Single-molecule localisation microscopy (SMLM), has allowed for optical microscopy to probe biological systems beyond the diffraction limit. The intrinsic 3D nature of biology has motivated the development of 3D-SMLM with novel techniques, including the double-helix point spread function (DHPSF). A bespoke microscope platform employing the DHPSF transformation was built, achieving ~10 nm lateral and ~20 nm axial localisation precision over a ~4 μm axial depth. Until recently, the DHPSF has been limited by spherical aberration present when imaging away from coverslip surfaces to the study of small volumes close to the coverslip. By matching the refractive index of the objective lens immersion liquid to that of the imaging media, this aberration can be minimised, facilitating large-volume imaging away from unphysiological flat surfaces. The work presented in this thesis illustrates the capabilities of the DHPSF for 3D-SMLM and single-particle tracking (SPT) in previously inaccessible areas of biological samples (e.g. in the nucleus and on the apical cell surface). Application of the DHPSF for SPT in eukaryotic cells are presented; tracking the motion of T-cell membrane proteins on the apical surface and components of the chromosome remodelling complex in the nucleus of embryonic stem cells. For these applications, meansquared displacement and jump distance diffusion analysis methodologies were extended into 3D and benchmarked against simulated datasets. A variety imaging applications that are facilitated by the extended depth of focus of the DHPSF are presented, focusing on quantification of T-cell membrane protein reorganisation upon immunological activation. Finally, the clustering distribution of the T-cell receptor is investigated by Ripley’s K analysis enabled by duel labelling of its position and the outer membrane in primary T cells.

In the past decade, DNA origami self-assembly has been widely applied for creating customised nanostructures with base-pair precision. In this technique, the unique chemical addressability of DNA can be harnessed to create programmable architectures, using components ranging from dye or protein molecules to metallic nanoparticles. In this thesis, we apply DNA nanotechnology for developing novel light-harvesting and optical voltage sensing nano-devices. We use the programmable positioning of dye molecules on a DNA origami plate as a mimic of a light-harvesting antenna complex required for photosynthesis. Such a structure allows us to systematically analyse optimal design concepts using different dye arrangements. Complementary to this, we use the resistive-pulse sensing technique in a range of electrolytes to characterise the mechanical responses of DNA origami structures to the electric field applied. Based on this knowledge, we assemble voltage responsive DNA origami structures labelled with a FRET pair. These undergo controlled structural changes upon application of an electric field that can be detected through a change in FRET efficiency. Such a DNA-based device could ultimately be used as a sensitive voltage sensor for live-cell imaging of transmembrane potentials.

Amyloidogenic neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are an important health issue. However, the underlying molecular mechanisms of the disease-related protein aggregates, that are present in humans, are only understood partially. I have used and developed biophysical methods to study the structural and biological properties of individual aggregates of Amyloid β peptide and α-Synuclein, proteins whose aggregation is associated with the development of Alzheimer's and Parkinson's disease respectively. I expanded the single aggregate visualisation through enhancement (SAVE) technique, which is a method based on the fluorescent dye Thioflavin T (ThT) that reversibly bind to the aggregates and whose fluorescence increases upon binding. I firstly explored the use of other dyes for these experiments and found that a ThT dimer has higher affinity to α-Synuclein aggregates in vitro. I then applied the SAVE method to the cerebral spinal fluid (CSF) of a cohort of AD patients and control CSF and observed no clear difference in aggregate number. However, these experiments provided insights into how antibodies bind the aggregates in human CSF. I could show, that despite altering the Ca2+ influx into both cells and vesicles, the antibody did not measurably affect the aggregate structure. To study the size specific effects of the Amyloid β 42 (Aβ42) peptide in more detail, I used and optimised gradient ultracentrifugation combined with single aggregate imaging to study the structural properties of the isolated aggregates. This aggregation kinetic independent method allowed me to compare the properties of fluorescently labelled and unlabelled Aβ42 and characterize the size dependent properties of aggregates in a single experiment. Since I could measure the relative concentration of different size aggregates it was also possible to compare the properties of single aggregates of different sizes. I then used biological assays to examine the ability of aggregates to permeabilise membranes resulting in the entry of calcium ions, and their ability to induce TNFα production in microglia cells. Both processes are thought to play key roles in the development of AD. I found that small soluble oligomers are most potent at inducing Ca2+ influx, whereas longer protofilaments are the most potent inducers of TNFα production. My results suggest that the mechanism by which aggregates damage cells changes as aggregation proceeds, as longer aggregates with different structures are formed. Protofilaments with a diameter of 1 nm or less have a structure that could make them particularly potent at causing the signalling of toll-like receptors, providing a molecular basis for their ability to induce TNFα production.

