In the past decades, numerous single-molecule techniques have been developed to investigate individual bio-molecules and cellular machines. While a lot is known about the structure, localization, and interaction partners of such molecules, much less is known about their mechanical properties. To investigate the weak, non-covalent interactions that give rise to the mechanics of and between proteins, an instrument capable of resolving sub-nanometer displacements and piconewton forces is necessary. One of the most prominent biophysical tool with such capabilities is an optical tweezers. Optical tweezers is a non-invasive all-optical technique in which typically a dielectric microsphere is held by a tightly focused laser beam. This microsphere acts like a microscopic, three-dimensional spring and is used as a handle to study the biological molecule of interest. By interferometric detection methods, the resolution of optical tweezers can be in the picometer range on millisecond time scales. However, on a time scale of seconds—at which many biological reactions take place—instrumental noise such as thermal drift often limits the resolution to a few nanometers. Such a resolution is insufficient to resolve, for example, the ångstrom-level, stepwise translocation of DNA-binding enzymes corresponding to distances between single basepairs of their substrate. To reduce drift and noise, differential measurements, feedback-based drift stabilization techniques, and ‘levitated’ experiments have been developed. Such methods have the drawback of complicated and expensive experimental equipment often coupled to a reduced throughput of experiments due to a complex and serial assembly of the molecular components of the experiments. We developed a high-resolution optical tweezers apparatus capable of resolving distances on the ångstrom-level over a time range of milliseconds to 10s of seconds in surface-coupled assays. Surface-coupled assays allow for a higher throughput because the molecular components are assembled in a parallel fashion on many probes. The high resolution was a collective result of a number of simple, easy-to-implement, and cost-efficient noise reduction solutions. In particular, we reduced thermal drift by implementing a temperature feedback system with millikelvin precision—a convenient solution for biological experiments since it minimizes drift in addition to enabling the control and stabilization of the experiment’s temperature. Furthermore, we found that expanding the laser beam to a size smaller than the objective’s exit pupil optimized the amount of laser power utilized in generating the trapping forces. With lower powers, biological samples are less susceptible to photo-damage or, vice versa, with the same laser power, higher trapping forces can be achieved. With motorized and automated procedures, our instrument is optimized for high-resolution, high-throughput surface-coupled experiments probing the mechanics of individual biomolecules. In the future, the combination of this setup with single-molecule fluorescence, super-resolution microscopy or torque detection will open up new possibilities for investigating the nanomechanics of biomolecules.

