Tesis por compendio / [ES] La necesidad de confinar y manipular micro-objetos tiene aplicaciones en múltiples áreas de la ciencia y tecnología. Actualmente, existen diversas técnicas para lograr este objetivo, y una de las más destacadas es el uso de las llamadas pinzas ópticas, que se han convertido en una herramienta ampliamente utilizada en laboratorios de todo el mundo. Este trabajo de investigación se centra en el fascinante campo del atrapamiento y manipulación de micro-objetos, con un enfoque destacado en la combinación de elementos ópticos difractivos y la técnica de pinzas ópticas. Esta combinación permite un aumento de la versatilidad de los sistemas experimentales de pinzas ópticas. Los avances presentados en esta tesis tienen aplicaciones en una amplia gama de campos, desde la nanotecnología hasta la biología celular. Como lentes difractivas implementadas en los sistemas de pinzas ópticas, se introducen las lentes difractivas Kinoform basadas en la secuencia aperiódica m-Bonacci. Estas lentes permiten atrapar múltiples partículas simultáneamente y manipularlas tridimensionalmente en dos planos focales diferentes, lo que amplía significativamente las posibilidades de investigación y desarrollo en diversas disciplinas. Además, se aborda la generación de múltiples trampas ópticas mediante lentes Kinoform cuadrifocales basadas en otra secuencia aperiódica conocida como Silver Mean, permitiendo atrapar partículas en cuatro planos focales de manera simultánea. Este avance mejora significativamente la versatilidad de los sistemas de pinzas ópticas. Adicionalmente el uso de vórtices multiplexados en un sistema de pinzas ópticas, permite atrapar de manera independiente múltiples partículas y transferir momento angular. Estos avances abren nuevas posibilidades en la construcción de micromotores y aplicaciones de micro-ensamblaje. Un efecto asociado a las trampas ópticas es la generación de microburbujas, en la actualidad estas se han convertido en objeto de estudio debido a la facilidad de generación y a sus posibles aplicaciones como agentes de transporte de partículas o micro-objetos. Aprovechando este efecto en esta tesis se implementa una técnica de atrapamiento que emplea fuerzas termoforéticas en la captura y manipulación de microburbujas en líquidos. Esto constituye otro avance importante en el campo del atrapamiento tridimensional. Por último, se desarrolla un laboratorio virtual utilizando COMSOL Multiphysics para simular el atrapamiento acústico, lo que permite a los estudiantes interactuar con el sistema y comprender mejor este fenómeno. Este enfoque educativo proporciona herramientas valiosas para la comprensión y análisis de la manipulación de partículas, lo que beneficia a los estudiantes de pre-grado y grado interesados en este campo. En conjunto, todos estos avances representan contribuciones significativas en el campo del atrapamiento y manipulación de partículas, en particular a través de las pinzas ópticas, promoviendo el progreso tecnológico y científico en diversas disciplinas y brindando oportunidades educativas para futuras generaciones de investigadores y científicos. A lo largo del desarrollo de esta tesis, se han creado nuevos elementos difractivos que superan ciertas limitaciones y aumentan las capacidades de las pinzas ópticas, abriendo nuevas perspectivas de aplicación para tecnologías preexistentes. / [CA] La necessitat de confinar i manipular microobjectes té aplicacions a múltiples àrees de la ciència i la tecnologia. En l'actualitat, hi ha diverses tècniques per assolir aquest objectiu, i una de les més destacades és l'ús de les anomenades pinces òptiques, que han esdevingut una eina molt utilitzada en laboratoris de tot el món. Aquest treball de recerca se centra en el fascinant camp de la captura i la manipulació de microobjectes, destacant la combinació d'elements òptics difractius i la tècnica de les pinces òptiques. Aquesta combinació permet augmentar la versatilitat dels sistemes experimentals de pinces òptiques. Els avenços presentats en aquesta Tesi tenen aplicacions en una àmplia gamma de camps, des de la nanotecnologia a la biologia cel·lular. Com a lents difractives implementades en sistemes de pinces òptiques, es presenten les lents difractives Kinoform basades en la seqüència aperiòdica m-Bonacci. Aquestes lents permeten atrapar simultàniament múltiples partícules i manipular-les tridimensionalment en dos plans focals diferents, fet que amplia significativament les possibilitats de recerca i desenvolupament en diverses disciplines. A més, s'aborda la generació de múltiples trampes òptiques utilitzant lents Kinoform quadrifocals basades en una altra seqüència aperiòdica coneguda com a Silver Mean, que permet atrapar partícules en quatre plans focals simultàniament. Aquest avenç millora significativament la versatilitat dels sistemes de pinces òptiques. A més, l'ús de vòrtexs multiplexats en un sistema de pinces òptiques permet atrapar múltiples partícules de manera independent i transferir el moment angular. Aquests avenços obren noves possibilitats en la construcció de micromotors i aplicacions de microassemblatge. Un efecte associat a les trampes òptiques és la generació de microbombolles, actualment aquestes s'han convertit en objecte d'estudi a causa de la facilitat de generació i de les seves potencials aplicacions com a agents de transport de partícules o microobjectes. Aprofitant aquest efecte, aquesta Tesi implementa una tècnica d'atrapament que utilitza forces termoforètiques en la captura i manipulació de microbombolles en líquids. Això constitueix un altre avenç important en el camp de l'atrapament tridimensional. Finalment, es desenvolupa un laboratori virtual utilitzant COMSOL Multiphysics per simular l'atrapament acústic, cosa que permet als estudiants interactuar amb el sistema i comprendre millor aquest fenomen. Aquest enfocament educatiu proporciona eines valuoses per a la comprensió i l'anàlisi de la manipulació de partícules, cosa