The profound simplicity and versatility of the molecule at the heart of all earth- bound life forms, DNA, continues to inspire new frontiers of scientific inquiry. Central to many of these, including the de novo design of novel DNA nanostructures and the use of DNA to probe the principles of biological self-assembly and the operation of cellular nanomachines, is the interaction of DNA with forces, both internal and external. This thesis comprises a survey of three key ways coarse-grained simulations using the oxDNA model can contribute to efforts to characterize these interactions. First, a non-equilibrium data analysis framework based on the Jarzynski equality from statistical physics is validated for use with oxDNA through the reconstruction of free energy landscapes for canonical DNA hairpin systems. We provide a framework for assessing errors in the method and apply it to study a system for which conventional equilibrium simulations would be impractical: DNA origami 'handles' proposed for use in force spectroscopy experiments. Next, we simulate the forcible unravelling of three DNA origami structures, the largest systems yet studied with simulated force spectroscopy. We combine these results with experimental AFM data to probe the mechanical response of origami in unprecedented detail, highlighting the effect of nanostructure design on unfolding behaviour. Lastly, we examine the validity of using widely-employed polymer elastic models to predict internal entropic forces in ssDNA. We develop a framework for measuring internal forces in the oxDNA coarse-grained model and apply it to analyze the pico-Newton range forces exerted by a recently proposed DNA origami force clamp, ultimately concluding that conventional means of estimating internal ssDNA forces are often inaccurate and should be supplemented with coarse-grained simulations. In addition to providing new insights about the DNA systems we present, our results highlight the significant fruits of complementing experimental studies with coarse-grained simulations.

The Rho family of guanosine triphosphatases (GTPases) function as binary molecular switches, which play an important role in the regulation of actin cytoskeleton rearrangement and are involved in several critical cellular processes including cell adhesion, division and migration. Rho GTPases are specifically activated by their associated guanine nucleotide exchange factors (RhoGEFs). Dysregulation of RhoGEFs function through mutation or overexpression has been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. T-cell lymphoma invasion and metastasis 1 (Tiam1) is a multi-domain Dbl family GEF protein and specifically activates Rho GTPase Rac1 through the catalytic Dbl homology and Pleckstrin homology (DH-PH) bi-domain. Previous works have shown that the nucleotide exchange function of the full-length Tiam1 is auto-inhibited and can be activated by N-terminal truncation, phosphorylation and protein-protein interactions. However, the molecular mechanisms of Tiam1 GEF auto-inhibition and activation have not yet been determined. In this study, the N-terminal PH-CC-Ex domain of Tiam1 is shown to directly inhibit the GEF function of the catalytic DH-PH domain in vitro. Using fluorescencebased kinetics experiments, we demonstrate that the auto-inhibition of Tiam1 GEF function occurs by a competitive inhibition model. In this model, the maximum velocity of catalytic activity remains unchanged, but the Michaelis-Menten constant of the auto-inhibited Tiam1 (the PH-PH fragment) on the substrate Rac1 is increased compared to the activated Tiam1 (the catalytic DH-PH domain alone). Through small angle X-ray scattering (SAXS), the structure of auto-inhibited Tiam1 (the PH-PH fragment) is shown to form a closed conformation in which the catalytic DH-PH domain is blocked by the N-terminal PH-CC-Ex domain. Taken together, these findings demonstrate the molecular mechanism of Tiam1 GEF autoinhibition in which the PH-CC-Ex domain of Tiam1 inhibits its GEF function by preventing the substrate Rho GTPase Rac1 from accessing the catalytic DH-PH bi-domain.

