Optical trapping and tweezing has been around for the last 30 years and since found its place in the fields of physics and biology. Over the years this technique has advanced exceedingly and is a unique tool to carry out research in the micrometre and nanometre scale regime. The aim of this dissertation was to illustrate that an optical trapping and tweezing system is an effective tool for the manipulation of micron sized particles and that using such a system allows one the ability to accurately and precisely measure optical forces in the piconewton scale. A custom built single gradient optical trapping system was built to illustrate the manipulation of micron sized particles. Here we will highlight some of the key components of such a system and give an explanation of how these components affect the optical trap. To enhance this system, we exploit the ability to shape light and in particular laser light to generate novel laser beams. This was achieved using a diffractive optical element known as a spatial light modulator (SLM). A spatial light modulator is an electronically addressed optical element which when incorporated into an optical system effectively manipulates the phase of light in order to generate various novel laser beams. In particular these novel laser beams include Laguerre-Gaussian, Bessel and recently proposed Bessel-like beams. Each of these beams contains interesting properties which can be beneficially exploited. Laguerre-Gaussian beams are particularly known as ‘donut’ shaped beams since they have a central dark hole. Increasing the order of these Laguerre-Gaussian beams leads to an increase in the central dark region. These beams are of particular interest since they carry orbital angular momentum. This is not easily observed; however, when incorporated into the optical trapping system, leads to the rotation of trapped particles due to the transfer of photons carrying orbital angular momentum. Bessel and Bessel-like beams on the other hand are classes of beam that possess interesting non-diffracting and self-reconstructive properties upon encountering an obstacle. Here the generation and properties of these novel laser beams will be discussed in detail. Furthermore it is well known that these novel laser beams prove highly useful when incorporated into an optical trapping system hence we will illustrate the effects on a trapped particle when incorporating a Laguerre-Gaussian beam carrying a topological charge of one. It is expected that the trapped particle should rotate due to the transfer of orbital angular momentum. The knowledge gained from beam shaping and the means to trap micron sized particles optically allows one the ability to incorporate this technique in a number of fields, including the promising field of microfluidics. This is an emerging field that deals with investigating fluid properties at the nano and microlitre regime. Optical tweezers integrated into a microfluidic device are beneficial since they are an adequate tool for measuring fluid flow using Stokes’ Law. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2011.

Gong, Xiangjun = 基於磁鑷和全反射顯微鏡的單粒子追踪微流變方法 / 龔湘君. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Gong, Xiangjun = Ji yu ci nie he quan fan she xian wei jing de dan li zi zhui zong wei liu bian fang fa / Gong Xiangjun.

