker, a solid state qubit, a single photon source, or, as is most relevant to this thesis, a nanoscale probe of many physical quantities. Nanoscale diamond crystals, known as nanodiamonds (NDs), are an ideal host for NV centre nanoscale sensors. The diminutive size ensures that the stand-off distance between the probe NV centre and the probed sample remains small. In order to best take advantage of their nanoscale size, techniques must be used to precisely control the position of the ND. A promising approach for precision manipulation of NDs is optical trapping. Optical tweezers use highly focused laser light to confine micro- and nanoscale particles in an aqueous medium, this makes them a particularly useful tool for biological applications. By trapping NV containing NDs using optical tweezers, the nanoscale probe can be manipulated in all three spatial dimensions, as well as controlling orientation. This thesis presents a suite of research on the combination of nanodiamonds with optical tweezers for highly versatile nanoscale sensing. Integrating these two technologies introduces new challenges, most notably the deleterious effect of the infrared trapping laser on common NV sensing protocols. As such, this work focuses on better understand ing the interaction between optical trapping and NV sensing, as well as the development of experimental methodologies which overcome any adverse effects.

Artificially engineered structures, namely metamaterials and their two-dimensional (2D) counterpart –metasurfaces, have been proven as promising platforms to realize unusual light-matter interactions. Such structures have enabled the efficient manipulation of electromagnetic waves in unprecedented ways that cannot be obtained using conventional materials and thus have triggered exciting applications such as hyperlensing, canalization of light, negative refraction, hyperbolic dispersion, cloaking, or the enhancement of the spontaneous emission rate of dipole emitters, amongst many others. Moreover, the discovery of graphene and other 2D materials, and their electrical tunability, have enabled the use of surface plasmon polaritons at terahertz and infrared frequencies in numerous nanophotonic applications.In this thesis, I propose novel nano-optical plasmonic tweezers based on hyperbolic and nonreciprocalmetasurfaces to efficiently trap and manipulate nanoparticles in the near field with superior performance compared to the state of the art. To this purpose, I develop a rigorous theoretical framework able to compute optical forces on dipolar Rayleigh nanoparticles located near the metasurfaces. The theoretical model is based on Lorentz force within the dipole approximation combined with the scattered dyadic Green’s function of the system. Analytical expressions show that the force strength is directly proportional to the fourth power of wavenumber of the supported surface plasmons. This tells that the strength can be dramatically enhanced by the proper choice of metasurfaces that support ultra-confined surface plasmons with larger wavenumber. One potential candidate to achieve such response is the use of hyperbolic metasurface that supports surface plasmons with wavenumbers up to ~200 times larger than the ones supported in free space. My theoretical and numerical results using full wave simulations show that the use of hyperbolic metasurfaces enables unusual enhancement of the force strength (up to 3 orders of magnitude) in comparison to the one obtained above conventional isotropic media. Importantly, such response enables stable lateral trapping and efficient manipulation of nanoparticles using low-power laser beam thus reducing the photodamage threat. However, these general optical tweezers are static in the sense that their response cannot be dynamically controlled.In this context, drift-biased nonreciprocal graphene has emerged as a promising platform to electrically tuneand manipulate the dispersion characteristic of the supported modes. I propose the use of this platform as a planar plasmonic hyperlens that provides ultra-subwavelength imaging with remarkable resolution over a broadband frequency range that cannot be obtained by other artificially engineered structure. In addition, drift-biased graphene can also readily be applied in the context of optical tweezers to provide novel responses: (i) particles can be manipulated unidirectionally independent to the direction of the incoming light, overcoming beam alignment challenges occurring in conventional optical tweezers; and (ii) the location of optical traps can efficiently be manipulated over a few microns range thanks to the electrically tunable response. In summary, I envisage that the proposed nano-optical hyperbolic and nonreciprocal plasmonic tweezers may open unprecedented venues for routing, trapping, and assembling nanoparticles and can effectively address some of the shortcomings of current techniques.

