Ce mémoire rapporte deux approches de la synthèse des protéines à l'échelle de la molécule unique. Nous utilisons la microscopie de fluorescence en onde évanescente pour sonder l'activité traductionnelle de deux types de ribosomes. Les premiers, issus d'E. Coli (organisme procaryote), sont mutés afin de les marquer d'un nanocristal semiconducteur (QD). La fin de la traduction, qui correspond au décrochage du ribosome de l'ARNm lorsque celui-ci atteint le codon stop, est alors mise en évidence par la disparition du QD de la surface de l'échantillon. Le deuxième type de ribosome étudié est quant à lui extrait de cellules de lapins (organisme eucaryote) et est dit "sauvage", c'est à dire qu'il n'a pas subi de modification, tandis qu'un oligonucléotide marqué d'un fluorophore est hybridé à l'ARNm. L'activité hélicase du ribosome lui permettant de séparer deux brins complémentaires, l'oligonucléotide et donc le fluorophore disparaissent en même temps que le ribosome parcourt l'ARNm, permettant ainsi de sonder l'activité du ribosome. Nous donnons pour ces deux types de ribosomes une vitesse moyenne de la traduction dans des milieux contenant les facteurs de la traduction issus d'extraits cellulaires.La deuxième approche de la synthèse des protéines porte sur les propriétés de l'ARNm, support de l'information génétique codant pour la séquence des protéines. Nous avons développé un montage de pince optique permettant de manipuler et caractériser les propriétés mécaniques d'oligonucléotides, ainsi qu'une méthode originale de calibration de ce piège optique. La cohérence de nos mesures sur l'étirement d'un double brin d'ADN avec la littérature nous permettra de poursuivre notre étude sur la mesure des forces nécessaires pour ouvrir une structure secondaire de l'ARNm. / We hereby report two approaches of the protein synthesis at the single molecule level. We use total internal reflection fluorescence microscopy to study the translation kinetic of two different types of ribosomes. The first ones, extracted from E. Coli (prokaryotic organism), are mutated in order to label them with a quantum dot (QD). The end of translation, which corresponds to the dissociation of the ribosome from the mRNA when the stop codon has been reached, is highlighted by the disparition of the QD from the surface. The second type of ribosome is extracted from rabbit cells (eukaryotic organism) and has not been modified (wild type), while a labeled oligonucleotide is hybridized on the mRNA. The helicase activity of the ribosome allowing the dissociation of two complementary strands, the oligonucleotide and so the label disappear at the same time while the ribosome moves along the mRNA and thus inform us about its activity. For these two types of ribosomes we measure their average translation speed in cell extracts.The second approach focuses on the properties of the mRNA, carrying the genetic code for the protein sequence. We developped an optical tweezer setup in order to manipulate and characterize the mechanical properties of nucleotides, as well as an original method to calibrate this optical trap. The consistency of our measurements with the litterature on the properties of a double stranded DNA will allow us to study secondary structures of mRNA.

Um sistema multi-feixe de pinças ópticas baseado na técnica de contraste de fase da ordem zero pode apresentar vantagens significativas sobre sistemas mecanicamente complexos e sensíveis ao alinhamento, e sobre tecnologias que, apesar de serem similares, requerem a customização de componentes ópticos. Porém, ao nosso conhecimento, este sistema até agora não tem sido implementado experimentalmente. Neste trabalho tem-se desenvolvido, como prova de princípio, o primeiro sistema baseado na técnica de contraste de fase da ordem zero gerador de múltiplas pinças ópticas. Esta técnica da óptica de Fourier utiliza conceitos do contraste de fase de Zernike e técnicas de codificação de dois pixels para gerar padrões de intensidade no plano da imagem que são diretamente relacionados a distribuições de fase no plano de entrada do sistema, o qual é formado por um modulador espacial de luz (SLM). Esta dissertação de mestrado descreve detalhadamente os passos tomados com o propósito de utilizar os campos estruturados de luz gerados pelo sistema de contraste de fase da ordem zero para aprisionar esferas de 2 µm de diâmetro de sílica fundida. Neste trabalho apresentamos os fundamentos teóricos do aprisionamento óptico e da técnica de contraste de fase da ordem zero, seguidos pela implementação de experimentos independentes em cada modalidade, e finalmente apresentamos a integração de ambos os sistemas dentro um sistema único de pinças ópticas multi-feixe. Apesar da baixa eficiência óptica do sistema, foi possível implementar um sistema de pinças ópticas duplas. Finalizamos o nosso trabalho na discussão detalhada das limitações do nosso arranjo óptico e comentamos sobre potenciais melhorias para aumentar a rigidez das pinças ópticas e a qualidade geral do sistema. / A multi-beam optical trapping system based on the zero order phase contrast technique may offer significant advantages over mechanically-complex, alignment-sensitive optical trapping systems, and over technologies that, though similar, require the customization of optics components. However, to our knowledge, such a system has not been yet implemented experimentally. We have developed, as a proof of principle, what we think is the first system based on the zero order phase contrast technique to successfully generate multiple optical traps. This Fourier optics technique makes use of existing concepts of Zernike phase contrast and two-pixel encoding techniques to generate intensity patterns in the image plane that are directly related to phase distributions in the input plane, which is comprised by a spatial light modulator (SLM). This master\'s dissertation describes in detail the steps taken towards using the structured light fields generated by a zero order phase contrast system to trap 2 µm diameter fused silica beads. We present the theoretical foundations of optical trapping and the zero order phase contrast technique, followed by the implementation of independent laboratory experiments in each modality, and finally integrate both systems into a single optical setup for multi-beam trapping. In spite of the low optical efficiency of the system, we were able to implement dual optical traps. We finalize by discussing in detail the limitations of our experimental setup in and comment on potential improvements to increase the stiffness of the optical traps and the overall quality of the system.

