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

This dissertation combines various holographic techniques with application on the two- and three-dimensional imaging of ophthalmic tissue, fingerprints, and microsphere samples with micrometer resolution. Digital interference holography (DIH) uses scanned wavelengths to synthesize short-coherence interference tomographic images. We used DIH for in vitro imaging of human optic nerve head and retina. Tomographic images were produced by superposition of holograms. Holograms were obtained with a signal-to-noise ratio of approximately 50 dB. Optic nerve head characteristics (shape, diameter, cup depth, and cup width) were quantified with a few micron resolution (4.06 -4.8 microns). Multiple layers were distinguishable in cross-sectional images of the macula. To our knowledge, this is the first report of DIH use to image human macular and optic nerve tissue. Holographic phase microscopy is used to produce images of thin film patterns left by latent fingerprints. Two or more holographic phase images with different wavelengths are combined for optical phase unwrapping of images of patent prints. We demonstrated digital interference holography images of a plastic print, and latent prints. These demonstrations point to significant contributions to biometry by using digital interference holography to identify and quantify Level 1 (pattern), Level 2 (minutia points), and Level 3 (pores and ridge contours). Quantitative studies of physical and biological processes and precise non-contact manipulation of nanometer/micrometer trapped objects can be effectuated with nanometer accuracy due to the development of optical tweezers. A three-dimensional gradient trap is produced at the focus position of a high NA microscope objective. Particles are trapped axially and laterally due to the gradient force. The particle is confined in a potential well and the trap acts as a harmonic spring. The elastic constant or the stiffness along any axis is determined from the particle displacements in time along each specific axis. Thus, we report the sensing of small particles using optical trapping in combination with the digital Gabor holography to calibrate the optical force and the position and of the copolymer microsphere in the x, y, z direction with nm precision.

Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis.

In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.

Colloidal particles are small particles ranging in size from nanometres to micrometres suspended in a fluid. Amongst many scientific and biological applications, they have been used to model crystallisation, vitrification, and particle interactions along with the use of colloidal model systems for the study of the fundamental nature of the fluid-crystal and fluid-glass phase transitions. It has been shown that colloidal particles can be trapped and manipulated using strongly-focused light beams known as optical tweezers, and this has paved the way for research into the area of micromanipulation using optical trapping. Holographic elements can replace multiple lenses in creating large numbers of optical tweezers and this is known as holographic optical trapping (HOT). A computer generated hologram can be designed to create large structured light fields, consisting of multiple foci, to enable trapping of multiple particles in arbitrary configurations. The overall aim of this project was to design, develop and test the suitability of a simple, inexpensive optical trapping arrangement suitable for multiple optical trapping. To achieve this, a theoretically-exact expression for the wavefront of a single point source was implemented in the coding scheme, allowing for the fast creation of multiple point sources suitable for holographic optical trapping experiments. Compensation for the spherical aberration present in the focusing optics was implemented into the coding scheme. Kodalith photographic film was chosen as the holographic recording medium for its high contrast and availability. The film has proven to be a successful medium, when used to record photographically-reduced images of high-quality printouts of the computed diffraction pattern, as it was able to successfully reproduce complex light fields. It is believed that this will be the first time that this film has been implemented for optical trapping purposes. The main limitations concerning the performance of the holograms recorded on Kodalith were the phase nonuniformities caused by unevenness in the film thickness which resulted in a failure to separately resolve light traps separated by less than about 5 (Mu)m. Index matching of the film between sheets of flat glass helped to compensate for these limitations. Holographic optical trapping was successfully observed using a variety of different initial beam powers, holographic aperture settings and light field configurations. Trapping experiments on of two types of particles (PMMA and polystyrene) were successfully conducted, with as little as ~ 150 (Mu)W per trap being required for multiple polystyrene trapping. However, particles were weakly trapped and were easily dislodged at these powers, and a higher power per trap of around 1 mW is preferred. The use of a relatively low numerical aperture (NA) 50 mm SLR lens for focusing the holographic optical traps was successful, proving that optical trapping can be conducted without the use of high NA microscope-objective lenses commonly used in other set ups. Holographic trapping of colloidal particles was successfully conducted at RMIT University for the first time proving the validity of the coding scheme, the recording method and the trapping arrangement.

Optical tweezers using highly focussed laser beams can be used to exert forces and torques and thus drive micromachines. This opens up a new field of microengineering, whose potential has yet to be fully realized. Until now, methods that have been used for modelling optical tweezers are limited to scatterers that are homogeneous or that have simple geometry. To aid in designing more general micromachines, I developed and implemented two main methods for modelling the micromachines that we use. These methods can be used for further proposed structures to be fabricated. The first is a FDFD/T-matrix hybrid method that incorporates the finite difference frequency domain (FDFD) method, which is used for inhomogeneous and anisotropic media, with vector spherical wave functions (VSWF) to formulate the T-matrix. The T-matrix is then used to calculate the torque of the trapped vaterite sphere, which is apparently composed of birefringent unit crystals but the bulk structure appears to be arranged in a sheaf-of-wheat fashion. The second method is formulating the T-matrix via discrete dipole approximation (DDA) of complex arbitrarily shaped mesoscale objects and implementing symmetry optimizations to allow calculations to be performed on high-end desktop PCs that are otherwise impractical due to memory requirements and calculation time. This method was applied to modelling microrotors. The T-matrix represents the scattering properties of an object for a given wavelength. Once it is calculated, subsequent calculations with different illumination conditions can be performed rapidly. This thesis also deals with studies of other light scattering phenomena including the modelling of scattered fields from protein molecules subsequently used to model FRET resonance, determining the limits of trappability, interferometric Brownian motion and the comparison between integral transforms by direct numerical integration and overdetermined point-matching.

