The thermal velocity fluctuations of microscopic particles mediate the transition from microscopic statistical mechanics to macroscopic long-time diffusion. Prior to this work, detection methods lacked the sensitivity necessary to resolve motion at the length and time scales at which thermal velocity fluctuations occur. This dissertation details two experiments which resulted in velocity measurement of the thermal motion of dielectric microspheres suspended by an optical trap in gases and liquids. First, optical tweezers were used to trap glass microspheres in air over a wide range of pressures and a detection system was developed to track the trapped microspheres' trajectories with MHz bandwidth and <100 fm/rt(Hz) position sensitivity. Low-noise trajectory measurements allowed for observation of fluctuations in the instantaneous velocity of a trapped particle with a signal to noise ratio (SNR) of 26 dB, and provided direct verification of the equipartition theorem and of the Maxwell-Boltzmann velocity distribution for a single Brownian particle. Next, the detection technology was further optimized and used to track optically trapped silica and barium titanate glass microspheres in water and acetone with >50 MHz bandwidth and <3 fm/rt(Hz) sensitivity. Brownian motion in a liquid is influenced by hydrodynamic, time-retarded coupling between the particle and the fluid flow its motion generates. Our measurements allowed for instantaneous velocity measurement with an SNR of up to 16 dB and confirmed the Maxwell Boltzmann distribution for Brownian motion in a liquid. The measurements also revealed several unusual features predicted for Brownian motion in the regime of hydrodynamic coupling, including faster-than-exponential decay of the velocity autocorrelation function, correlation of the thermal force and non-zero cross-correlation between the particle's velocity and the thermal force preceding it. / text

Mechanics and biological function of nucleic acids are intimately coupled. The DNA double helix must be opened to allow base pairing of RNA during transcription; RNA must bend and fold in its many cellular functions. Presented in this dissertation are two investigations of mechanical deformations of nucleic acids, conducted with optical tweezers.In the introduction, the mechanical properties of DNA and RNA and their relevance to their cellular functions are introduced, to give the reader context for the results presented in the Chapters 2 and 3. This is followed by an introduction to the theory of semiflexible polymer elasticity. The optical tweezers instruments used in conducting these investigations are then presented, along with calibration procedures and a short introduction to optical trapping physics.Chapter 2 presents an investigation of the effect of downstream DNA tension on initiation by T7 RNA polymerase. A hidden Markov model is fit to force-dependent lifetimes obtained from optical tweezers experiments, allowing us to identify which steps in initiation are force-dependent and estimate rates and transition state distances. We find that 1-2 pN of tension is sufficient to turn o gene expression by causing transcription bubble collapse and destabilizing the bound state. Our force-dependence scheme and estimated transition distances provide independent supportfor the \scrunching" model of initiation.The effects of cation binding and screening on single-stranded helix formation in poly(A) RNA are presented in Chapter 3. Magnesium and calcium bind to poly(A), stabilize the helix, and change its mechanical properties. A new model of helix-coil transitions is presented and used to estimate energetics and mechanical properties.Chapter 4 presents the first fully objective algorithm for use in analyzing the noisy staircaselike data that is often produced by single-molecule fluorescence experiments. A test based on the SIC (BIC) statistic is used in conjunction with a progressive step-placement scheme to locate changepoints (steps) in noisy data. Its performance is compared to other step detection algorithms in use by biophysicists by repeating tests performed in a recent review.Experimental protocols and computer codes used in these investigations are presentedin detail in the appendices.

Single atoms are promising candidates for physically implementing quantum bits, the fundamental unit of quantum information. We have built an apparatus for cooling, trapping and imaging single rubidium atoms in microscopic optical tweezers. The traps are formed from a tightly focused off-resonant laser beam, which traps atoms using the optical dipole force. The traps have a diameter of ~1 &mu;m and a depth of ~1 mK. The novelty of our approach is the use a digital mirror device (DMD) to generate multiple independently movable tweezers from a single laser beam. The DMD consists of an array of micro-mirrors that can be switched on and off, thus acting as a binary amplitude modulator. We use the DMD to imprint a computer-generated hologram on the laser beam, which is converted in to the desired arrangement of traps in the focal plane of a lens. We have developed fast algorithms for calculating binary holograms suitable for the DMD. In addition, we use this method to measure and correct for errors in the phase of the wavefront caused by optical aberrations, which is necessary for producing diffraction-limited focal spots. Using this apparatus, we have trapped arrays of up to 20 atoms with arbitrary geometrical arrangements. We exploit light-assisted collisions between atoms to ensure there is at most one atom per trapping site. We measure the temperature of the atoms in the traps to be 12 &mu;K, and their lifetime to be 1.4 s. Finally, we demonstrate the ability to select individual atoms from an array and transport them over a distance of 14μm with laser cooling, and 5 &mu;m without.