que beneficia els estudiants de grau i postgrau interessats en aquest camp. En conjunt, tots aquests avenços representen contribucions significatives al camp de l'atrapament i la manipulació de partícules, particularment a través de pinces òptiques, promovent el progrés tecnològic i científic en diverses disciplines i proporcionant oportunitats educatives per a futures generacions d'investigadors i científics. Al llarg del desenvolupament d'aquesta Tesi, s'han creat elements difractius nous que superen certes limitacions i augmenten les capacitats de les pinces òptiques, obrint noves perspectives d'aplicació per a tecnologies preexistents. / [EN] The need to confine and manipulate micro-objects has applications in multiple areas of science and technology. Currently, there are several techniques to achieve this goal, and one of the most prominent is the use of the so-called optical tweezers, which have become a widely used tool in laboratories around the world. This research work focuses on the fascinating field of micro-object capture and manipulation, highlighting the combination of diffractive optical elements and the optical tweezers technique. This combination allows to increase the versatility of the experimental optical tweezers systems. The advances presented in this thesis have applications in a wide range of fields, from nanotechnology to cell biology. As diffractive lenses implemented in optical tweezers systems, Kinoform diffractive lenses based on the aperiodic m-Bonacci sequence are presented. These lenses allow multiple particles to be trapped simultaneously and manipulated three-dimensionally in two different focal planes, which significantly expands the possibilities for research and development in various disciplines. In addition, the generation of multiple optical traps is addressed using quadrifocal Kinoform lenses based on another aperiodic sequence known as Silver Mean, which allows particles to be trapped in four focal planes simultaneously. This advance significantly improves the versatility of optical tweezer systems. In addition, the use of multiplexed vortices in an optical tweezer system allows multiple particles to be trapped independently and angular momentum to be transferred. These advances open up new possibilities in micromotor construction and microassembly applications. One effect associated with optical traps is the generation of microbubbles, currently these have become an object of study due to the ease of generation and their potential applications as transport agents for particles or micro-objects. Taking advantage of this effect, this thesis implements an trapping technique that employs thermophoretic forces in the capture and manipulation of microbubbles in liquids. This constitutes another important advance in the field of three-dimensional trapping. Finally, a virtual laboratory is developed using COMSOL Multiphysics to simulate acoustic trapping, allowing students to interact with the system and better understand this phenomenon. This educational approach provides valuable tools for the understanding and analysis of particle manipulation, benefiting undergraduate and graduate students interested in this field. Taken together, all of these advances represent significant contributions to the field of particle trapping and manipulation, particularly through optical tweezers, promoting technological and scientific progress in various disciplines and providing educational opportunities for future generations of researchers and scientists. Throughout the development of this thesis, new diffractive elements have been created that overcome certain limitations and increase the capabilities of optical tweezers, opening new application perspectives for pre-existing technologies. / We acknowledge the financial support from Ministerio de Ciencia e Innovación (grants PID2019-107391RB-I00 and PID2022-1142407NB-I00), Generalitat Valenciana (grant PROMETEO/2019/048 and CI-PROM/2022/30), and Universitat Politècnica de València (PAID-01-20-25), Spain. We acknowledge the financial support from CONACyT (grant A1-S-28440). / Muñoz Pérez, FM. (2024). Diseño y caracterización experimental de sistemas de atrapamiento y manipulación de micro-objetos mediante técnicas ópticas, térmicas y acústicas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203436 / Compendio

The experiment discussed in the next chapter was to confirm the aforementioned bystander effect. In the first experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this confirms the effect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting affected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of effect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander effect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseff08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct confirmation of the same. The next study was to find out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander effect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to find out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to find out the same. We took clinical samples from BMRI for patients having a confirmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong confirmation about the previously observed bystander effect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot differentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more confirmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander effect and tried to find out the released substances which are responsible for this effect. We have shown that this technique can use the bystander effect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the effects of drugs and to find out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to find out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to find out the effect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can find out any difference between healthy and unhealthy cells.