Single molecule studies can provide information of biological molecules which otherwise is lost in ensemble studies. A wide variety of fluorescence-based techniques are utilized for single molecule studies. While these tools have been widely applied for imaging soluble proteins, single molecule studies of transmembrane proteins are much more complicated. A primary reason for this is that, unlike membrane proteins, soluble proteins can be easily isolated from the cellular environment. One approach to isolate membrane proteins into single molecule level involves a very low label expression of the protein in cells. However, cells generate background fluorescence leading to a very low signal to noise ratio. An alternative approach involves isolating membrane proteins in artificial membrane derived vesicles. This approach is limited to proteins which can be solubilized or stabilized in detergent solution. This intermediate step endangers the structural integrity of proteins with multiple subunits. Hence, we isolated transmembrane proteins into cell-derived vesicles which maintain the proteins in their physiological membrane without compromising their functional integrity. We studied the stoichiometric assembly of α3β4 nicotinic receptors which are pentameric receptor with possible stoichiometry of (α3)2(β4)3 and (α3)3(β4)2. We found that (α3)2(β4)3 is the predominant stoichiometry, and we have verified our finding with both single and double color experiments. We have also demonstrated that cell-derived vesicles can be utilized to study ligand receptor interactions. Cell-derived vesicles generated from cellular preparations provide a method to study the overall structural and functional properties of membrane proteins. However, organelle specific information is not available in this approach. Alternatively, separating vesicles based on their original organelle could provide information on the assembly and trafficking of membrane proteins. For example, it has been hypothesized that nicotine acts as a pharmacological chaperone of α4β2 nicotinic receptors and nicotine alters the assembly of the nicotinic receptors towards the high sensitivity isoform in the ER. To validate this hypothesis, we isolated α4β2 nicotinic receptors located on vesicles derived from the ER and plasma membrane origins and utilized single molecule studies to determine the stoichiometric assembly of the receptor. The data suggested that the ER has a higher percentage of the low sensitivity isoform ((α4)3(β2)2) than the plasma membrane indicating that the high sensitivity isoform trafficked more efficiently to the cell surface. When nicotine was added, the distribution of nicotinic receptors changes in those compartments. In both the ER and plasma membrane, the percentage of high sensitivity isoform was greater than the sample without the presence of nicotine. The results suggested that nicotine altered the assembly of nicotinic receptors to form the high sensitivity isoform in the ER and the altered assembly trafficked to the plasma membrane efficiently increasing the ratio of this isoform in the plasma membrane. The cell derived vesicles we utilized to isolate single receptors are structurally similar to liposomes, an FDA approved drug delivery system, which is spherical vesicles composed of at least one lipid bilayer. Hence, cell-derived vesicles possess potential to be utilized as drug delivery vehicles. I explored the applicability of cell-derived vesicles as general delivery vehicles to cultured cells. Additionally, we implanted xenografts into immune compromised nude mice and prepared cell derived vesicles labeled with dye molecules. The vesicles were injected in a mouse containing a xenograft to monitor whether these vesicles can reach to the xenograft. Our data suggested that cell-derived vesicles can successfully reach the xenograft and thus have potential to be utilized as a drug delivery vehicle.