Molecular machines are small assemblies harnessed to perform mechanical work in cells. These motors convert the chemical energy of ATP to move cargoes, transcribe RNA, and pump protons, to name a few examples. The study of these nanomachines is at the border of physics and biology, interesting to both fields for its implications of energy transfer at these length scales and their importance to cellular function, respectively. Optical tweezers is a natural choice as the tool to investigate these machines, as the technique has very high spatio-temporal resolution, enough to resolve the quick, small steps that these motors can take, and the applied force can be used to investigate the energetics and force generation properties of the system.The DNA packaging motor of Bacillus subtilis phage φ29 is a model system for studying biological nanomachines. The role of this homopentameric ring motor is to package the DNA genome into capsids during viral replication. This ATPase is a member of the additional strand, conserved glutamate (ASCE) superfamily, meaning the study of this motor can reveal insights to the function of other members in the family. Benefits to studying φ29 include the simplicity of performing experiments, only requiring the motor-capsid complex, DNA, and ATP, its relatively large step size (0.85nm), and moderate speed (40nm/s) which makes observation of motor stepping well-suited for optical tweezers experiments. Single-molecule optical tweezers studies of this motor have revealed great detail about its mechanochemical cycle and packaging mechanism, including interesting symmetry breaking from this homomeric ring, in which one of the five subunits is special in that it does not perform a mechanical task, but a regulatory one. This motor operates under a dwell-burst cycle, where the motor, starting from an ADP-filled ring, first exchanges ADP for ATP in an ordered, sequential fashion (during the dwell). After all five have exchanged, the special subunit hydrolyzes its ATP, which then causes the other four subunits to sequentially hydrolyze their ATPs and translocate 0.85nm (2.5bp) of substrate each (during the burst), resulting in the packaging of 10bp. The delineation between the one special and four translocatory subunits shows a division of labor, where one subunit takes on a different role from the others, despite their identical protein sequences. The similarity between the amount of DNA packaged in one cycle (10bp) and the pitch of DNA (10.4bp) is not just a coincidence, previous studies suggest that the event that assigns the identity of the special subunit is that it contacts two adjacent phosphates every pitch, and that this identity is preserved cycle after cycle. Despite this knowledge, the actual motions of the motor subunits that translate into substrate translocation are unknown. One proposed translocation model for p29 involves a dehydration-induced B-form DNA to A-form DNA transition. This scrunchworm model explains the 0.85nm step size as the difference in contour length of 10bp B-form and 10bp A-form DNA. We can test if this model is correct by making the motor package dsRNA, a substrate that can only adopt an A-form structure. In addition, this substrate has a different pitch than DNA, so we can see if the burst size changes if the pitch of the substrate changes. We found that, when challenged with dsRNA, the motor is indeed able to package it, invalidating the scrunchworm model. Packaging of dsRNA and RNA:DNA hybrid combinations reveals that the motor conforms its burst size to the periodicity of its substrate. Hence, we conclude that the motor’s burst size is determined by the pitch of the substrate. However, we observe that the step size of the motor is still the 0.85nm it is on DNA, with one step shortened to accommodate the shorter total burst size, suggesting that the step size of the motor is innate and not determined by the substrate. Because the structure of the ATP-full motor was found to exist in an open lock-washer state by cryo-EM, we propose a model of translocation in which the ring is planar at the start of the dwell and successively opens up upon nucleotide exchange during the dwell. Thus, at the end of the dwell, and at the beginning of the burst, the motor adopts a lock-washer conformation. The open-ring structure is the mechanism by which the burst size of the motor adapts to the pitch of its substrate. During the burst, nucleotide hydrolysis successively closes the ring, translocating the substrate and reverting the motor to a planar conformation. The step size is determined by the motion of the “hinges” formed by the adjacent subunits in the spiral. This translocation mechanism is an interesting example of a motor adapting to the periodicity of its substrate, while taking advantage of its repeating chemical moieties by using them as rest points like the rungs of a ladder.