In the past decades, numerous single-molecule techniques have been developed to investigate individual bio-molecules and cellular machines. While a lot is known about the structure, localization, and interaction partners of such molecules, much less is known about their mechanical properties. To investigate the weak, non-covalent interactions that give rise to the mechanics of and between proteins, an instrument capable of resolving sub-nanometer displacements and piconewton forces is necessary. One of the most prominent biophysical tool with such capabilities is an optical tweezers. Optical tweezers is a non-invasive all-optical technique in which typically a dielectric microsphere is held by a tightly focused laser beam. This microsphere acts like a microscopic, three-dimensional spring and is used as a handle to study the biological molecule of interest. By interferometric detection methods, the resolution of optical tweezers can be in the picometer range on millisecond time scales. However, on a time scale of seconds—at which many biological reactions take place—instrumental noise such as thermal drift often limits the resolution to a few nanometers. Such a resolution is insufficient to resolve, for example, the ångstrom-level, stepwise translocation of DNA-binding enzymes corresponding to distances between single basepairs of their substrate. To reduce drift and noise, differential measurements, feedback-based drift stabilization techniques, and ‘levitated’ experiments have been developed. Such methods have the drawback of complicated and expensive experimental equipment often coupled to a reduced throughput of experiments due to a complex and serial assembly of the molecular components of the experiments. We developed a high-resolution optical tweezers apparatus capable of resolving distances on the ångstrom-level over a time range of milliseconds to 10s of seconds in surface-coupled assays. Surface-coupled assays allow for a higher throughput because the molecular components are assembled in a parallel fashion on many probes. The high resolution was a collective result of a number of simple, easy-to-implement, and cost-efficient noise reduction solutions. In particular, we reduced thermal drift by implementing a temperature feedback system with millikelvin precision—a convenient solution for biological experiments since it minimizes drift in addition to enabling the control and stabilization of the experiment’s temperature. Furthermore, we found that expanding the laser beam to a size smaller than the objective’s exit pupil optimized the amount of laser power utilized in generating the trapping forces. With lower powers, biological samples are less susceptible to photo-damage or, vice versa, with the same laser power, higher trapping forces can be achieved. With motorized and automated procedures, our instrument is optimized for high-resolution, high-throughput surface-coupled experiments probing the mechanics of individual biomolecules. In the future, the combination of this setup with single-molecule fluorescence, super-resolution microscopy or torque detection will open up new possibilities for investigating the nanomechanics of biomolecules.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: A magneto optical trap (MOT) is capable of trapping a vapor cloud consisting of atoms cooled down to the micro Kelvin range. Three orthogonal pairs of counter-propagating laser beams of the correct circular polarisation form an optical molasses which facilitates the cooling of neutral atoms. Additionally a spatially non-uniform magnetic field produced by two current carrying coils in a Maxwell gradient configuration is used to trap the cooled atoms. In this report the effects of the trap parameters, including the laser beam intensity and frequency detuning, beam diameter and magnetic field gradient, on the number of trapped atoms are discussed. Secondly the development of an experimental setup for laser cooling and trapping of 87Rb atoms in vacuum with the aid of a MOT is presented. All trap components were implemented and characterised. The vacuum system and trapping chamber in which the cooling takes place were designed and constructed. A rubidium getter to act as a source of atoms was integrated into the vacuum system. The two external cavity diode lasers used for trapping and optical re-pumping were characterised. The optical setup required for the optical molasses was designed, constructed and characterised. Saturated absorption spectroscopy was performed to investigate the hyperfine structure of 87Rb and to frequency lock the lasers. We report on the current status of the project with regards to progress, results and future work. / AFRIKAANSE OPSOMMING: 'n Magneto-optiese val (magneto optical trap, MOT) kan 'n dampwolk van atome vang en afkoel tot in die mikro Kelvin bereik. Drie ortogonale pare laserbundels, elke paar voortplantend in teenoorgestelde rigtings, met die korrekte sirkelvormige polarisasie vorm 'n sogenaamde optiese molasse wat die afkoeling van neutrale atome moontlik maak. Bykomend word 'n ruimtelik nie-uniforme magneetveld geproduseer deur twee stroomdraende spoele in 'n Maxwell gradient-opstelling gebruik om die afgekoelde atome te vang. In hierdie verslag word die invloed van die val parameters, insluitend die laserbundel intensiteit en frekwensie afstemming, die laserbundel deursnit en magneetveld gradiënt, op die aantal atome in die val bespreek. Tweedens word die ontwikkeling van 'n eksperimentele opstelling vir laser afkoeling en vang van 87Rb atome in vakuum met die hulp van 'n MOT voorgelê. Alle komponente van die val is geïmplementeer en gekarakteriseer. Die vakuumsisteem en val-kamer waarin die afkoeling plaasvind is ontwerp en gebou. 'n Rubidium gasbinder is in die vakuumsisteem ingebou om as 'n bron van atome te dien. Die twee eksterne resonator diodelasers wat gebruik is vir die val en die optiese terugpomp is gekarakteriseer. Die optiese opstelling wat nodig is vir die optiese molasse is ontwerp, gebou en gekarakteriseer. Versadigde absorpsiespektroskopie is uitgevoer om die hiperfynstruktuur van 87Rb te ondersoek en om die lasers se frekwensies te stabiliseer. Verslag word gedoen oor die huidige stand van die projek wat betref vordering, resultate en toekomstige werk.