Optical tweezers using highly focussed laser beams can be used to exert forces and torques and thus drive micromachines. This opens up a new field of microengineering, whose potential has yet to be fully realized. Until now, methods that have been used for modelling optical tweezers are limited to scatterers that are homogeneous or that have simple geometry. To aid in designing more general micromachines, I developed and implemented two main methods for modelling the micromachines that we use. These methods can be used for further proposed structures to be fabricated. The first is a FDFD/T-matrix hybrid method that incorporates the finite difference frequency domain (FDFD) method, which is used for inhomogeneous and anisotropic media, with vector spherical wave functions (VSWF) to formulate the T-matrix. The T-matrix is then used to calculate the torque of the trapped vaterite sphere, which is apparently composed of birefringent unit crystals but the bulk structure appears to be arranged in a sheaf-of-wheat fashion. The second method is formulating the T-matrix via discrete dipole approximation (DDA) of complex arbitrarily shaped mesoscale objects and implementing symmetry optimizations to allow calculations to be performed on high-end desktop PCs that are otherwise impractical due to memory requirements and calculation time. This method was applied to modelling microrotors. The T-matrix represents the scattering properties of an object for a given wavelength. Once it is calculated, subsequent calculations with different illumination conditions can be performed rapidly. This thesis also deals with studies of other light scattering phenomena including the modelling of scattered fields from protein molecules subsequently used to model FRET resonance, determining the limits of trappability, interferometric Brownian motion and the comparison between integral transforms by direct numerical integration and overdetermined point-matching.

Following the first demonstration in 1987 by Arthur Ashkin of trapping of biological objects with infrared laser light, optical tweezers have become increasingly useful and versatile tool in a variety of non-contact micromanipulation experiments in biological applications. In this thesis we demonstrated various applications of optical tweezers in botanical sciences, chemical engineering and anatomical sciences. The investigation of the three-dimensional shape of spinach chloroplasts has been accomplished. This was done using a steerable and a stationary trap system. A trapped rotating calcite crystal positioned close to a chloroplast provided means for inducing the rotation and orientation of chloroplast. The utility of rotating birefringent particles is demonstrated for the first time in biological applications. The stirrer method is a versatile method in orienting any biological object to study its shape and/or structure. Also, we demonstrated the ability of optical tweezers to fix and displace chloroplasts inside a living spinach plant cell. In the second part of the work described in this thesis, the steerable trap was used to study the viscoelastic properties of a polymeric filament that connects a single bacterium to an activated sludge floc. Also we estimated the minimum bonding force that can cause a weak interaction between the bacterium surface and the filament using optical tweezers as a transducer. This force was estimated to be at least 10 pN. These measurements are of value in improving activated sludge flocculation and ultimately the wastewater treatment process. In addition, the steerable trap was used to move small organelles inside large bacteria cells. The repositioning of organelles resulted in creating new internal cell structure. In the final part of the thesis, experiments are described where the laser tweezers system was combined with a cw argon-ion laser microbeam to investigate the fusion of smooth muscle cells and macrophages. In order to minimize the optical damage to the cells, a special arrangement was established to create short pulses for cutting the contact of the cell membrane of the two-fusion cell partners. The effectiveness of the cutting function of the pulsed system when used at 488 nm wavelength varied from cell to cell. The laser parameters such as laser power, pulse duration and repetition rate were varied in order to obtain the best working function of the setup. But overall the results indicate that the relatively long (ms) pulses possible may not be well suited to such applications.

xii, 138 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Controlling colloidal interactions continues to receive a great deal of attention due both to basic scientific interests as well as industrial applications. However, many aspects of interactions between microparticles remain poorly understood, including the attraction observed between particles with the same kind of charge (like-charge attraction). This situation hinders progress in the generation of colloidal self-assembled structures. This thesis focuses on measurements of pair interactions of functionalized silica microspheres and the resulting insights into colloidal interactions. Silica microparticles were functionalized in two ways. For one method, each particle was coated with a lipid inlayer membrane. The charge density of the particle surface can thereby be easily tuned by controlling the type or amount of charged lipids. For the other method, the cholera toxin subunit B protein (CTB) was bound to lipid-functionalized microparticles. To measure pair interactions, we invented a line optical trap that enables nearly free one-dimensional Brownian motion of particles. Pair interaction energies of functionalized particles above the bottom of the experimental chamber can be extracted via a Boltzmann relationship. Both lipid-only and lipid-plus-protein functionalized microparticles show tunable, attractive pair interactions. For lipid-only coatings, the attraction becomes stronger by increasing the fraction of positively charged lipids. There is a linear relationship between pair potential and molar percentage of positively charged lipids. For lipid-plus-protein coatings, attractive potentials were weakened monotonically by binding more CTB. Decompositions of potential curves allow identification of directly charge-dependent and charge-independent contributions to colloidal like-charge attraction. Analysis shows that the correlations between attraction strength and range are opposite in these two sets of particles. Moreover, the correlations between particle-wall separation and attraction strength in lipid-only and lipid-plus-protein functionalized particles are also opposite. These comparisons show that like-charge attraction may result from more than one mechanism. Finally, we measured pair potential energies of lipid functionalized silica particles above a lipid functionalized glass chamber bottom, which exhibit a quadratic relationship between the attraction strength and the fraction of positively charged lipids. Compared with the situation of particle functionalization only, this relation indicates that confinement-induced like-charge attraction can be modulated by altering electrostatic properties of the confining wall. / Committee in charge: Dr. Stephen D. Kevan, Chair; Dr. Raghuveer Parthasarathy, Advisor; Dr. Hailin Wang; Dr. Miriam Deutsch; Dr. Marina G. Guenza

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.