Este trabalho é uma contribuição às análises de forças de aprisionamento óptico exercidas sobre partículas esféricas por superposições discretas de feixes de Bessel. Um estudo teórico-numérico foi realizado no regime de óptica geométrica, completando as pesquisas já realizadas com tais feixes e no caso escalar, tanto no regime de Rayleigh quanto através de um formalismo eletromagnético completo. Investigam-se padrões longitudinais de intensidade de possível interesse prático, com potenciais de fornecer múltiplas armadilhas ópticas simultâneas utilizando dois métodos de análise. O primeiro parte da observação de que no regime paraxial todos os raios associados aos feixes se encontram quase paralelos entre si quando se toma como aproximação uma superposição de raios paralelos que incidem completamente sobre um hemisfério do espalhador. Tal método, entretanto, é naturalmente restrito pelas forças transversais. O segundo, que torna mais confiáveis e precisas as predições acerca da componente longitudinal de força, adota procedimentos mais robustos que levam em consideração tanto a contribuição da pressão de radiação (força de espalhamento) quanto a força gradiente devido a gradientes locais de intensidade associados a raios não-paralelos. Assim, acredita-se que este trabalho traz como contribuição um reforço a esta classe específica e promissora de feixes não difrativos como feixes de luz interessantes para aplicações em aprisionamento e micromanipulação óptica. / This work is a contribution to the analyses of optical trapping forces exerted on spherical particles by discrete superpositions of Bessel beams. A theoretical-numerical study has been carried out in the ray optics regime, completing the pre-existing research performed with such beams, in the scalar case, both in the Rayleigh regime and through a complete electromagnetic formalism. We investigate longitudinal intensity patterns of possible practical interest with potential to provide multiple simultaneous optical traps, by using two methods of analysis. The first assumes that, in the paraxial regime, all rays associated to the beams are almost parallel to each other, taking a superposition of parallel rays that are completely incident on a hemisphere of the scatterer as a suitable approximation. Such a method, however, is naturally constrained by the transverse forces. The second one, which makes the predictions about the longitudinal force component more reliable and accurate, adopts more robust procedures that take into consideration the contribution of the radiation pressure (scattering force) as well as the gradient force due to local intensity gradients associated to non-parallel rays. Thus it is hoped that this work will contribute to reinforce this specific and promising class of non-diffractive beams as interesting light beams for applications in optical trapping and micromanipulation.

