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Title: Development of optical tools for biological applications based on acousto-optic technology
Author: Bola Sampol, Raúl
Director/Tutor: Montes Usategui, Mario
Martín Badosa, Estela
Keywords: Òptica
Microscòpia de fluorescència
Fluorescence microscopy
Issue Date: 17-Jun-2021
Publisher: Universitat de Barcelona
Abstract: [eng] To ensure progress in the field of biomedicine and drug development, it is essential to keep improving the equipment needed to carry out most of the experiments. With better tools, scientists can be more efficient, increasing the quality and volume of collected data from biological samples. At the same time, these tools should offer enough flexibility to adapt to new protocols and experiments. Among all the tools used in cell biology laboratories, photonic technology is the most popular, since it is considered a non-invasive technique, being fully compatible with living samples. The work done in this thesis focuses on the development of two optical tools with great applicability in the field of cell biology: an optical trapping and force measurement system and a novel and flexible confocal microscopy unit. The combination of both apparatus will allow biologists to manipulate and measure forces inside living cells, while providing high contrast visualization of the specimen in real time. Both technologies share the same light modulator element: an acousto-optic deflector (AOD). AODs are diffractive devices that use mechanical waves to deflect an incident beam of light with extreme precision and speed (in the kilohertz to megahertz range). Despite being developed in the 30s, their full potential has not been exploited until now. During the thesis, I have carried out a thorough study of the AOD properties, culminating in a new way to understand and use these devices: the acousto-optic holography (AOH). With slight modifications in the control electronics, these devices can be used in the same way as a full complex spatial light modulator. With this new approach, AODs can project arbitrary light patterns and scanning schemes, going beyond their main application as pure beam deflectors. Incorporating AODs in an optical trapping system allows generating multiple stable optical traps through time multiplexing a single laser beam, catching and manipulating a plurality of objects at the same time. This scheme also allows synchronizing the laser position with a force measurement system based on direct momentum changes, being able to address single object information. The optical trapping system developed in the first half of this thesis has been used to perform controlled oscillations, as well as measure the response force, in a variety of situations. It has been employed to obtain information about the mechanical properties of some biological structures, such as the cellular cytoskeleton or in active gels of tubulin bundles. Second, focusing on acousto-optic holography, the thesis presents a new confocal microscope concept: the programmable array microscope, which serves as the starting point for a new generation of solid-state digital microscopes. This new concept proposes eliminating all the mechanical and mobile elements of conventional microscopes and replacing them with fully programmable elements. Specifically, it proposes eliminating the physical ”pinhole” and motorized scanning systems, thus resulting in a very flexible device. The prototype allows the projection of an infinity of structured light patterns at high speeds to produce high-quality reconstructions. Given the high degree of flexibility, this new solid-state microscope can implement multiple imaging modes that can adapt to the needs of each experiment and/or sample. Apart from implementing already existing techniques, the prototype allows the investigation of new imaging modes and smart scanning schemes. These new modes aim to extract sample information more efficiently, faster, or at a higher resolution. The thesis details the entire development process of the prototype, both in the optomechanical design, the generation of lighting patterns and scanning schemes. Regarding the confocal filtering part, we present two different solutions. First, we present a set of new image processing algorithms that take advantage of our flexible illumination system. Then, we provide the development of a custom and flexible camera module that allows arbitrary pixel reading. Finally, with the same technology, two different paths have been explored in the field of super-resolution. On the one hand, the confocal system has been adapted for the parallelization of STED microscopy, speeding up the capture process and presenting promising first results. On the other hand, we have explored computational strategies based on deep learning that allow the recovery of high frequencies. This allows the observation of fine structures well beyond the diffraction limit barrier.
[cat] Per poder garantir l’avanç de les ciències de la vida, amb la gran repercussió a la salut mundial que això comporta, és important anar millorant les eines amb les quals els científics (especialment els biòlegs) desenvolupen els experiments, podent així millorar la quantitat i el volum de les dades extretes. Amb noves eines, els investigadors poden desenvolupar i dissenyar nous experiments que proporcionin informació rellevant sobre l’estudi i la comprensió de molts processos cel·lulars i malalties. La tesi tracta sobre el desenvolupament de dos sistemes òptics amb gran aplicació en el camp de la biologia, ambdues basades en un element modulador de llum comú, els deflectors acusto-òptics (AODs). El AODs són dispositius totalment analògics, on s’utilitzen ones acústiques per poder modular i deflectar un làser amb una gran precisió i velocitat. A la primera part, s’explica el desenvolupament d’un sistema d’atrapament òptic i mesura de força. El sistema permet atrapar i manipular, de manera estable, múltiples objectes, així com realitzar oscil·lacions controlades. Al mateix temps, el sistema és totalment compatible amb mesura de forces per canvis de moment. Tot això permet paral·lelitzar experiments a l’interior cel·lular de manera totalment invasiva, oferint informació sobre les propietats mecàniques de diverses estructures biològiques. A la segona part, es presenta una nova forma d’entendre i utilitzar aquests dispositius: l’holografia acusto-òptica. Mitjançant la generació de senyals acústiques complexes, els AODs permeten projectar patrons de llum arbitraris, més enllà del seu ús principal com a deflectors làser. Això porta al desenvolupament d’un nou microscopi confocal, totalment programable i sense elements mecànics o mòbils. El microscopi permet projectar una infinitat de patrons de llum estructurada, per tal d’obtenir reconstruccions d’alta qualitat a centenars d’imatges per segon. Aquesta nova plataforma de microscòpia d’estat sòlid, permet investigar i implementar una infinitat de maneres d’imatge, per adaptar-se a les necessitats de cada experiment i / o mostra.
Appears in Collections:Tesis Doctorals - Facultat - Física

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