In the atmosphere, cloud droplets can remain in a supercooled liquid phase at temperatures as low as -40 °C. Above this temperature, cloud droplets freeze via heterogeneous ice nucleation whereby a rare and poorly understood subset of atmospheric particles catalyze the ice phase transition. As the phase state of clouds is critical in determining their radiative properties and lifetime, deficiencies in our understanding of heterogeneous ice nucleation poses a large uncertainty on our efforts to predict human induced global climate change. Experimental challenges in properly simulating particle-induced freezing processes under atmospherically relevant conditions have largely contributed to the absence of a well-established model and parameterizations that accurately predict heterogeneous ice nucleation. Conversely, the sparsity of reliable measurement techniques available struggle to be interpreted by a single consistent theoretical or empirical framework, which results in layers of uncertainty when attempting to extrapolate useful information regarding ice nucleation for use in atmospheric cloud models. In this dissertation a new framework for describing heterogeneous ice nucleation is developed. Starting from classical nucleation theory, the surface of an ice nucleating particle is treated as a continuum of heterogeneous ice nucleating activity and a particle specific distribution of this activity g is derived. It is hypothesized that an individual particle species exhibits a critical surface area. Above this critical area the ice nucleating activity of a particle species can be described by one g distribution, 𝑔, while below it 𝑔 expresses itself expresses externally resulting in particle to particle variability in ice nucleating activity. The framework is supported by cold plate droplet freezing measurements for dust and biological particles in which the total surface area of particle material available is varied. Freezing spectra above a certain surface area are shown to be successfully fitted with 𝑔 while a process of random sampling from 𝑔 can predict the freezing behavior below the identified critical surface area threshold. The framework is then extended to account for droplets composed of multiple particle species and successfully applied to predict the freezing spectra of a mixed proxy for an atmospheric dust-biological particle system. The contact freezing mode of ice nucleation, whereby a particle induces freezing upon collision with a droplet, is thought to be more efficient than particle initiated immersion freezing from within the droplet bulk. However, it has been a decades’ long challenge to accurately measure this ice nucleation mode, since it necessitates reliably measuring the rate at which particles hit a droplet surface combined with direct determination of freezing onset. In an effort to remedy this longstanding deficiency a temperature controlled chilled aerosol optical tweezers capable of stably isolating water droplets in air at subzero temperatures has been designed and implemented. The new temperature controlled system retains the powerful capabilities of traditional aerosol optical tweezers: retrieval of a cavity enhanced Raman spectrum which could be used to accurately determine the size and refractive index of a trapped droplet. With these capabilities, it is estimated that the design can achieve ice supersaturation conditions at the droplet surface. It was also found that a KCl aqueous droplet simultaneously cooling and evaporating exhibited a significantly higher measured refractive index at its surface than when it was held at a steady state temperature. This implies the potential of a “salting out” process. Sensitivity of the cavity enhanced Raman spectrum as well as the visual image of a trapped droplet to dust particle collisions is shown, an important step in measuring collision frequencies of dust particles with a trapped droplet. These results may pave the way for future experiments of the exceptionally poorly understood contact freezing mode of ice nucleation.

This thesis describes the construction of an Optical Tweezer apparatus to be used in conjunction with a confocal Raman spectrometer. The tweezer utilizes an infrared (λ=1064 nm) laser directed into an inverted microscope with NA=1.4 oil immersion 100x objective lens that strongly focuses the laser light into a sample to function as a single-beam gradient force trap. The long term goal of this research program is to develop a single molecule Raman tweezers apparatus that allows one to control the position of a Raman nanoplasmonic amplifier. This thesis describes the construction of the Raman tweezer apparatus along with several Raman spectra obtained from optically trapped samples of polystyrene fluorescent orange, amine-modified latex beads. In addition, I explored the Raman spectra of bulk cytochrome c mixed with or injected onto Ag aggregates for SERs enhancement.