Ce travail de thèse est principalement axé sur le développement d’une nouvelle méthode de manipulation de vortex d’Abrikosov individuels dans les supraconducteurs de type II. Cette méthode, rapide, efficace et précise, est basée sur l’optique en champ lointain et repose sur l’échauffement local du supraconducteur sous l’action d’un faisceau laser focalisé. Elle apporte une excellente alternative aux techniques existantes de manipulation de vortex, toutes basées sur l’utilisation de sondes locales, et donc intrinsèquement lentes et difficiles à mettre en oeuvre dans un environnement cryogénique. La combinaison de cette méthode à une technique d’imagerie magnéto-optique performante permet de déplacer des vortex individuels avec un taux de réussite de 100% et sur de grandes échelles limitées uniquement par le champ de l’objectif de microscope. Les vitesses de manipulation atteintes sont élevées, de l’ordre de 10 mm.s-1, mais encore limitées par l’instrumentation utilisée et loin des limites fondamentales offertes par cette méthode, estimées au km.s-1. La méthode de manipulation optique permet aussi de mesurer la distribution des forces de piégeage de chaque vortex d’un échantillon. En utilisant des puissances de chauffage laser permettant de dépasser localement la température critique, nous avons également pu étudier la pénétration des vortex à l’interface entre une zone normale et une zone supraconductrice.Durant ces travaux, nous avons aussi eu l’opportunité de mettre en évidence, par spectroscopie de molécules uniques, l’effet flexomagnétoélectrique dans un matériau multiferroïque, en employant un supraconducteur de type I comme générateur de champ magnétique inhomogène. Enfin, nous proposons à la fin de ce mémoire un concept de jonction Josephson créée tout optiquement, et dont les propriétés seraient contrôlables en temps réel par laser. / This thesis focuses on the development of a new manipulation technique to handle single Abrikosov vortices in type II superconductors. This fast, efficient and precise method is based on far field optics and rests on the local temperature elevation produced by a focused laser beam. It brings an excellent alternative to the existing techniques which are all based on local probes and thus heavy to implement in a cryogenic environment. The combination of this method with an efficient magneto-optical imaging system allows us to manipulate single vortices with a 100% rate of success on a large scale only limited by the field of view of the microscope objective. Manipulation speeds are high, of the order of 10 mm.s-1, but still limited by our setup and far from the fundamental limits offered by this technique, estimated to the km.s-1. This manipulation technique also allows to measure the pinning force of any single vortex in a superconducting sample. By using a high enough laser power which locally pushes the temperature above the critical temperature, we could also study the vortex penetration at the interface between normal and superconducting areas.In the course of this work, we also evidenced, with single molecule spectroscopy, the flexomagnetoelectric effect in a multiferoic material, by using a type I superconductor as a source of inhomogeneous magnetic field. Finally, we propose at the end of the manuscript the new concept of an optically created Josephson junctions, whose properties could be controlled in real time just with a laser beam.

After an intense development of optical tweezers as a biophysical tool during the last decades, quantitative experiments in living cells have not found in this technique its best ally, due, in part, to the lack of a reliable method to measure forces in complex environments. The attempts to overcome this problem either require complicated in situ calibrations, which make their use impossible in the study of dynamic processes, or they are inaccurate. Using a different approach, Steven Smith at Carlos Bustamante’s lab at the University of Berkeley developed a method based on the direct measurement of the momentum change of the trapping beam. However, its diffusion has been modest mainly because it requires a counter-propagating optical trapping system, which is difficult to implement and combine with other techniques. Although it has not been used for this purpose yet, it seems a more suitable method for in vivo experiments since the measurement depends only on some properties of the sensor apparatus but not on the experiment itself. On the other hand, the use of holographic optical tweezers in molecular biology experiments involving force and position measurements is still far from established. The existence of different effects associated to the use of spatial light modulators to create the optical traps has restricted their use. In this thesis, I present the work that I carried out in the Optical Trapping Lab – Grup de Biofotònica at the University of Barcelona related to these two subjects. During these years, I have focused on the implementation of the force detection method based on the conservation of the light momentum in single-beam optical traps, and on the analysis of several aspects of holographic tweezers oriented to their use in quantitative experiments.

This dissertation details our experiments on studying the Brownian motion of an optically trapped microsphere with ultrahigh resolution, and cooling of its motion towards the quantum ground state. We have trapped glass microspheres in water, air and vacuum with optical tweezers. We developed a detection system that can monitor the position of a trapped microsphere with Angstrom spatial resolution and microsecond temporal resolution. We studied the Brownian motion of a trapped microsphere in air over a wide range of pressures. We measured the instantaneous velocity of a Brownian particle. Our results provide direct verification of the Maxwell-Boltzmann velocity distribution and the energy equipartition theorem for a Brownian particle. For short time scales, the ballistic regime of Brownian motion is observed, in contrast to the usual diffusive regime. We are currently developing a new detection system to measure the instantaneous velocity of a Brownian particle in water. In vacuum, we have used active feedback to cool the three center-of-mass vibration modes of a trapped microsphere from room temperature to millikelvin temperatures with a minimum mode temperature of 1.5 mK, which corresponds to the reduction of the root mean square (rms) amplitude of the microsphere from 6.7 nm to 15 pm for that mode. The mean thermal occupation number of that mode is reduced from about 6.8$\times 10^8$ at 297 K to about 3400 at 1.5 mK. / text

