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

Orientador: Carlos Lenz Cesar / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-11T02:36:11Z (GMT). No. of bitstreams: 1 Neves_AntonioAlvaroRanha_D.pdf: 9709693 bytes, checksum: 0a918ae7eeaa5895a391c7465942bdd2 (MD5) Previous issue date: 2006 / Resumo: A pinça óptica é um instrumento capaz de manipular e aprisionar partículas dielétricas por meio da pressão de radiação. Suas aplicações nas ciências da vida e biofísica cresceram exponencialmente após a demonstração de que ela permitia manter vivos microorganismos capturados por longos tempos. Informações obtidas destes experimentos requerem um transdutor de força, para o qual se utiliza o deslocamento de uma microesfera capturada. Estamos portanto trabalhando no limite dos regimes de óptica geométrica e Rayleigh, que geralmente são utilizados para simplificar a força óptica. Até hoje não existe consenso entre as teorias das forças, para um regime de tamanho arbitrário, nas pinças ópticas nem para sistemas de alta simetria como microesferas, muito menos em geometrias mais complicadas. Uma das maiores dificuldades encontradas nesse aspecto é a ausência de boas medidas experimentais das forças ópticas, independentes de modelos. Por isso grande parte do trabalho dessa tese foi o desenvolvimento de um sistema de medidas de forças ópticas utilizando pinças duplas para obtenção de toda uma curva da força em função do deslocamento tridimensional da partícula capturada, e não apenas os valores da força em posições fixas. A segunda grande contribuição vem da descrição teórica da força óptica. A grande dificuldade nesse aspecto é a descrição de um feixe incidente de grande abertura numérica e sua decomposição em ondas parciais. É nesse contexto que se encaixa esse trabalho de tese. Acreditamos ter dado uma contribuição extremamente valiosa resolvendo de forma analítica e exata o problema da decomposição de um feixe convergente em ondas parciais relativas a qualquer origem do sistema de coordenadas em três dimensões. / Abstract: The optical tweezers is an instrument capable of manipulating and trapping dielectric particles through the radiation pressure. Their applications in the life sciences and biophysics increased exponentially after it has been demonstrated that it allowed to microorganisms to be maintained alive trapped for long times. Information obtained from these experiments requires a force transducer, for which the displacement of the captured micro sphere is used. We are therefore working in the limit of the geometrical optics and Rayleigh regime, which are usually used to simplify the optical force. Until today consensus fails to exist among the theories of forces for optical tweezers, of an arbitrary size regime, neither for systems of high symmetry as micro spheres, much less in more complicated geometries. One of the greatest difficulties encountered in this aspect is the absence of good experimental measurements of optical forces, independent of models. Therefore great part of this thesis was the development of a system capable of measuring optical forces using a double tweezers setup to obtain an entire curve of the force as a function of the three-dimensional displacement of the trapped particle, and not just the values of the force for fixed positions. The next grand contribution comes from the theoretical description of the optical force. The large difficulty in this aspect is the description of incident beams of great numerical aperture and its decomposition in partial waves. It is in this context that this thesis fits in. We believed to have given an extremely valuable contribution, solving in an analytical and exact way the problem of the decomposition of a convergent beam partial waves relative the any origin of the three dimensional coordinate system. / Doutorado / Física / Doutor em Ciências