Tuberculosis (TB) is an airborne disease that kills 1.5 million people and affects ~ 9 million worldwide. Its etiological agent, Mycobacterium tuberculosis (Mtb), persists in a third of the general population. There is also an emergent problem of drug and multidrug resistance in Mtb that creates the need for novel drugs. In the treatment against TB, one target of antibiotics is the prokaryotic RNA polymerase (RNAP), an essential multitask enzyme that catalyzes RNA synthesis during transcription. Rifampicin, the first-line antibiotic anti-TB, binds and inhibits the MtbRNAP.Consequently, combatting TB requires a better understanding of the primary mechanism of transcription by MtbRNAP. In fact, during Mtb transcription, unique and essential factors might affect the MtbRNAP dynamics and regulate its activity. That regulation plays a significant role in the adaptation and pathogenesis of the parasite. Therefore, understanding how these factors and new antibiotics modulate together the MtbRNAP activity will contribute valid information to address TB. To this end, we use a single-molecule approach based on high-resolution optical tweezers to study transcription elongation by MtbRNAP in the context of its DNA elements, transcriptional factors, and antibiotics.

Understanding complex interactions between a cell and its extracellular matrix (ECM) lies at the core of mechanobiology. For instance, stiffness of the ECM was previously shown to be highly heterogenous and deregulated in wound healing or during cancer progression. However, tools available for measuring and dynamically altering peri-cellular stiffness have been lacking and bulk measurements do not probe stiffness sensed by the cells. Thus, we have developed an optical tweezers active microrheology (AMR) system capable of multi-axes stiffness measurements in the peri-cellular region. In this thesis, multi-axes AMR system was used to investigate highly heterogenous and anisotropic stiffness landscapes established by dermal fibroblasts, human breast cancer and fibrosarcoma cells. Peri-cellular stiffness and anisotropy are shown to vary between the tested cell lines and with different treatments modifying cell behavior. Further, stiffness landscape and cell response to ECM vary with hydrogel source, concentration and fiber architecture of the local ECM. These studies underscore the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. Additionally, the method of patterned crosslinking is used to alter ECM topography by inducing fiber alignment and peri-cellular stiffness landscape. Following localized crosslinking, human breast cancer cells undergo contact guidance and durotaxis, as indicated by cell migration in a direction of fiber alignment and along an off-axis stiffness gradient, respectively. While these phenomena are widely known and studied on 2D substrates or in 3D synthetic hydrogels, patterned crosslinking allows for better understanding of processes governing directed migration of individual cells embedded inside naturally-derived fibrous hydrogels. Collectively, this thesis work investigates how cells respond to and regulate their local ECM stiffness based on a variety of different factors. Local ECM stiffness landscape established by the cells and probed using multi-axes AMR differs with the addition of different treatments regulating cell behavior and it is also strongly dependent on biochemical, mechanical and topographical properties of the ECM.

How biological fibrous materials respond to forces and changes at the cellular scale is important for understanding what cells `feel' from their extracellular environment. The local mechanical property within a fibrin fiber network is dependent on the local structure of the network. Elastic force transmission through hydrogel depends on the arrangement of the fiber. A novel method for visualizing force transmission through this network is measuring the pixel fluctuation of individual fibers in the presence of an actively applied oscillation using embedded microbeads and optical tweezers. Optical tweezers can also be used for measuring local stiffness through active microrheology. After micropatterned crosslinking, fibrin hydrogels were shown to produce local stiffening directly and indirectly through strain hardening. This stiffening resulted in stiffness anisotropy and fiber alignment which MDA-231 cells were shown to respond to. Finally, a model of elastically connected nodes recapitulates some of the experimental observations such as fiber force transmission, strain hardening, and stiffness anisotropy when strained. Manipulating, measuring, and modelling of local stiffness changes in response to applied forces offers further insight into how cells sense their surroundings.

Abstract: Folding is the process by which biomolecules spontaneously self-assemble into specific, complex, three-dimensional structures from simple one-dimensional polypeptide chains. Folding is a critical process in biology as there exists a tight link between the structure and function of biomolecules, and misfolding into the wrong structure often leads to disease. Traditional studies of folding have used ensemble measurements to monitor how the statistics of the folded and unfolded states, respond to perturbations to the stability of the states. Ensemble studies have yielded important results and indeed, are the primary basis for our current understanding of folding reactions. However, due to the asynchronous nature of folding reactions, such studies are incapable of probing transition paths. Transition paths comprise those parts of a folding trajectory where the molecule passes through the high-energy transition states separating the folded and unfolded states. The transition states determine the folding kinetics and mechanism but are difficult to observe because of their brief duration. Single-molecule experiments have in recent years, begun to characterize transition paths in folding reactions, allowing the microscopic conformational dynamics that occur as a molecule traverses the energy barriers, to be probed directly. In this thesis, I present the first direct measurements of transition-path trajectories. I then show how, using single-molecule force spectroscopy, I have been able to make the first-ever measurements of several transition-path properties including: the local velocity along the paths, the path shapes, and the transition state dynamics inferred by them. I discuss how these measurements have been related to theories of folding as diffusion over an energy landscape, to deduce properties such as the diffusion coefficient, and how they further our understanding of folding. The richly detailed information available from transition path measurements holds great promise for an improved understanding of microscopic mechanisms in folding.