La microscopie de molécule unique (single-molecule microscopy, SMM) regroupe un ensemble de techniques pour la biologie moléculaire et cellulaire permettant de visualiser le mouvement de molécules biologiques individuelles. Néanmoins, les techniques SMM imposent de fortes contraintes en ce qui concerne les luminophores utilisés. Récemment, un nouveau luminophore appelé «particule à conversion ascendante» (upconverting nanoparticles, UCNP) a attiré l'attention de la communauté scientifique en raison de son émission efficace de lumière visible après une excitation par de la lumière infrarouge. Cette propriété fait des UCNPs un luminophore très intéressant pour les applications biologiques : l'excitation infrarouge permet d'éliminer l’autofluorescence, généralement associé à une excitation dans la gamme du visible. De plus, la photostabilité extrême des UCNP et l’absence de photoclignotement sont également de précieux atouts pour les expériences SMM. L’objectif de cette thèse était d’adapter les UCNPs aux applications SMM, avec le but ultime d’exploiter leurs propriétés uniques pour améliorer les performances des expériences SMM. Au cours du projet, les protocoles de dispersion des UCNPs dans des tampons aqueux ont été optimisées pour conserver une bonne monodispersité des particules; l'efficacité des UCNPs dans les expériences de transfert résonant d'énergie en particule unique a été estimée; des protocoles pour l'imagerie d'UCNPs uniques ont été développés; et la preuve de concept de l'utilisation des UCNPs dans des expériences de suivi de molécules uniques à la surface de cellules vivantes a été réalisée. Finalement, ces résultats forment une base solide pour de futures expériences SMM utilisant les UCNPs. / Single-molecule microscopy (SMM) is a powerful set of techniques for molecular and cell biology that allows visualizing the movement of individual biological molecules, but has strict requirements towards the utilized luminophores. Recently, a new luminophore called upconverting particles (UCNPs) gained attention of the research community due to their efficient emission of visible light upon excitation with infrared light. This property makes UCNPs a valuable luminophore for biological applications due to the elimination of autofluorescence background, commonly associated with regular visible light excitation. Extreme photostability of UCNPs and absence of sporadic photoswitching are also valuable for SMM experiments. The objective of this thesis was to adapt UCNPs to SMM applications, with the ultimate goal of exploiting their unique properties towards superior performance of SMM experiments. During the project, protocols for dispersing UCNPs in aqueous buffers were streamlined to provide superior particle monodispersity; the efficiency of UCNPs in single-molecule resonance energy transfer experiments was estimated; protocols for single-molecule imaging with UCNPs were developed; and a proof-of-concept system for targeted single-molecule tracking with UCNPs in live cells was demonstrated. Overall, these findings will serve as a foundation towards robust SMM assays based on UCNPs.

Hydrophobic interactions guide important molecular self-assembly processes such as protein folding. On the macroscale, hydrophobic interactions consist of the aggregation of "oil-like" objects in water by minimizing the interfacial energy. However, the hydration mechanism of small hydrophobic molecules on the nanoscale (~1 nm) differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic cross-over length scale is critically important to hydrophobic interactions in polymers, proteins and other macromolecules. Accurate experimental determination of hydration mechanisms and their interaction strengths are needed to understand protein folding. This thesis reports the development of experimental and analytical techniques that allow for direct measurements of hydrophobic interactions in a single molecule. Using single molecule force spectroscopy, the mechanical unfolding of a single hydrophobic homopolymer was identified and modeled. Two experiments examined how hydrophobicity at the molecular scale differ from the macroscopic scale. The first experiment identifies macroscopic interfacial tension as a critical parameter governing the molecular hydrophobic hydration strength. This experiment shows that the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways, consistent with theoretical predictions. The second experiment probes the hydrophobic size effect by studying how the size of a non-polar side-chain affects the thermal signatures of hydration. Our experimental results reveal a cross-over length scale of approximately 1 nm that bridges the transition from entropically driven microscopic hydration mechanism to enthalpically driven macroscopic hydration mechanism. These results indicate that hydrophobic interactions at the molecular scale differ from macroscopic scale, pointing to potential ways to improve our understanding and predictions of molecular interactions. The system established in this thesis forms the foundation for further investigation of polymer hydrophobicity.