Orientador: René Alfonso Nome Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T04:23:53Z (GMT). No. of bitstreams: 1 Zornio_BrunoFedosse_M.pdf: 1493558 bytes, checksum: 07565e7077eefa5f9f91f03dc9cc90d4 (MD5) Previous issue date: 2014 / Resumo: No final do século XIX Van¿tHoff empiricamente estabeleceu que a constante de velocidade de uma reação química é função exponencial da razão entre energia de ativação da reação pela energia térmica do ambiente (e portanto função da temperatura). Há uma variada ordem nas escalas de tempos reacionais, em especial, a constante de velocidade de reações lentas (por exemplo, reações bioquímicas não catalisadas) é difícil de determinar. Uma partícula difundindo em um meio viscoso que apresenta movimento aleatório ¿ com posição média (em um intervalo de tempo suficientemente grande) nula, e a variância da posição linearmente dependente em função do tempo ¿ é dita browniana, e quando submetida a um potencial bi quadrático é um bom modelo para descrição de reações químicas. A partir da dinâmica de Langevin (que serve para descrever a dinâmica de uma partícula browniana) é derivada a teoria de Kramers para meio viscosos - que relaciona o formato da curva potencial com a constante de taxa de reações químicas -. Experimentalmente pode-se recriar esse modelo utilizando pinças ópticas. Pinças ópticas são capazes de aprisionar partículas da ordem micrométricas em suspensão, pode-se recriar um potencial bi estávelutilizando uma pinça óptica dupla (com dois pontos de aprisionamento). Este estudo tem como objetivo avaliar a constante de taxa de um processo de transição entre poços de potencial de partículas brownianas teoricamente utilizando uma simulação de dinâmica de Langevin para sistemas em equilíbrio tanto quanto para sistemas antes de atingir o equilíbrio, assim como determinar experimentalmente utilizando microscopia ocoeficiente de difusão a partir da trajetória temporal de uma única partícula. Os resultados teóricos obtidos são bastante condizentes com os resultados experimentais descritos na literatura, assim como as predições da constante de taxa para tempos antes do equilíbrio apresentam correlação com o sistema em equilíbrio. Com relação à estimativa do coeficiente de difusão apresenta um erro sistemático associado ao tamanho da trajetória temporal de uma única partícula / Abstract: By the end of the XIX century, Van¿t¿ Hoff has empirically established that the rate constant of some chemical reaction is exponentially dependent by the ratio between the reaction activation energy and the environment thermal energy (and so on function of temperature). There is a wide variety in the reaction time scales, in particular, the rate constant of slow reactions (such as uncatalysed biochemical reactions) is difficult to determine. A diffusing particle in a viscous media which exhibit random motion ¿ with mean position (in a sufficiently large time series) is zero, and the position variance is linearly time dependent ¿ is called Brownian, and when is submitted in a biquadratic potential it¿s a good model to describe chemical reactions. By the Langevin dynamics (which serves to describe the Brownian particle motion) the Kramers theory for viscous media is derived ¿ that theory connects the potential energy shape with the chemical rate constant -. Experimentally it is possible to create this model using optical tweezes. Optical tweezers where capable to trap micrometrical beads in suspension, it can generate a bi-stable using a double optical tweezers (that is with two trapping points). The main objective of this essay is evaluate the rate constant a Brownian particles jumping between potential wells theoretically using Langevin dynamics simulations for the system at equilibrium and before reach the equilibrium, as determinate experimentally the diffusion coefficient of single particle time path using microscopy. The theoretical results is very consistent with experimental results described in literature, as well as the prediction of the rate constant for the system before reaches equilibrium are correlated with the rate constant for the system at equilibrium. For the diffusion coefficient estimative it was observed that there is a systematical source of errors, and its is related with the length of the time series of the single particle path / Mestrado / Físico-Química / Mestre em Química