In this thesis, I outline the development, implementation and evaluation of new sensor-side techniques in Optical tweezers that improve current capabilities in hydrodynamics, colloid-colloid studies and tweezers-based field scanning, by focussing on the high-fidelity Brownian tracking possible with photodiode sensing. Utilising a Field Programmable Gate Array (FPGA) to perform ultra-fast sensor interrogation, pre-processing and trap control, I am able to achieve multi-colloidal confinement with arrangement capacity, spatial and temporal measurement exceeding present standards, while also enabling and exploring new post-processing methods that stand to substantially improve the performance of Optically Trapped Probe Microscopy (OTPM). This thesis begins with a review of the present state-of-the-art in optical tweezers-based tracking and control, and an outline of current and emerging statistical analyses of Brownian motion. It continues with an investigation into the effects of trap-beam aberrations on measurements of Brownian motion, necessary to show that individual positional measurements in an imperfect system still possess enough information/certainty to be used in future chapters. Following this, a rapid trap-multiplexing method of multi-trap control is introduced, characterised and evaluated, whereby the FPGA is utilised as a genericised trap positioning and sensing platform, allowing arbitrary, fast-moving configurations of optical tweezers and simultaneous high-fidelity measurement. Finally, the utility of this high-bandwidth sensing is further explored with the development of a novel technique to correct for Brownian-based broadening in optical tweezer-based scanning images. The correction technique is demonstrated experimentally and using a Brownian dynamics simulation, providing both proof of concept results and promising directions for further development.

DNA packaging is a major step in the process of assembly of many viruses, including many dsDNA bacteriophages (bacterial viruses). An ATP-powered molecular motor translocates a dsDNA molecule into the viral prohead shell where it is confined to near-crystalline density. Our studies concern efforts to understand how this multi-subunit nanoscale motor functions to transfer ATP chemical energy to mechanical work as needed to translocate DNA against resisting load forces. In this dissertation, optical tweezers, a method for real-time, high force, and small displacement measurements on single DNA molecules, was employed to conduct a series of experiments on bacteriophage T4, φ29 and Lambda DNA packaging mechanisms. Improved instrument calibration methods and novel data analysis methods were also introduced. We used measurements of DNA elasticity in the linear, high-force regime to determine trap compliance more-reliably, simultaneously with other calibration parameters. New methods for pause and slip detection, data segmentation, and motor velocity determination were also explored. A major focus was investigation into whether substrate DNA sequence affects motor function. Evidence against a ‘B-A Scrunchworm’ model was obtained by showing T4 translocation dynamics are insensitive to A-philic DNA sequences. Preliminary data was also obtained supporting the same result for the φ29 motor. For T4, which exhibits large motor velocity fluctuations and pausing/slipping, more general evidence against sequence-dependent motor function was obtained from analyses looking for correlations across many events recorded with the same or different sequences. Phage lambda packaging was measured with improved resolution and with translocation interrupted by a putative ADP release inhibitor (Na3VO4) previously found with φ29 to cause isolated bursts of translocation steps ~10 bp in size. For lambda, preliminary evidence was obtained suggesting a different quantized translocation size of ~5 bp and a distinct packaging-slip-pause behavior. Lastly, preliminary studies of the T4 motor found no significant slowing with a high concentration of added phosphate, suggesting that phosphate release following ATP hydrolysis is essentially irreversible, consistent with prior reported findings on φ29 packaging. Preliminary T4 measurements with Na3VO4 revealed significantly slowed packaging but quantized translocation steps could not be detected because all translocation events were interrupted by variable size slips.