Statistical physics in soft matter systems, physical properties of bio-inspired systems and the mechanical manipulations of nano-systems have been studied using optical tweezers to form the basis of this doctoral Thesis. The first two chapters are on a general introduction about optical tweezers and detailed description of the setup used along with its calibrations. The next three chapters describe studies of statistical properties in soft matter systems, namely, out-of-equilibrium microrheology in a worm-like micellar system, irreversibility to reversibility crossover in the non-equilibrium trajectories of an optically trapped particle with the verification of fluctuation theorems even for non-ergodic descriptions of the system and high velocity Brownian vortexes at the liquid-air interface. The mechanical manipulation of the nano-systems, i.e. optically driven nano-rotors and the trapping, as well as transportation of palladium decorated single wall carbon nanotubes using optical tweezers have been discussed in the next two chapters. In the next chapter, the study of physical property of a bio-inspired system -the cell membrane deformability of human erythrocytes with increasing calcium ion concentration has been described. This Thesis is an endeavor to understand different mesoscopic systems using optical trapping and manipulation. Chapter 1 gives an introduction on optical tweezers. The working principle of optical trapping and manipulation are discussed along with their applicability in different fields of physics. Chapter 2 discusses the experimental setup in detail. The setup used for the experiments is a dual optical trap around an inverted microscope. The formation of the traps, the technique to steer the trapping beams and to place the traps at the desired positions in 3D without affecting the symmetry or stiffness are described. Instantaneous position tracking of the trapped particle is a very crucial part of optical trapping experiments. A tracking beam is used for this purpose and the trapped bead is imaged on a quadrant photo diode which provides the current signals that corresponds to the particle’s position in the focal plane. Then the calibration of the setup using various calibration methods are explained. Calibration of the setup includes the calibration of the position sensing devices, e.g. the quadrant photo diode and the CCD camera attached to the microscope, calibration of the electronic devices, e.g. the stage nano-positioner, nano-tilt mirror mount etc., and finally calibration of the trap stiffnesses (in both X and Y ) at varying laser powers. Precautions taken during the experiments to minimize the artifacts are also mentioned. In Chapter 3, a nonlinear microrheology experiment to probe directional viscoelasticity of a sheared worm-like micellar system has been described. Many wormlike micellar systems exhibit appreciable shear thinning due to shear induced alignment. As the micelles get aligned, introducing directionality in the system, the viscoelastic properties no longer remain isotropic. An optical tweezers based technique enables us to probe the out-of-equilibrium rheological properties of CTAT (cetyltrimethylammonium tosylate, cationic surfactant) system simultaneously along two orthogonal directions -parallel to the applied shear, as well as perpendicular to it. A trapped bead is dragged through the medium (1 wt% CTAT) and the position fluctuations of the bead, along the direction of motion (X) and perpendicular to it (Y ), are recorded in both ‘drive on’ and ‘drive off’ states. While the displacement of the bead along X -in response to the active drag force -carry signature of conventional shear thinning, its spontaneous position fluctuations along Y , following the fluctuation dissipation theorem, provide the loss modulus (G∗∗ along Y ) which manifests a dramatic orthogonal shear thickening, an effect hitherto unobserved. Chapter 4 describes an irreversibility to reversibility crossover in the transient response of a particle in optical trap; and the verification of the fluctuation theorem for a non-ergodic description of this system. The transient position fluctuations of a colloidal bead is studied as it approaches equilibrium after being released from varying heights (by using an additional very strong optical trap) in the potential energy landscape created by a weak optical trap. The time evolution of the system shows dramatic changes as the release point energy is decreased. Starting from a small-time-reversible to long-time-irreversible transition for a higher energy release, a time independent completely reversible state could be reached just by lowering the initial potential energy a bit. For an even lower energy release, the system shows an anomalous irreversibility. In this state, it