Optical manipulation covers a wide range of techniques to guide and trap cells using only the forces exerted by light. Another optical tool is photoporation, the technique of injecting membrane-impermeable molecules using light, which has become an important alternative to other injection techniques. Together they provided sterile tools for manipulation and molecule delivery at the single-cell level. In this thesis, the properties of low Reynolds fluid flows are exploited to guide cells though a femtosecond Bessel beam. This design allows for high-throughput optical injection of cells without the need to individually target cells. A method of 'off-chip' hydrodynamic focusing was evaluated and was found to confine 95.6% of the sample within a region which would receive a femtosecond dose compared to 20% without any hydrodynamic focusing. The system was tested using two cell lines to optically inject the membrane-impermeable dye, propidium iodide. This resulted in an increase of throughput by an order of magnitude compared to the previous microfluidic design (to up to 10 cells per second). Next optical trapping and photoporation were combined to create a multimodal workstation. The system provides 3D beam control using spatial light modulators integrated into a custom user interface. The efficiency of optical injection of adherent cells and trapping capabilities were tested. The development of the system provides the groundwork for exploration of the parameters required for photoporation of non-adherent cells. Finally optical trapping is combined with temporally focused multiphoton illumination for scanless imaging. The axial resolution of the system was measured using different microscope objectives before imaging cells stained with calcein. Both single and a pair of recently trypsinised cells were optically trapped and imaged. The position of the trapped cells was manipulated using a spatial light modulator in order to obtain a z-stack of images without adjusting the objective position.

Le ribosome est un organite essentiel de la cellule qui assure la synthèse des protéines. C'est une structure très conservée, composée d'ARN et de protéines ribosomiques organisés en deux sous-unités. Les expériences de reconstitution in vitro du ribosome d'E. coli ont montré que l'assemblage est un processus coordonné impliquant de nombreuses interactions entre les différents constituants. En particulier, les premières étapes de l'assemblage de la grande sous-unité dépendent fortement de la fixation coopérative de cinq protéines ribosomiques à l'ARN 23S, mais les mécanismes moléculaires sous-jacents sont mal connus.Cette étude à l'échelle de la molécule unique vise à préciser ces mécanismes et porte sur un fragment constitué des hélices H18, H19 et H20 du domaine I de l'ARN ribosomique 23S contenant les sites de fixation des protéines uL24 et uL4. Ce fragment d'ARN a été préparé dans une configuration qui permet la mesure de force via un double piège optique. Les courbes de force obtenues ont permis de dresser une cartographie de la stabilité des structures du fragment d'ARN.Ces cartes ont été comparées en absence et en présence des protéines ribosomiques uL24 et/ou uL4, démontrant ainsi que le fragment d'ARN est stabilisé par la fixation des protéines uL24 et/ou uL4. Leur fixation est coopérative et la présence conjointe des deux protéines sur-stabilise les structures du fragment d'ARN.Ces résultats sont discutés dans la perspective de préciser le rôle du fragment d'ARN et des protéines ribosomiques uL24 et uL4 dans l'assemblage de la grande sous-unité du ribosome. / Ribosomes are essential organelles of the cell responsible for the synthesis of proteins. Their well conserved structure made of RNA and proteins is organized into two subunits. In vitro reconstitution of E. coli ribosomes showed that their assembly is a coordinated process which involves many interactions between the components. More specifically, the early stages of the large subunit assembly depend strongly on the cooperative binding of five ribosomal proteins to the 23S RNA. The underlying molecular mechanisms however remain poorly understood.The aim of this study is to shine new light on these mechanisms at the single molecule level. It focuses on a 23S ribosomal RNA fragment composed of the helices H18, H19 and H20 in domain I which encompasses the binding sites of the ribosomal proteins uL24 and uL4. This RNA fragment has been prepared in a dumbbell configuration and force versus displacement measurements have been performed using a dual optical trap. From these measurements, a map summarizing the mechanical stability of the RNA fragment has been determined.The maps obtained in absence and in presence of the ribosomal proteins uL24 and/or uL4 have been compared consequently demonstrating mechanical stabilization of the RNA fragment induced by the binding of uL24 and/or uL4. Moreover, their binding is cooperative and when both are present, the mechanical stabilization of the RNA fragment is enhanced.These results are discussed to specify the role of the RNA fragment and proteins uL24 and uL4 in the large ribosomal subunit assembly.