About a decade ago, optically levitated nanoparticles have been proposed for macroscopic tests of quantum mechanics. For such tests, the thermal motion of the particle’s center of mass is required to be close to its ground state of energy. Ever since these proposals, research groups around the world try to achieve ground-state cooling of optically levitated glass particles. In this dissertation, we cool the center-of-mass motion of a nanoparticle in an optical trap. Based on the position measurement of the particle, we apply a damping force in proportion to the particle’s speed, which leads to a cooling effect. We find that the cooling performance of our cold damping scheme is limited by the measurement imprecision. We analyze our detection principle theoretically and find an ideal detection scheme whose imprecision is at the fundamental noise level dictated by quantum mechanics. Such a Heisenberg-limited detection would, in principle, allow for ground-state feedback cooling. With these insights applied to our experiment, we cool the motion of our particle to an average of four quanta. Moreover, we resolve an asymmetry between the Stokes and anti-Stokes scattered light from the particle. This quantum effect allows us to calibrate the system to the ground state energy. Our work advances the research field of levitated optomechanics toward quantum control and therefore toward macroscopic tests of quantum mechanics.

Motor proteins take part in the organization and division of eukaryotic cells by using their ability to move unidirectionally along the cytoskeletal tracks. While kinesin and myosin motor families have members that move towards either end of actin and microtubules, respectively, all dynein motors exclusively move towards the minus-end of microtubules. Previous studies reported that dynein asymmetrically responds to external forces, moving faster when pulled forward, while resisting backward movement under hindering forces. I hypothesized that this asymmetry enables dynein to harness energy from external force fluctuations for faster movement towards the minus-end.In my doctoral work, I have shown that dynein can harness energy from cytoskeletal fluctuations. Using optical trapping techniques, I have characterized how external forces affect the velocity of dynein motility in the presence and absence of ATP. The results demonstrated that dynein forms an asymmetric slip bond with the microtubule. Using an oscillatory optical trapping assay, I showed that dynein can rectify force fluctuations to move towards the microtubule minus-end in the absence of ATP. Dynein was capable of moving towards the minus-end, even when the net force is in the plus-end direction. In the presence of ATP, dynein was able to move faster, generate power from force fluctuations, and stand against higher resistive forces. I developed a mathematical model that connects the force-induced release rate of dynein monomers with the force-velocity relationship of dynein dimers to describe dynein’s reaction to force.

Abstract: Programmed ribosomal frameshifting (PRF) represents an important mechanism for translational genetic recoding, especially in viruses. The components of a PRF stimulator have been well characterized, though accounting for the variation in the frameshift stimulating efficiency has thus far been elusive. Frameshift efficiencies at known PRF sites vary from a few percent to 70-80%, and several studies have been undertaken to determine what distinguishes a high efficiency PRF site from a low efficiency PRF site via structural characterization of the stimulatory structure. Observations suggest that conformational plasticity, the ability of a certain sequence to adopt multiple conformations, is correlated with frameshift efficiency. We examine a very high efficiency (70%) PRF stimulatory structure responsible for the NS1′ frameshift in West Nile virus (WNV) to determine its characteristics. We find a high degree of structural plasticity and heterogeneity; the PRF signal exhibits multiple different starting states and unfolds via two main pathways. Furthermore, we characterize the structures involved in these pathways, and find that they correspond to predicted structures using bioinformatic predictions and SHAPE analysis. Moreover, we suggest a new operational metric of conformational plasticity, one that obviates two existing problems with the previous method for defining conformational plasticity, namely the requirement to specify a native state, and the insensitivity to multiple conformations. Additionally, we extend this definition to be force dependent, and find that the value of this conformational plasticity metric in the force range of ribosomal stalling correlates highly with frameshifting efficiency. These results may elucidate the process of frameshifting by illustrating the relationship between conformational plasticity within a specific force range and frameshift efficiency. In addition, the characterization of a high efficiency frameshift signal allows for a better understanding of the structural dynamics underlying frameshifting.