progressively extracts useful work from the thermal fluctuations and surprisingly goes to a higher energy phase point. Highlighting the competition between the micro-reversibility and the irreversible dissipative loss in determining the long-time system behavior, this study exhibits the prominent emergence of a completely reversible state even at long time, in between the two irreversible states of opposite kind. The Transient Fluctuation Theorem (TFT) and the Integrated Transient Fluctuation Theorem (ITFT) which are defined to be valid only for ergodic systems, have been verified even for non-ergodic descriptions (separately for different release points) of this system. Chapter 5 illustrates the study of high velocity Brownian vortex at the liquid-air interface. A general kind of Brownian vortexes are constituted by applying an external non-conservative force field to a colloidal particle bound by a conservative optical trapping force at a liquid-air interface. As the liquid medium is translated at a constant velocity with the bead trapped at the interface, the drag force near the surface provide enough rotational component to bias the particle’s thermal fluctuations in a circulatory motion. The frequency of that circular motion increases linearly with the stage velocity, while an increment in the trapping laser power shows the opposite effect. The properties of these Brownian vortexes have been studied extensively to demonstrate how the thermal fluctuations and the advection of the bead play their role in the vortex motions, with an inference that the angular velocity of the circulatory motions offer a comparative measure of the interface fluctuations. In Chapter 6 the optical manipulation of asymmetric nanorods that constitutes optically driven nanorotors are described. The light force, irrespective of its polarization, is used to run a simple nanorotor. While the gradient force of a single beam optical trap holds an asymmetric nanorod, the scattering force is utilized to generate a non-zero torque on the nanorod making it rotate about the optic axis. The inherent textural irregularities or morphological asymmetries of the nanorods give birth to chirality which is responsible for generation of the torque under the radiation pressure. A farther study on nanorotors that are more transparent to infra-red (trapping beam) confirms that the scattering force is indeed the origin of the torque. A model is proposed to explain the rotational motion of the nanorods and estimate the speed of rotation. If the nanorods are not fairly transparent to the laser beam, even a small surface irregularity with non-zero chirality is sufficient to produce enough torque for moderate rotational speed. Different sized rotors can be used to set the speed of rotation over a wide range, with fine tuning possible through the variation of the laser power. Chapter 7 discuses optical trapping and transportation of palladium decorated single wall carbon nanotubes (Pd-SWNT). Individual carbon nanotubes being substantially smaller than the wavelength of light are not much responsive to optical manipulation. Decorating those single-walled carbon nanotubes with palladium particles changes that scenario dramatically, making the optical trapping and manipulation much easier. Palladium decorated nanotubes (Pd/SWNTs) have higher effective dielectric constant and are trapped at much lower laser power level with greater ease. In addition to that, an asymmetric line trap makes it possible to transport the Pd decorated SWNTs to a desired distant location in the sample cell. In the asymmetric line trap the Pd/SWNTs are first get attracted by the gradient force and then the scattering force push them away towards the other end of the line trap. In Chapter 8, how the rotational motion of crenated erythrocytes in an optical trap can be used to probe their membrane deformability is explained. When placed in a hypertonic buffer medium, discocytic human erythrocytes are subjected to crenation and take deformed shapes. The deformation of the cells brings in chirality and asymmetries in shape that make them rotate under the scattering force of a linearly polarized optical trap. A change in the deformability of the erythrocytes, due to any internal or environmental factor, is reflected in the rotational speed of the trapped crenated cells. Therefore the average rotational speed and the probability of rotation of the crenated erythrocytes in an optical trap can be considered as a direct signature of their membrane deformability. As an example, the relative increment in erythrocyte membrane rigidity with adsorption of Ca++ ions is examined quantitatively through this approach. The Thesis concludes with a summary of the main results and a brief discussion of the scope of future work in Chapter 9.