This thesis investigates the use of Raman tweezers microspectroscopy to interrogate the biochemistry of single biological cells. Raman tweezers microspectroscopy is a powerful technique, which combines traditional Raman microspectroscopy and optical trapping, allowing the manipulation and environmental isolation of a biological cell of interest whilst simultaneously probing its biochemistry gleaning a wealth of pertinent information. The studies carried out in this thesis can be split into two broad categories: firstly, the exploitation of Raman tweezers microspectroscopy to study biological cells and secondly developments to the Raman tweezers microspectroscopy technique that extend its capabilities and the range of samples that can be studied. In the application of Raman tweezers, the stacking and interrogation of multiple cells is reported allowing a rapid representative Raman signal to be recorded from a small cell population with improved signal to noise. Also demonstrated is the ability of Raman spectroscopy to identify and grade the development of Human Papillomavirus induced cervical neoplasia with sensitivities of up to 96 %. These studies demonstrate the potential of Raman spectroscopy to study biological cells but it was noted that the traditional Raman tweezers system struggled to manipulate large cells thus a decoupled Raman tweezers microspectroscopy system is presented where a dual beam fibre optical trap is used to perform the trapping function and a separate Raman probe is introduced to probe the biochemical nature of the trapped cell. This development allowed the trapping and examination of very large cells whilst opening up the possibility of creating Raman maps of trapped objects. Raman tweezers microspectroscopy could potentially become an important clinical diagnostic and biological monitoring tool but is held back by the long signal integration times required due to the weak nature of Raman scattering. The final study presented in this thesis examines the potential of wavelength modulated Raman spectroscopy to improve signal to noise ratios and reduce integration times. All these studies aim to demonstrate the potential and extend the performance of Raman tweezers microspectroscopy.

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.

Desde a sua origem nos laboratórios do Bell Labs, durante a década de 70, pinças ópticas vêm sendo investigadas, desenvolvidas e diversificadas por vários grupos nacionais e internacionais incluindo, nos últimos anos, o nosso departamento (Departamento de Engenharia Elétrica e de Computação - SEL/EESC/USP) e, em particular, o Grupo de Telecomunicações a ele vinculado, com propostas de aplicações em óptica biomédica e medicina. Dada a importância deste assunto, o principal objetivo deste trabalho é estender a análise teórica de forças ópticas utilizando uma descrição simples, porém eficaz, e analítica de um feixe de Bessel truncado, isto é, gerado por abertura finita. Até onde sabemos, esta é a primeira tentativa de introduzir esta investigação dos feixes originais de Durnin na literatura, e, deste modo, temos a possibilidade de contribuir com uma análise inédita de um feixe de Bessel com características físicas reais. Com a modelagem matemática dos feixe de Bessel truncados em mãos, aplicamos o cálculo de forças nos regimes de óptica geométrica e de Rayleigh e analisamos o seu comportamento quando os feixes incidem em espalhadores dielétricos esféricos. Como resultado direto deste trabalho, publicamos dois artigos descrevendo as forças ópticas no regime de óptica geométrica e de Rayleigh que um feixe de Bessel truncado causa em uma partícula dielétrica esférica. Como resultados indiretos, publicamos dois artigos com os mesmos cálculos de forças, adotando classe de feixes chamada Frozen-Waves, que é uma superposição de feixes de Bessel de mesma frequência. / Since its origins at Bell Labs laboratories, during the 1970s, optical tweezers have been researched, developed and diversified by several research groups, both nationally and internationally, including in our department (Department of Electrical and Computing Engineering - SEL/EESC/USP), in particular the Telecom Group associated to it, with several proposals with applications of optical tweezers in different fields from medicine to biomedical optics. Given the importance of this field, our main goal is to extend the theoretical analysis of optical forces using a simplest - but effective - analytical description of truncated Bessel beams (i.e. beams that are generated by finite apertures). As far we know, this is the first attempt to introduce such analysis of the originals Durnin beams in the literature, thus, we may have the chance to contribute to an unpublished study of a truncated Bessel beam with real characteristics. With such mathematical model at hands, we calculate the forces exerted by the truncated Bessel beam in ray optics and Rayleigh regime and analyze their behavior when hits dielectric spherical particles. As direct result of this work, we have published two papers describing the optical forces in ray optics and Rayleigh regime that a truncated Bessel beam causes in a spherical dielectric particle. As indirect results, we have published two papers with the same force considerations, adopting a beam class called Frozen-Waves, which is a superposition of same frequency Bessel beams.