The most common B-form Deoxyribonucleic acid (DNA) consists of two polymeric nucleic acid chains held together by Watson-Crick base pair (bp) with a right-handed twist at every 10.5 bp. In the nucleus, the DNA is subjected to spatial constraint and hence undergoes supercoiling (ς) to relieve stress through either overwinding (positive superhelicity), unwinding (negative superhelicity), DNA bending or melting. Similar topological changes observed during many cellular processes such as transcription, replication, etc. have a significant effect on the rate of these processes. Apart from the duplex DNA, many other non-B DNA structures exist in cells such as hairpin, G-quadruplex, i-motif, triplex, cruciform, etc. There is a lack of information on the topology of these structures under torsional stress. My research work focused on addressing the population dynamics of non-B DNA tetraplex structures under torsionally constrained physiological conditions at the molecular level. For this study, we developed magneto-optical tweezers by combining dual-beam optical tweezers and the manipulation of magnetic tweezers for rotation. The effect of torsional stress on the topology of non-B DNA tetraplexes was studied by varying the superhelicity of the template DNA through rotation of the magnets. For this study, we analyzed the sequence [d(ACAGGGGTGTGGGG)2] from a promoter region of Insulin Linked Polymorphic Region and quantified the molecular population dynamics of DNA tetraplexes under various chemical (ions and pH) and mechanical (template superhelicity and molecular crowding) conditions by single-molecule mechanical unfolding methods. By mechanical unfolding of individual tetraplexes, we found that ions and pH have the most substantial effect on the formation of G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding condition. While chemical effects are specific to tetraplex species mechanical factors show generic influences. This result provides substantial evidence for topology-based transcription modulation at the molecular level. Understanding the effect of torsional stress on the topology of secondary structures found in the chromosomes will serve as a deep insight into various cellular functions.

In the first half of this thesis, we describe our study of the elongation dynamics of E. coli RNA polymerase using optical tweezers. Optical tweezers constitute an important tool in modern biophysical research, as they allow the manipulation and tracking of individual molecules, such as enzymes that carry out diverse biological functions by converting chemical energy into mechanical work. Improvements to the spatio-temporal resolution and accuracy of optical tweezers therefore directly impact our ability to probe the tiniest and fastest motions of such enzymes.RNA polymerase is a central enzyme present in all organisms, that transcribes the genetic information encoded in DNA into RNA, one nucleotide at a time. This process constitutes the first step of gene expression, and is highly regulated at all its stages: initiation, elongation, and termination. In particular, elongation—i.e., the processive polymerization of the nascent RNA chain—does not occur in a continuous fashion, but consists of periods of active translocation interspersed by long-lived, sequence-dependent pauses, that have been implicated in various biological roles.While optical tweezers have long been able to observe such long-lived pausing events, many questions remained open, due to the limited spatio-temporal resolution of the technique. Here, we demonstrate algorithmic and instrumental developments that improve our ability to probe the transcription cycle at the finest level. Improvements in spatial resolution allowed us to robustly observe individual translocation events over long distances, and thus record the distribution of the dwell times spent at each position by the enzyme. Improvements in temporal resolution and spatial accuracy allowed us to understand the dynamics of the enzymes immediately as it reaches a "pause site". Specifically, we were able to show that transcription through a pause site is always accompanied by a decrease of the forward transcription rate. We established that entry into "backtracked" pauses occurs in a stepwise fashion, with a relatively slow entry into deeply backtracked states. We also probed the effect of nascent RNA structures on RNAP dynamics, and found that, depending on the sequence context, such structures could either enhance or attenuate pre-existing pauses.In the second half of this thesis, we review another fundamental single-molecule technique: super-resolution microscopy. Unlike optical tweezers, optical microscopy allows us to observe cellular processes in vivo or in situ; and the recent development of super-resolution microscopy has greatly enhanced the field of application of the technique. However, super-resolution microscopy also yields data that is much more difficult to interpret than classical ("diffraction-limited") microscopy. We discuss recent developments in our ability, not only to localize molecules with high accuracy, but also to quantify them. Finally, we present a fluorescent protein engineering work, regarding the development of a split-photoactivatable fluorescent protein system, towards the goal of studying protein-protein interaction at high resolution.