Les assemblages supramoléculaires font parties des réactions biochimiques de base dans les fonctions cellulaires (ex: réplication de l'ADN ou réponse immunitaire). Les calix[n]arènes ont été décrit pour interagir avec une large gamme de biomolécules. En conséquence, ils peuvent être trouvés dans de nombreuses applications biologiques tel qu'en diagnostic ou en traitement thérapeutique. Leurs fonctionnalisations sur des nanoparticules d'argent ont produit de nouveaux nano-composés hybrides ayant des propriétés optique, électrique et biologique uniques. Cette thèse a été dédiée à l'étude de ces nano systèmes pour la bio-détection et leurs potentielles applications biomédicales. Le développement de capteur analytique à bas coût, portable et ultra-sensible représente une des attentes majeures dans les applications des calix[n]arènes sur nanoparticules d'argent. Dans cette thèse, ces nano-composés ont été étudiés selon leurs capacités à suivre la micellisation de surfactant mixtes, et pour discriminer un type moléculaire tel qu'une famille d'acide nucléique ou une espèce d'albumine sérique. Dans une deuxième partie, ces nanoparticules hybrides ont été évaluées pour une série d'activités biologiques. Ils ont montré des facultés antibiotiques et anti-oxydantes, de transporter des Ingrédients Pharmaceutiques Actifs, et d'atteindre des cibles antivirales et anticancéreuses / Supramolecular assemblies are among the basic biochemical reactions in the cellular functions (e.g. DNA replication, immune response). Calix[n]arenes are macrocyclic molecules that have been reported for interacting with a wide range of biomolecules. As a consequence, they can be found in many biological applications from diagnosis to therapeutic treatment. Their functionalization on silver nanoparticles have produced new nano hybrid compounds with unique optical, electrical and biochemical properties. This thesis has been dedicated to the study of these nano-systems for bio-sensing and for their potent biomedical applications. Cost effective, portable and ultra-sensitive analytical tools are one of the major expectations of the applications of silver nanoparticles capped with calix[n]arenes. Calix[n]arenes nanoparticles have been reported here for following the micellisation process of mixed surfactants or for discriminating a type of molecule such nucleic acid or a serum albumin specie. In a second part, these hybrid nanoparticles have been evaluated for series of biological activities. They’ve been shown to possess anti-oxidant and antibacterial activities, to transport Active Pharmaceutical Ingredient and to reach antiviral and anti-cancer targets

Over the last few decades, the use of light to control and manipulate microscopic particles has become widespread. These methods are enabling new areas of research to flourish across the physical and biological sciences. This thesis describes investigations into both optical trapping and the closely related field of optical sorting. It documents the development of a variety of new techniques. The thesis begins with a short review of optical trapping and existing methods for sorting mixtures of microscopic particles. The first half of this chapter highlights some of the reasons behind optical trapping's rapid growth in popularity. By reviewing an array of methods for sorting particles and discussing the relative merits of each, the case for optical sorting is established. The second chapter describes research into using a spatial light modulator to create three-dimensional optically trapped colloidal structures using the time-sharing technique. Limiting factors inherent in the technology are discussed in detail. The third chapter reviews a sophisticated particle-tracking software package that has proved to be a considerable success. It was developed explicitly with colloidal microscopy in mind and experimental plots produced by the software are used throughout the thesis. Experimental studies have been performed into the behaviour of microscopic particles moving under the influence of two classes of propagation-invariant beams: Mathieu beams and Bessel beams. The Bessel beam studies have been complimented by a theoretical model and have led ultimately to a new method for the static optical sorting of both solid particles and biological cells, with particular emphasis on human blood. The fifth and final chapter describes how re-configurable optical devices can be implemented to spatially separate different colloidal species. A new method for creating arbitrary optical landscapes using an acousto-optic modulator is reported. This new technique is then used to optically sort four particle species simultaneously - the first experimental demonstration of polydisperse optical fractionation. Additionally, experiments are reported that demonstrate controlled, static optical sorting using a spatial light modulator.