The thesis details the design and characterization of an innovative optical tweezer system. Optical tweezers provide a relatively new technique for non-contact manipulation of micron-scale particles. They employ a laser beam to hold such particles at the laser’s focus. Optical tweezers are used for many scientific purposes, such as: measuring the mechanical properties of bio-molecules, cell and molecule sorting, stiction-less micro-manipulators, and fundamental research in physics. Typically, trap location has been controlled using steer-mirrors or spatial light modulators, operating without beam quality feedback. Here, an innovative trap control system has been developed, featuring a closed-loop adaptive optics system. The prototype system employs a deformable mirror and wavefront sensor to control trap position in three dimensions, while simultaneously removing beam aberrations. The performance of this system is investigated in terms of controllable range of trap motion, trap stiffness, and trap position stability.

No description available.

Dans la première partie de cette thèse, nous étudions la cinétique de la complexation d'un double brin d'ADN par la protéine NCp7. Pour ce faire, nous étudions l'évolution des propriétés mécaniques de l'ADN au fur et à mesure de sa complexation, en étirant la complexe ADN/NCp7 à l'aide d'un montage de piégeage optique. Nous avons observé que la longueur de persistance du complexe diminue au fur et à mesure de la complexation. En utilisant un modèle statistique décrivant l'évolution de la flexibilité de l'ADN complexé par NCp7. Notre principal résultat est que la fraction//phi de paires de bases ayant réagi n'est pas une fonction linéaire du temps aux faibles //phi. Nous interprétons nos résultats en supposant que l'adsorption de NCp7 sur l'ADN est fortement coopérative. Dans deuxième chapitre, nous décrivons la dynamique de particules sondes dans une suspension vitreuse colloïdale de Laponite. La Laponite est une particule colloïdale discoïdale de 25nm de diamètre et de 0.92 nm d'épaisseur. Nous utilisons une expérience de microscopie en onde évanescente, et suivons le mouvement de particules fluorescentes de latex. Nous imageons ensuite ces particules. Nous montrons que, pour un mouvement possédant une seule échelle de temps caractéristique, elle est simplement une fonction linéaire du temps. Nous obtenons que, quelle que soit leur taille, le mouvement des particules sondes peut être décrit par une succession de deux modes dynamiques, où le mode le plus rapide correspond à la diffusion des particules dans un fluide viscoélastique. / In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.

Made available in DSpace on 2015-03-26T13:35:18Z (GMT). No. of bitstreams: 1 texto completo.pdf: 5613341 bytes, checksum: b10dc863b3516fce568d5d4513b2ee67 (MD5) Previous issue date: 2012-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this work, by using the optical tweezers technique, it was possible to study the changes on the mechanical properties of DNA - cisplatin complexes, as a function of some parameters such as the drug diffusion time and its concentration in the sample. A model was proposed in order to explain the behavior of the persistent length as a function of drug concentration, using only the data obtained from single molecule stretching experiments. Such analysis allow us to show that cisplatin binds cooperatively to the DNA molecule. In addition, DNA molecule compactation by the action of the drug, was characterized from studying the kinetics of some mechanical properties such as the radius of gyration and the maximum average end - to - end distance. / Neste trabalho, utilizando a técnica de pinçamento óptico, foi possível estudar as mudanças nas propriedades mecânicas dos complexos DNA - cisplatina, em função de alguns parâmetros de interesse, tais como: o tempo de difusão do fármaco e a concentração do fármaco na amostra. Um modelo foi proposto para explicar o comportamento do comprimento de persistência em função da concentração do fármaco, utilizando-se apenas os dados obtidos a partir dos experimentos de estiramento. Tal análise nos permitiu mostrar que a cisplatina se liga cooperativamente à molécula de DNA. Além disso, a compactação da molécula de DNA pela ação da cisplatina foi caracterizada a partir do estudo da cinética de algumas propriedades mecânicas, tais como: o raio de giro e a distância máxima ponta - à - ponta dos complexos.