The ability of cytoskeletal motors to move unidirectionally along linear tracks is central to their cellular roles. While kinesin and myosin motor families have members that move in opposite directions, all dyneins studied to date exclusively move towards the microtubule minus-end. The source of dynein’s directionality remains as the central unresolved question about the mechanism of its motility. In my doctoral work, I focused on understanding the underlying mechanism of dynein’s directional motility along the microtubules in three dimensions. I used a protein engineering approach, guided by all-atom molecular dynamics simulations and high-resolution cryoEM imaging, along with the tools of single-molecule biophysics to dissect the elements of dynein motility. We successfully engineered a plus-end-directed dynein for the first time and revealed the mechanism of its directionality. This work has three major outcomes. First, by altering the length of the coiled-coil stalk that connects the dynein motor domain to the microtubule, we controlled the handedness of the helical motility of dynein around the circumference of the microtubule. This experiment showed that the stalk length of native dynein is critical for restricting sideways movement and directing dynein motility along the microtubule axis. Second, we altered the angle and length of dynein’s stalk. Remarkably, these modifications reversed the direction by which the linker swings relative to the microtubule and directed the motility towards the plus-end. Finally, similar to native dynein, the plus-end directed mutant maintains its preference to release from the microtubule when pulled towards the minus-end by an optical trap. Our results provide direct evidence for the linker swing model in which the direction the linker swings determines the direction of dynein motility. Our results also rule out the asymmetric release model that the directionality is proposed to be determined by the direction dynein favors for release from the microtubule under tension. Two critical features of dynein’s stalk, the length of its antiparallel coiled-coils and two proline residues located at its base, control the directionality by directing the linker swing towards the MT minus-end. Because both features of the stalk are fully conserved in cytoplasmic and ciliary dyneins across species, this mechanism explains the minus-end directionality of all dyneins.

Intracellular transport along microtubules is a process critical to cell health, andone whose breakdown is associated with neurodegenerative disorders. In this thesis, wedescribe new technologies that will enable and improve investigations of multiple typesof intracellular transport on microtubules.Although the single molecule properties of many motor proteins have been well characterized,their behavior when transporting biological cargoes as a group of motors is stillnot well understood due to measurement challenges, as well as a lack of a model systemfor systematically studying collective motor behavior in vitro. In this work, we discussthe construction of an optical trapping setup capable of applying and measuring forceswith 2 pN accuracy. We report our design of a biomimetic droplet system that reproducesthe relevant surface properties of biological cargoes while allowing the droplets tobe used as optical trapping probes. We also present a new optical trapping calibrationtechnique that allows experimenters to utilize the nonlinear range of the trapping forceprofile, and thereby measure the high forces developed by groups of motor proteins.Finally, we investigate the distributions of diffusion coeffcients found for MicrotubuleAssociated Proteins diffusing on microtubules through Monte Carlo simulations, andexamine the subtleties of interpreting and reporting single molecule diffusion data.

The purpose of this research is to increase the speed with which holographic optical tweezers (HOT) can individually assemble microscale particles for fabricating useful volumes of mechanical metamaterials in reasonable build times. HOT systems are unique among other additive manufacturing approaches in their ability to create true-3D structures with multiple materials, microscale features, and overhanging geometries. To scale up HOT-based fabrication, this research focuses on four primary goals: (1) scaling the number of particles that can be handled simultaneously using scanning holographic optical tweezers (SHOT), (2) developing efficient path planning and automation algorithms for efficiently delivering particles to their destinations within the fabricated structure, (3) modeling the dynamics of discretely-stepped optically-trapped particles to reliably handle them at high speeds, and (4) efficiently joining adjacent particles to produce a permanent structure. In addition, this work also demonstrates a proof-of-concept system that combines the HOT approach with the two-photon lithography approach, which allows for simultaneous fabrication and manipulation of microscale mechanisms in the same system for the first time. By improving the number and speed of particles that can be handled with the HOT approach, this work provides a promising path towards automated and scalable manufacturing of materials with extraordinary properties, such as microgranular lattices or microstructures with embedded strain energy.