Bacterial resistance to antibiotics is increasing at a high rate in both developing and developed countries. To circumvent the problem of drug-resistant bacterial pathogens, we need to develop new effective methods, substances, and materials that can disarm and prevent them from causing infections. However, to do this we first need to find new possible targets in bacteria to approach and novel strategies to apply.Escherichia coli (E. coli) bacteria is a normal member of the intestinal microflora of humans and mammals, but frequently cause diverse intestinal and external diseases by means of virulence factors, which leads to hundreds of million sick people each year with a high mortality rate. An E. coli bacterial infection starts with adhesion to a host cell using cell surface expressed adhesion polymers, called adhesion pili. Depending on the local environment different types of pili are expressed by the bacteria. For example, bacteria found in the gastrointestinal tract commonly express different pili in comparison to those found in the urinary tract and respiratory tract. These pili, which are vital for bacterial adhesion, thereby serve as a new possible approach in the fight against bacterial infections by targeting and disabling these structures using novel chemicals. However, in order to develop such chemicals, better understanding of these pili is needed.Optical tweezers (OT) can measure and apply forces up to a few hundred pN with sub-pN force resolution and have shown to be an excellent tool for investigating mechanical properties of adhesion pili. It has been found that pili expressed by E. coli have a unique and complex force-extension response that is assumed to be important for the ability of bacteria to initiate and maintain attachment to the host cells. However, their mechanical functions and the advantage of specific mechanical functions, especially in the initial attachment process, have not yet been fully understood.In this work, a detailed description of the pili mechanics and their role during cell adhesion is presented. By using results from optical tweezers force spectroscopy experiments in combination with physical modeling and numerical simulations, we investigated how pili can act as “shock absorbers” through uncoiling and thereby lower the fluid force acting on a bacterium. Our result demonstrate that the dynamic uncoiling capability of the helical part of the adhesion pili modulate the force to fit the optimal lifetime of its adhesin (the protein that binds to the receptor on the host cell), ensuring a high survival probability of the bond.iiiSince the attachment process is in proximity of a surface we also investigated the influence of tether properties and the importance of different surface corrections and additional force components to the Stokes drag force during simulations. The investigation showed that the surface corrections to the Stokes drag force and the Basset force cannot be neglected when simulating survival probability of a bond, since that can overestimate the probability by more than an order of magnitude.Finally, a theoretical and experimental framework for two separate methods was developed. The first method can detect the presence of pili on single cells using optical tweezers. We verified the method using silica microspheres coated with a polymer brush and E. coli bacteria expressing; no pili, P pili, and type 1 pili, respectively. The second method was based on digital holography microscopy. Using the diffraction of semi-transparent object such as red blood cells, we showed that this method can reconstruct the axial position and detect morphological changes of cells.

In this thesis, we report a novel symmetry breaking system in single-beam optical trap. The breaking of symmetry is observed in Brownian dynamics of a linked pair of beads with substantially differing radii (500nm and 100nm). Such composite beads were originally conceived as a manipulation means to study of Brownian interactions between mesoscopic biological agents of the order of 100 – 200 nm (viruses or bacteria) with cell surfaces. During the initial testing of the composite bead system, we discovered that the system displayed thermally activated transitions and energetics of symmetry breaking. This thesis, while making a brief overview of the biological relevance of the composite bead system, focuses primarily on the analysis and experimentation that reveals the complex dynamics observed in the system. First, we theoretically analyze the origin of the observed symmetry breaking using electromagnetic theory under both Gaussian beam approximation and full Debye-type integral representation. The theory predicts that attachment of a small particle to a trapped microsphere results in creation of a bistable rotational potential with thermally activated transitions. The theoretical results are then verified using optical trapping experiments. We first quantify the top-down symmetry breaking based on measurement of the kinetic transition rates. The rotational potential is then explored using an experiment employing a novel algorithm to track rotational state of the composite bead. The results of the theory and experiments are compared with results of a Brownian dynamics simulation based on Smart Monte Carlo algorithm.

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.

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.

Nanoaperture optical tweezers are emerging as useful tools for the detection and identification of biological molecules and their interactions at the single molecule level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward, high-speed platform for single molecule studies without the need to use tethers or labeling. This thesis gives a general description of conventional optical tweezers and how they are limited in terms of their capability to trapping biological molecules. It also looks at nanoaperture-based optical tweezers which have been suggested to overcome the limitations of conventional optical tweezers. The thesis then focuses on the double nanohole optical tweezer as a tool for trapping biological molecules and studying their behaviour and interactions with other molecules. The double nanohole aperture trap integrated with microfluidic channels has been used to detect single protein binding. In that experiment a double-syringe pump was used to deliver biotin-coated polystyrene particles to the double nanohole trapping site. Once stable trapping of biotin-coated polystyrene particle was achieved, the double-syringe pump was used to flow in streptavidin solution to the trapping site and binding was detected by measuring the transmission through the double nanohole aperture. In addition, the double nanohole optical tweezer has been used to observe the real-time dynamic variations in protein-small molecule interaction (PSMI) with the primary focus on the effect of single and multiple binding events on the dynamics of the protein in the trap. Time traces of the bare form of the streptavidin showed slower timescale dynamics as compared to the biotinylated forms of the protein. Furthermore, the double nanohole aperture tweezer has been used to study the real-time binding kinetics of PSMIs and to determine their disassociation constants. The interaction of blood protein human serum albumin (HSA) with tolbutamide and phenytoin was considered in that study. The dissociation constants of the interaction of HSA with tolbutamide and phenytoin obtained using our technique were in good agreement with the values reported in the literature. These results would open up new windows for studying real-time binding kinetics of protein-small molecule interactions in a label-free, free-solution environment, which will be of interest to future studies including drug discovery. / Graduate

Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas. DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient. The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores.

Arthur Ashkin's Nobel Prize-winning single-beam gradient force optical tweezers have revolutionized research in many fields of science. The invention has enabled various atomic and single molecular studies, proving to be an essential tool for observing and understanding nature at the nanoscale. This thesis showcases the uniqueness of single-beam gradient force traps and the advances necessary to overcome the limitations inherent in conventional techniques of optical trapping. With decreasing particle sizes, the power required for a stable trap increases and could potentially damage a particle. This is a significant limitation for studying biomolecules using conventional optical traps. Plasmonic nanoaperture optical trapping using double-nanohole apertures is introduced as a solution to overcoming these limitations. Achievements in double-nanohole optical trapping made possible by the pioneering work of Gordon et. al are highlighted as well. This thesis focuses on the advances in nanoaperture fabrication methods and improvements to experimental techniques adopted in single molecular optical trapping studies. The technique of colloidal lithography is discussed as a cost-effective high-throughput alternative method for nanofabrication. The limitation in using this technique for producing double-nanohole apertures with feature sizes essential for optical trapping is analyzed. Improvements to enable tuning of aperture diameter and cusp separation is one of the main achievements of the work detailed in this thesis. Furthermore, the thesis explains the modified fabrication process tailor-made for designing double-nanohole apertures optimized for optical trapping. Transmission characterization of various apertures fabricated using colloidal lithography is carried out experimentally and estimated by computational electrodynamics simulations using the finite-difference time-domain (FDTD) method. Optical trapping with double-nanohole apertures fabricated using colloidal lithography is demonstrated with distinct results revealing trapping of a single polystyrene molecule, a rubisco enzyme and a bovine serum albumin (BSA) protein. / Graduate

Mechanical forces regulate biological processes in unique and unexpected ways, but many biochemical methods are unable to reproduce the vectorial stretching experienced by proteins in cells. Force spectroscopy techniques remedy these shortcomings by utilizing microscopic force probes to stretch and relax single protein, DNA, and RNA molecules. The central focus of this thesis is the development and implementation of a custom-built protein magnetic tweezers for unfolding and refolding Ig domains from titin, a critical filament of the sarcomere, and the longest continuous peptide in the human body. Suspended from the Z-disc to the tip of the thick filament, titin sustains the brunt of intracellular forces during muscle elongation. Since the discovery of titin, it has been widely debated whether Ig domain unfolding contributes to muscle mechanics. A combination of single quantum dot tracking in myofibrils extracted from rabbit muscle and single molecule magnetic tweezers experiments on recombinant titin fragments confirms, for the first time, the presence of titin Ig domain unfolding and refolding at physiological sarcomere lengths and stretching forces. The magnetic tweezers experiments show the surprising ability of titin Ig domains to generate piconewton level forces during folding, and we advance the hypothesis that titin folding is an important source of energy during muscle contraction. Muscle elongation recruits Ig domains to the unfolded state, whereby folding is initiated through reduction of force on titin upon actomyosin crossbridges formation. Magnetic tweezers measurements demonstrate that titin Ig folding generates peak work, velocity, and power output of 64 zeptojoules, 1.9 microns per second, and 6,000 zeptowatts, matching or exceeding the equivalent single molecule measurements from single molecule myosin II powerstrokes. The forces generated by protein folding are therefore likely to be an integral part of the contractile process of animal muscle.