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

I advanced the understanding of particle morphology and its implications for the behavior and effects of atmospheric aerosol particles. I have developed new experimental methods for the Aerosol Optical Tweezers (AOT) system and expanded the AOT’s application into studying realistic secondary organic aerosol (SOA) particle phases. The AOT is a highly accurate system developed to study individual particles in real-time for prolonged periods of time. While previous AOT studies have focused on binary or ternary chemical systems, I have investigated complex SOA, and how they interact with other chemical phases, and the surrounding gas-phase. This work has led to new insights into liquid-liquid phase separation and the resulting particle morphology, the surface tension, solubility, and volatility of SOA, and diffusion coefficients of SOA phases. I designed a new aerosol optical tweezers chamber for delivering a uniformly mixed aerosol flow to the trapped droplet’s position. I used this chamber to determine the phase-separation morphology and resulting properties of complex mixed droplets. A series of experiments using simple compounds are presented to establish my ability to use the cavity enhanced Raman spectra to distinguish between homogenous single-phase, and phase-separated core-shell or partially-engulfed morphologies. I have developed a new algorithm for the analysis of whispering gallery modes (WGMs) present in the cavity enhanced Raman spectra retrieved from droplets trapped in the AOT. My algorithm improves the computational scaling when analyzing core-shell droplets (i.e. phase-separated or biphasic droplets) in the AOT, making it computationally practical to analyze spectra collected over many hours at a few Hz. I then demonstrate for the first time the capture and analysis of SOA on a droplet suspended in an AOT. I examined three initial chemical systems of aqueous NaCl, aqueous glycerol, and squalane at ~ 75% relative humidity. For each system I added α-pinene SOA – generated directly in the AOT chamber – to the trapped droplet. The resulting morphology was always observed to be a core of the initial droplet surrounded by a shell of the added SOA. By combining my AOT observations of particle morphology with results from SOA smog chamber experiments, I conclude that the α-pinene SOA shell creates no major diffusion limitations for water, glycerol, and squalane under humid conditions. My AOT experiments highlight the prominence of phase-separated core-shell morphologies for secondary organic aerosols interacting with a range of other chemical phases. The unique analytical capabilities of the aerosol optical tweezers provide a new approach for advancing the understanding of the chemical and physical evolution of complex atmospheric particulate matter, and the important environmental impacts of aerosols on atmospheric chemistry, air quality, human health, and climate change.

L'ARN s'est longtemps vu attribuer un simple role de transmission entre l'ADN, garant de l'information genetique, et les proteines, assurant les fonctions et donc la survie cellulaire. Ce n'est qu'avec les decouvertes des ARNs de transfert dans les annees 70, puis des ribozymes dans les annees 80, qu'il a ete realise que l'ARN pouvait assurer ces deux roles : l'information genetique est stockee dans sa sequence lineaire, et l'adoption de structures tridimensionnelles complexes rend possible une activite catalytique. Depuis, de nouvelles fonctions de l'ARN n'ont cesse d'etre decouvertes, a tous les niveaux de regulation de l'expression genique entre autres. La majorite de ces fonctions repose sur la structuration tridimensionnelle d'ARNs simple brin.Dans ce travail, differents aspects de la structuration de l'ARN sont abordes, toujours en utilisant la technique de mesure de forces sur molecules uniques par piegeage optique. Dans un premier temps, une etude comparative d'une structure secondaire modele, le hairpin dans ses formes ARN et ADN, a ete realisee. La question de l'interaction d'une structure secondaire avec une proteine helicase (DbpA) a ensuite ete abordee. Enfin, dans le cadre plus general d'une etude sur l'assemblage du ribosome, nous avons debute le developpement d'une nouvelle methode d'analyse des structures secondaires. Cette methode repose sur le suretirement d'un hybride ARN ribosomique / ADN. / Traditionally, RNA has been considered as a mere intermediate between DNA, keeper of the genetic information, and proteins, which assume cells self-sustenance. With the discoveries of the transfert RNA in the 70s, and of the ribozymes in the 80s, RNA took on both roles: it can store information in its linear sequence, and tridimensional structuration enables catalytic functions. Since then, numerous roles devoted to RNA have been discovered, particularly for gene expression regulation. Most of these functions rely on tridimensional structuration of single stranded RNA. In this work, we used an optical tweezers setup to study several aspects of RNA structuration by single molecule force measurement. In a first part, we compared the dynamic behaviour of a model secondary structure made of either RNA or DNA, the hairpin. Then we considered the interaction of a secondary structure with a protein, the RNA helicase DbpA. Finally, within a wider study of ribosome assembly, we worked on the development of a new method to study tridimensional structuration. This method relies on the overstretching of a hybrid ribosomal RNA / DNA molecule.