While tissue stiffness is thought to play a role in regulation of cellular behavior, for the most part, stiffness is measured at the bulk level. The bulk measurement masks microscale dynamics within the fibrous extracellular matrix (ECM) and is insensitive to changes as cells remodel their local ECM. In order to investigate, cell-ECM dynamics I have developed an automated active microrheology (AMR) system and used it to probe the ECM near both single, isolated cells and multi-cell angiogenic sprouts, quantifying the pericellular distribution of stiffness. Additionally, I developed a new technique to modify stiffness within the ECM, at a scale relevant to the pericellular distribution of stiffness. My work shows that both human fibroblasts and smooth muscle cells establish a complex heterogeneous pericellular stiffness landscape. As expected, cell contraction strain hardened the matrix, but surprisingly, cells must also be competent in ECM proteolysis, which is to say the matrix must be broken down for cell-mediated stiffening. My findings suggest pericellular stiffness distributions should be considered in the study of cell-ECM interactions. In collaboration with Professor Andrew Putnam, I measured the evolution of stiffness change within a capillary morphogenesis model over time. We applied both bulk rheology and AMR to measure stiffness at different length scales. This data highlighted that bulk rheology was dominated by the activity of supportive stromal cells but blinded to the stiffness heterogeneity found proximal to vessels via AMR. These findings underscore that characterizing ECM mechanics across length scales can provide a deeper understanding of the microenvironment’s role within these complex processes. Lastly, I developed and evaluated a method to modify stiffness within fibrin matrices at the micron-scale. This method allows for a patterning of stiffness at a spatial scale and magnitude similar to that observed by cell-mediated stiffening. By using ruthenium-catalyzed photo-crosslinking coupled with our laser scanning confocal microscope, we can selectively illuminate and thereby selectively crosslink regions of interest within the volume of a hydrogel. This results in a stiffness increase of up to 25X, with a steep stiffness gradient in the surrounding area. Selective crosslinking could be of great utility in creating more complex patterns of stiffness, which could be invaluable for the investigation of mechanotransduction within a natural 3D ECM context. Collectively, these works show that the mechanical topography surrounding cells within ECM is varied and must be considered in future study of mechanically driven hypotheses. Microrheology in combination with selective photo-crosslinking provides a new tool to better understand roles for tissue stiffness in cell regulation, and vice versa.

Abstract: The formation of an abnormal form of proteins in cells can cause aggregation and neurodegenerative pathology, such as Alzheimer’s, Parkinson’s and prion diseases, which affects both humans and animals. Nowadays, the understanding of the mechanism of prion misfolding and propagation, including effective ways of treating prion diseases, is not clearly identified. This may result from the complexity of the biological ensembles. Optical tweezers, one of the single-molecule force spectroscopy methods, can be used to better understand mechanisms of protein misfolding and dynamics by manipulating small beads attached to single protein molecules, resulting in the mechanical denaturation of protein structure. Previous work studying the mechanical denaturation of structure in hamster prion protein (PrP) by laser tweezers, for example, found that the folding pathways of the native protein were composed of two states of folding and unfolding (Yu et al., 2012; 2013). Although the sequence of PrP is very highly conserved between species, there are a few differences in amino acid residues between PrP in different species. Furthermore, mouse PrP, a model system for studying prion diseases, has a lower susceptibility to the misfolding underlying prion diseases than hamster PrP. Thus, the differences in the protein sequences may lead to differences in the folding and misfolding which may relate to the different susceptibilities. This work is the first single-molecule study of mouse PrP folding; the prion proteins from mice were attached to DNA handles using click chemistry and were studied by optical tweezers in order to compare my results to those of the previous studies on hamster PrP. As a result of the successful attachments, the force- extension curves of mouse prion proteins had close similarities in unfolding behavior compared to those of hamsters. Furthermore, according to the data analysis of the curves, the contour lengths of the mouse prion proteins from the combination of rips were found to be closely matched to the expected length in the protein structure. However, it was challenging to avoid problems caused by attaching the DNA handles to the wrong cysteine residues in the protein. For a more complete understanding of protein misfolding, future work involving the study of protein structure is required. Single-molecule studies of mouse prion proteins can also allow one to study the energy landscapes, specifically the folding pathways of proteins in different species. Information regarding protein structure may be useful for the development of specific therapeutic drugs. Future work on the molecular mechanisms of the prion proteins will not only provide a better understanding of prion diseases, but also other related neurodegenerative disease that share similar mechanisms of protein aggregation.