Single-molecule spectroscopy has provided a means to uncover pathways and heterogeneities that were previously hidden beneath the ensemble average. Such heterogeneity, however, is often obscured by the artifacts of experimental noise and the occurrence of undesired processes within the experimental medium. This has subsequently caused in the need for new analytical methodologies. It is particularly important that objectivity be maintained in the development of new analytical methodology so that bias is not introduced and the results improperly characterized. The research presented herein identifies two such sources of experimental uncertainty, and constructs objective approaches to reduce their effects in the experimental results. The first, photoblinking, arises from the occupation of dark electronic states within the probe molecule, resulting in experimental data that is distorted by its contribution. A method based in Bayesian inference is developed, and is found to nearly eliminate photoblinks from the experimental data while minimally affecting the remaining data and maintaining objectivity. The second source of uncertainty is electronic shot-noise, which arises as a result of Poissonian photon collection. A method based in wavelet decomposition is constructed and applied to simulated and experimental data. It is iii found that, while making only one assumption, that photon collection is indeed a Poisson process, up to 75% of the shot-noise contribution may be removed from the experimental signal by the wavelet-based procedure. Lastly, in an effort to connect model-based approaches such as molecular dynamics simulation to model-free approaches that rely solely on the experimental data, a coarse-grained molecular model of a molecular ionic fluorophore diffusing within an electrostatically charged polymer brush is constructed and characterized. It is found that, while the characteristics of the coarse-grained simulation compare well with atomistic simulations, the model is lacking in its representation of the electrostatically-driven behavior of the experimental system.

Our group has developed a family of single molecules termed nanocars, which are aimed at performing controllable motion on surfaces. In this work, a series of light-activated motorized nanomachines incorporated with a MHz frequency light-activated unidirectional rotary motor were designed and synthesized. We hope the light-activated motor can serve as the powering unit for the nanomachines, and perform controllable translational motion on surfaces or in solution. A series of motorized nanovehicles intended for scanning tunneling microscopy (STM) imaging were designed and synthesized. A p-carborane-wheeled motorized nanocar was synthesized and monitored by STM. Single-molecule imaging was accomplished on a Cu(111) surface. However, further manipulations did lead to motor induced lateral motion. We attributed this result to the strong molecule-surface interactions between the p-carborane-wheeled nanocar and the Cu(111) surface. To fine-tune the molecule-surface interactions, an adamantane-wheeled motorized nanocar and a three-wheel nanoroadster were designed and synthesized. In addition, the STM substrates will be varied and different combinations of molecule-surface interactions will be studied. As a complimentary imaging method to STM, single-molecule fluorescence microscopy (SMFM) also provides single-molecule level resolution. Unlike STM experiment requires ultra-high vacuum and conductive substrate, SMFM experiment is conducted at ambient conditions and uses non-conductive substrate. This imaging method allows us to study another category of molecule-surface interactions. We plan to design a fluorescent motorized nanocar that is suitable for SMFM studies. However, both the motor and fluorophore are photochemically active molecules. In proximity, some undesired energy transfer or interference could occur. A cyanine 5- (cy5-) tagged motorized nanocar incorporated with the MHz motor was designed and synthesized in order to minimize the potential energy transfer or interference between the motor and the fluorophore. The SMFM study of this cy5-tagged motorized nanocar is currently undergoing. The design of light-activated motorized nanocar inspired the design of nanosubmarines. We used fluorescence quenching and fluorescence correlation spectroscopy (FCS) to study the diffusion of single molecules. The fluorescence quenching experiments of Ru(bpy)3+2 by a quenching nanosubmarine was conducted, but no motor induced acceleration of the molecule were observed. Another fluorescent nanosubmarine was monitored by FCS, and no increase of diffusion coefficient was found. Finally, a 1-D channel approach was adopted for decreasing the effects of Brownian motion, and acceleration of nanosubmarine was observed.