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.

The main focus of this thesis is on photochemical modulation of molecular recognition in various host-guest systems. This involves the design, synthesis and integration of light-triggered conformational switches into peptidomimetic guests and molecular tweezer hosts. The impact of the switches on guest and host structures has been assessed by spectroscopic and computational conformational analysis. Effects of photochemical structure modulation on molecular recognition in protein-ligand and supramolecular host-guest systems are discussed. Phototriggerable peptidomimetic inhibitors of the enzyme M. tuberculosis ribonucleotide reductase (RNR) were obtained by incorporation of a stilbene based amino acid moiety into oligopeptides between 3-9 residues long (Paper I). Interstrand hydrogen bond probability in the E and Z forms of the peptidomimetics was used as a tool for predicting conformational preferences. Considerable differences in inhibitory potency for the E and Z photoisomers were demonstrated in a binding assay. In order to advance the concept of photomodulable inhibitors, synthetic routes towards amino acid derivatives based on the more rigid stiff-stilbene chromophore were developed (Paper II).  The effect of E-Z isomerization on the conformational properties of peptidomimetic inhibitors incorporating the stiff-stilbene chromophore was also assessed computationally (Paper III). It was indicated that inhibitors with the more rigid amino acid derivative should display larger conformational divergence between photoisomers than corresponding stilbene derivatives. Bisporphyrin tweezers with enediyne and stiff-stilbene spacers have been synthesized, and the conformational characteristics imposed by the spacers have been studied and compared to a glycoluril linked tweezer. The effects of spacers on tweezer binding of diamine guests and helicity induction by chiral guests have been investigated (Paper IV). Connections between spacer flexibility and host-guest binding strength have been established. The structural properties of the stiff-stilbene spaced tweezer made it particularly susceptible to helicity induction by both monotopic and bitopic chiral guests. Finally, the possibility of photochemical bite-size variation of tweezers with photoswitchable spacers has been assessed. Initial studies have shown that photoisomerization of the tweezers is possible without photochemical decomposition. Conformational analyses indicate that isomerization should impact binding characteristics of the tweezers to a significant extent (Paper V).

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.

Proteomic studies at the single molecular level could provide better understanding of the protein’s behaviour and the effects of its interactions with other biomolecules. This could have an impact on drug development methods, disease diagnosis, and targeted therapy. Aperture assisted optical trapping is a proven technique for isolating single proteins in solution without the use of tethers or labels, and without denaturing them. Thus enabling studies of protein-protein interactions, protein-small molecule interactions, and protein-DNA interactions. In this work, double nanohole (DNH) optical tweezers were used to analyze the protein composition of heterogeneous mixtures. The trapped proteins were grouped by molecular mass based on two metrics: standard deviation of the trapping laser intensity fluctuations, and the time constant of the autocorrelation function of these fluctuations. The quantitative analysis is demonstrated first for two separate standard-size proteins, then for a mixed solution of both. Finally, the approach is applied to real unprocessed egg white solution. The results correspond well with the known protein composition of egg white found in the literature. The DNH optical tweezers’ ability to distinguish proteins in unpurified heterogeneous mixtures, can progress this technique to the next level, allowing for single biomolecular studies of unprocessed physiological solutions like blood, urine, or saliva. / Graduate

Orientador: Hugo Enrique Hernandez Figueroa, Michel zamboni Rached / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-14T09:52:49Z (GMT). No. of bitstreams: 1 Ambrosio_LeonardoAndre_D.pdf: 7134713 bytes, checksum: 36a77f1e2e7f49fb16e793969325d64e (MD5) Previous issue date: 2009 / Resumo: Nesta tese, abordamos aplicações de feixes localizados em FSO - Free Space Optics - e em pinças ópticas, com ênfase maior para o segundo. No primeiro caso, mostramos que é possível pré-determinar o padrão de intensidade longitudinal através de elementos ópticos adequadamente modelados em suas funções de fase: os áxicons. Assim, estes feixes poderiam ser usados para alinhar o link. No caso de pinças ópticas, exploramos a idéia de que, em breve, será possível a contrução de partículas esféricas homogêneas, na escala micrométrica, com índice de refração negativo (as chamadas DNG particles, ou Double-Negative particles), e verificamos as propriedades de aprisionamento óptico tanto para feixes gaussianos quanto para feixes localizados, no regime de óptica geométrica e no caso mais geral da teoria eletromagnética. A idéia de que partículas são atraídas para regiões de alta intensidade quando seu índice de refração é maior do que o do meio, e para regiões de baixa intensidade quando este índice é menor, embora válida para partículas convencionais - aquelas com índice de refração positivo -, deve ser revista para partículas DNG. / Abstract: In this thesis, we explore some applications of localized beams in FSO - Free Space Optics - and optical tweezers, greater emphasis been given to the second one. For FSO, we show that it is possible to choose the desired longitudinal intensity pattern by using optical elements adequately modeled in their phase functions: the axicons. In this way, these beams could be uses for optical alignment of the link. In the case of optical tweezers, we investigate the possibility that it will soon be possible to design and build homogeneous spherical particles, in the micron scale, with negative refractive index (the so called DNG particles, or Double-Negative particles), and we verify some properties related to optical trapping, both for Gaussian and Bessel beams, in the optics ray regime and in the more general electromagnetic case. The idea that particles with refractive index higher than the medium in which it is immersed is attracted to regions of high intensity, whereas it is attracted to regions of low intensity when its refractive index is lower than the medium, although valid for conventional particles - those with positive refractive index - must be revisited for DNG particles. / Doutorado / Telecomunicações e Telemática / Mestre em Engenharia Elétrica

Orientadores: Maria Lourdes Barjas-Castro, Carlos Lenz Cesar / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T15:31:46Z (GMT). No. of bitstreams: 1 Fernandes_HeloisePockel_M.pdf: 11930391 bytes, checksum: ee8655790477f44b4f7a68132905dc0c (MD5) Previous issue date: 2009 / Resumo: A membrana eritrocitária contém proteínas e glicoproteínas imersas em uma bicamada lipídica que possui um comportamento viscoelástico. Algumas glicoproteínas contém ácido siálico, que é o principal responsável pelas cargas negativas na superfície da hemácia que quando em solução cria um potencial elétrico (Ç) repulsivo. A carga elétrica negativa da superfície eritrocitária influencia na distribuição dos íons da solução ao redor da célula formando uma dupla camada de íons. A primeira, conhecida como camada compacta de cargas ou "Stern" é formada por íons rigidamente ligados à hemácia e a segunda camada é composta por íons distribuídos difusamente e conhecida como camada difusa. O objetivo deste estudo foi medir o potencial zeta (Ç), a espessura da dupla camada das cargas iónicas (DLC) ao redor da hemácia, e a força de agregação eritrocitária com diferentes meios potencializadores da aglutinação utilizando pinça óptica. (...continua) / Abstract: The red blood cell (RBC) membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that confers viscoelastic behavior. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface, which creates a repulsive electric zeta potential (Z) between the cells.The compact layer of charge or Stern consists of ions rigidly bonded to the cell and the double layer includes ions diffusely distributed around the cell. The aim of this study was to measure the RBC double layer thickness of the charge (DLC) around the cell, zeta potential (Z) and cell aggregation force in agglutination potentiator solutions, using optical tweezers. (¿to be continue) / Mestrado / Mestre em Farmacologia