This dissertation presents the implementation of a technique to generate atomic Fock states of Lithium 6 with ultra-high fidelity, called laser culling. Fock states, atomic states with a definite number of particles, are a mandatory step for studying few-body quantum phenomena such as quantum tunneling, quantum entanglement, and serve as building blocks for quantum simulators. The creation of ultra-high fidelity Fock states begins with a degenerate Fermi gas in an optical dipole trap. Being fermions, lithium-6 atoms fill the energy levels of the dipole trap with 2 atoms per energy level. Introducing a magnetic field gradient creates a linear potential that tilts the potential produced by the optical dipole trap. The initially bound energy levels become quasi-bound states, each with a different lifetime. By exploiting the difference between these lifetimes, one can generate a single pair of atoms in the ground state of the trap with fidelities that can exceed 99.9%. This dissertation first presents the details of the design and construction of an apparatus for laser culling, and then reports on the progress made towards the creation of atomic Fock states with ultra-high fidelity. / text

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

This dissertation presents subwavelength optics with focus on the theory and applications of subwavelength apertures in a metal film. Two main issues regarding the optics with subwavelength apertures are investigated. As the first issue, the extraordinary optical transmission (EOT) through a single hole in a metallic waveguide is presented. A total transmission through a single subwavelength aperture is theoretically predicted for a perfect electric conductor regardless of the aperture size, without relying on aperture arrays and surface corrugations as presented in previous works. The waveguide EOT is then applied to boost the optical throughput of an apertured near-field scanning optical microscope (NSOM) probe. Using a new structure for the apertured NSOM probe which allows for waveguide EOT, the optical throughput and the damage threshold are boosted by 100× and 40× as compared to a conventional structure, and the experimental findings are backed-up by comprehensive finite-difference time-domain (FDTD) simulations. Single fluorescent molecules are scanned using the EOT apertured NSOM probe, and a spatial resolution of 62 nm is achieved. As the second issue, subwavelength apertures are found useful for optical trapping. A small dielectric particle can significantly change the optical transmission through an aperture by dielectric loading, and subsequently, a large optical force is induced which favors trapping. A self-induced back-action (SIBA) optical trap is designed using a circular nanohole in a gold film. Trapping of 50 nm polystyrene particle is experimentally achieved, which is not possible using a conventional single beam optical tweezers. The circular nanohole SIBA trap works beyond the perturbative regime, as proven by FDTD simulations and a Maxwell stress tensor analysis. We further improve the nanohole trapping using a double-nanohole, which is more sensitive for small dielectric changes due to the intense local field enhancement between its two sharp tips. A single 12 nm silica sphere is experimentally trapped using the double-nanohole, as the smallest trapped dielectric particle reported. We also achieve the trapping of a single protein – a bovine serum albumin (BSA) protein with a hydrodynamic radius of 3.4 nm in the folded form. The trapped BSA is also unfolded by the large optical force, as confirmed by experiments with changing optical power and changing pH. The high signal-to-noise ratio of 33 in monitoring single protein trapping and unfolding shows a tremendous potential for using the double-nanohole as a sensor for protein binding events at a single molecule level. / Graduate

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.