The behaviour of colloidal particles in two-dimensional (2D) systems is addressed in real space and time using magnetic fields, optical tweezers and optical video microscopy. First, the fluctuations of a grain boundary in a 2D colloidal crystal are analysed. A real space analogue of the capillary fluctuation method is derived and successfully employed to extract the key parameters that characterise the grain boundary. Good agreement is also found with a fluctuation-dissipation based method recently suggested in simulation. Following on from analysis of the interface fluctuations, the properties of the individual grain boundary particles are analysed to investigate the long standing hypothesis that suggests that grain boundary particle dynamics are similar to those in supercooled liquids. The grain boundary particle dynamics display cage breaking at long times, highly heterogeneous particle dynamics and the formation of cooperatively moving regions along the interface, all typical behaviour of a supercooled liquid. Next, the frustration induced by confining colloidal particles inside a pentagonal environment is investigated. The state of the system is adjusted via two separate control parameters: the inter-particle interaction potential and the number density. A gradual crystalline to confined liquid-like transition is observed as the repulsive inter-particle interaction potential is decreased. In contrast, re-entrant orientational ordering and dynamical effects result as the number density of the confined colloidal particles is increased. Finally, the dynamics of colloidal particles distributed amongst a random array of fixed obstacle particles is probed as a function of both the mobile particle and fixed obstacle particle number densities. Increasing the mobile and the obstacle particle number density drives the system towards a glass transition. The dynamics of the free particles are shown to behave in a similar way to the normal glass transition at low obstacle density and more analogous to a localisation glass transition at high obstacle density.

Pendant le développement d'un organisme, les tissus subissent, génèrent des changements morphologiques drastiques nécessaires à l'obtention d'une forme finale ou intermédiaire spécifique et fonctionnelle. On comprend cette acquisition de formes comme un phénomène émergent résultant de l'interaction mécanique entre toutes les cellules composant le tissu. On sait que les structures de protéines du cytosquelette sont capables aussi bien de générer des forces ou de changer les propriétés mécaniques des cellules i.e de changer la réaction de celles-ci à un stress mécanique. Ces phénomènes émergents font l'objet de nombreuses études et mesures aussi bien lors d'expériences in vitro (solution de protéines purifiées) que sur cellules de cultures. Le travail décrit dans cette thèse s'est attaché à mesurer quantitativement les forces et propriétés mécaniques in vivo durant le développement d'un organisme. À terme, ces mesures sont utiles à l'élaboration d'un modèle mécanique qui amènerait une meilleure compréhension des phénomènes morphogénétiques.Pour réaliser ces mesures, un banc de mesures optiques a été développé. Il permet de réaliser des mesures de microrhéologie passives et actives durant l'extension de la bandelette germinale de l'embryon de drosophile. Des mesures quantitatives de viscosité, de raideur et de force jonctionnelle ont été réalisées. / Pendant le développement d'un organisme, les tissus subissent, génèrent des changements morphologiques drastiques nécessaires à l'obtention d'une forme finale ou intermédiaire spécifique et fonctionnelle. On comprend cette acquisition de formes comme un phénomène émergent résultant de l'interaction mécanique entre toutes les cellules composant le tissu. On sait que les structures de protéines du cytosquelette sont capables aussi bien de générer des forces ou de changer les propriétés mécaniques des cellules i.e de changer la réaction de celles-ci à un stress mécanique. Ces phénomènes émergents font l'objet de nombreuses études et mesures aussi bien lors d'expériences in vitro (solution de protéines purifiées) que sur cellules de cultures. Le travail décrit dans cette thèse s'est attaché à mesurer quantitativement les forces et propriétés mécaniques in vivo durant le développement d'un organisme. À terme, ces mesures sont utiles à l'élaboration d'un modèle mécanique qui amènerait une meilleure compréhension des phénomènes morphogénétiques.Pour réaliser ces mesures, un banc de mesures optiques a été développé. Il permet de réaliser desmesures de microrhéologie passives et actives durant l'extension de la bandelette germinale de l'embryon de drosophile. Des mesures quantitatives de viscosité, de raideur et de force jonctionnelle ont été réalisées.

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.