Desde o desenvolvimento dos primeiros métodos de controle do movimento e posição de partículas usando lasers, ainda no início da década de 1970, até o reconhecimento com o prêmio Nobel de Física de 2018, uma das principais e mais versáteis ferramentas de manipulação óptica, as chamadas pinças ópticas, têm sido usadas majoritariamente para explorar objetos em dois regimes de tamanho: o limite das partículas sub-nanométricas (átomos e moléculas simples) e o limite das partículas micrométricas (com aplicações especialmente em sistemas biológicos). Nesse trabalho, foi desenvolvido e construído um aparato experimental para aprisionar micro e nanopartículas numa pinça óptica, que pode ser controlada de forma dinâmica usando modulação acusto-óptica do feixe de aprisionamento. A calibração da pinça óptica foi feita por diversos métodos, incluindo o método de equipartição de energia e análise do potencial óptico, resultando em forças de aprisionamento da ordem de piconewtons por micrometros. Ademais, simulações computacionais de modelos estocásticos foram realizadas com o intuito de comparar os resultados experimentais com àqueles previstos teoricamente e guiar estudos futuros. / Since the development of early methods for controlling the motion and position of particles using lasers, in the 1970s, to the recognition with the 2018 Nobel Prize for Physics, one of the most versatile optical manipulation tools, the so-called optical tweezers, have been used mostly to explore objects in two limits of sizes: the sub nanometric particles (atoms and simple molecules) and the micrometric particles (with applications especially in biological systems). In this work, an experimental apparatus was developed and built to trap micro and nanoparticles in an optical tweezer that can be dynamically controlled, using acoustic-optical modulation of the trapping beam. The calibration of the optical tweezer was done using several methods, including the energy equipartition method and optical potential analysis, resulting in trapping forces on the order of piconewtons per micrometers. In addition, computational simulations of stochastic models were performed with the purpose of comparing the experimental results with those predicted theoretically and guiding future studies.

In an effort to develop an optical-acoustical understanding of ultrasound contrast agent microbubble dynamics in a micro-environment that resembles blood vessels, this thesis presents experimental work on optical trapping and acoustical probing of ultrasound contrast agent microbubbles confined in regenerated cellulose capillaries. First, we showed by acoustical means that the pressure threshold of an individual microbubble shell rupture increases significantly when confined in regenerated cellulose capillaries. We report that the shell rupture threshold in regenerated cellulose capillaries increased by at least 0.3 MPa from 0.8 MPa for unconfined microbubbles. Second, we achieved optical trapping and manipulation of ultrasound contrast agent microbubbles confined in capillaries using Hermite-Gaussian laser beams. / Graduation date: 2012

In dieser Arbeit wurden Nanopartikel mit einer Laserpinzette gespeichert und untersucht. Ein Schwerpunkt war speziell die Untersuchung der bei der Speicherung beteiligten Kräfte. Diese Abhängigkeiten wurden bei variablen Drücken im Bereich von einigen mbar betrachtet. Als Testpartikel dienten µm große Agglomerate aus Nanodiamanten. Aus den gemachten Beobachtungen wurden Erkenntnisse über das Verhalten und die Wechselwirkung von Streukraft, Gradientenkraft und photophoretischer Kraft gewonnen. Ein zweiter Schwerpunkt war die Charakterisierung der Partikel. Mit einem einfachen optischen Aufbau konnten Fluoreszenzspektren von Nanodiamanten mit N-V-Zentren aufgenommen werden. Zusätzlich wurden einige der Partikel mit dem Laserfarbstoff Rhodamin 6G benetzt und spektroskopiert.