There are a growing variety of research fields requiring non-contact micro- manipulation. An increasing number of these fields are turning to optical tweezers as a solution, owing to their high spatial and temporal resolution. Optical tweezers have the ability to quantitively exert and measure forces on the piconewton scale, a convenient force scale for soft biological materials, and are hugely versatile due to the wide assortment of beam shaping techniques that can be employed. The work in this thesis can be broadly divided into two main themes: that quantifying the optical trapping forces in shaped beams; and bringing control and simplification of complex systems to non-expert users who may utilise optical tweezers as part of interdisciplinary collaborations. Static beam shaping is used to generate a conically refracted optical trap and the trapping properties are characterised. It is shown that trapping in the lower Raman spot gives full, 3D gradient trapping, while the upper Raman spot allows for particle guiding due to its levitation properties. Particles in the Lloyd/Poggendorff rings experience a lower trap stiffness than particles in the lower Raman spot but benefit from rotational control. Dynamic beam shaping techniques are exploited for the simplification of complex systems through the development and testing of the HoloHands program. This software allows a holographic optical tweezers experiment to be controlled by gestures that are detected by a Microsoft Kinect. Multiple particle manipulation is demonstrated, as well as a calibration of the tweezers system. Application of trapping forces is demonstrated through an examination of integrin – ligand bond strength. Both wild type effector T cells and those with a kindlin-3 binding site mutation similar to that found in neutrophils from Leukocyte Adhesion Deficiency sufferers are investigated. Through the use of back focal plane interferometry, a bond rupture force of (17.9 ± 0.6) pN at a force loading rate of (30 ± 4) pN/s, was measured for single integrins expressed on wild type cells. As expected, a significant drop in rupture force of bonds was found for mutated cells, with a measured rupture force of (10.1 ± 0.9) pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects bond strength of single integrin-ligand bonds. An experimental system for studying these cells under more physiologically relevant conditions is also presented. Additionally, a low-cost optical micromanipulation system that makes use of simple microfabricated components coupled to a smartphone camera for imaging is proposed and demonstrated. Through the layering of hanging droplets of polydimethylsiloxane (PDMS) on microscope coverslips, lenses capable of optical trapping are created. Combination of PDMS with Sudan II dye led to the fabrication of long pass filters. An extension of this low-cost system into the life sciences is proposed through the adaptive use of bubble wrap, which allows for the culturing of cells in a chamber compatible with optical trapping.

This dissertation describes the development of a device capable of suspending a microscale object in a controlled flow. The uPIVOT is a system integrating two laser-based techniques: micron particle image velocimetry (uPIV) and optical tweezers (OT). The OT allows the suspension and manipulation of micron-sized objects such as microspheres or biological cells. uPIV provides imaging of the suspended object and velocity measurements from which fluid induced stresses can be determined. Using this device, we measured fluid velocities around an optically suspended polystyrene microsphere (an experimental first) and studied the interaction between two particles suspended in a uniform flow. The results were consistent with theoretical low Reynolds number, Newtonian flow predictions. Additionally, we analyzed a single cell's mechanical response to a controlled and measurable multiaxial external force (fluid flow) without the cell being physically attached to a surface. The cell's mechanical response was monitored by observing its morphology and measuring its deformation. The results show significant deformations of optically suspended cells at substantially smaller stresses than previously reported and demonstrate the opportunity to optically distinguish a cell by its trapping efficiency. These initial applications of the uPIVOT demonstrate the potential of this unique device as a research tool for novel studies in the fields of fluid/particle(s) interactions, non-Newtonian fluid mechanics, and single cell biomechanics.

In the late 19th and early 20th centuries famous scientists like Boltzmann, Loschmidt, Maxwell and Einstein tried, unsuccessfully, to find the link between the time-reversible equations of motion of individual molecules and irreversible thermodynamics. The solution to this puzzle was found in 1993, and the link is now known as the Fluctuation Theorem (FT). In the decade that followed theory and computer simulation tested the FT and, in 2002, an experiment indirectly demonstrated the FT.¶ This thesis describes original experiments that demonstrate the FT directly using Optical Tweezers. A related expression, known as the Kawasaki Identity, is also experimentally demonstrated. These experimental results provide a rigorous demonstration that irreversible dynamics can be obtained from a system with time-reversible dynamics.

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed. The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve ngstrm-level resolution through careful design to reduce noise.

This study proposes a novel microfluidic system based on a computer controlled digital image processing (DIP) technique and optical tweezers for automatic cell/microparticle recognition, counting and sorting in a continuous flow environment. In the proposed system, the cells/microparticles are focused electrokinetically into a narrow sample stream and are then driven through the region of interest (ROI), where they are recognized and traced in real time using a proprietary DIP system. Synchronized control signals generated by the DIP system are then used to actuate a focused IR laser beam to displace the target cells from the main sample stream into a neighboring sheath flow, which carries them to a downstream collection channel where they are automatically counted. The proposed approach makes possible the continuous sorting and counting of cell samples without the need for any moving parts or embedded transducers. The experimental results show that the proposed system is capable of sorting 5 £gm or 10 £gm PS bead from a mixture of 5 £gm and 10 £gm samples in the flow speed 300 £gm/sec. The proposed system provides a simple, low-cost, high-performance solution for cell manipulation in microfluidic devices.

Die heterobimetallischen Pi-Pinzetten können erfolgreich als Grundbaustein für die Darstellung von mehrkernigen Übergangsmetallkomplexen, in denen die Metallzentren M(I) (M = Cu, Ag) über unterschiedliche stickstoffhaltige organische sigma-Donor-Liganden verbrückt sind, verwendet werden. In Abhängigkeit von der Natur der korrespondierenden organometallischen Pi-Pinzetten und der organischen Brückeneinheiten konnten verschiedene mehrkernige Übergangsmetallkomplexe synthetisiert werden. Diese Komplexe wurden mittels IR, 1H- und 13C-NMR-Spektroskopie sowie MS charakterisiert. Die Festkörperstrukturen ausgewählter Komplexe konnten durch Einkristall-Röntgenstrukturanalyse erhalten werden. Die Wechselwirkungen der Kupfer(I)- sowie Silber(I)-Ionen, über die koordinativ gebundenen stickstoffhaltigen. Stichworte: Organometallischen À-Pinzetten; Bis(alkinyl)titanocene; Titanium; À- Konjugierte; Mehrkernigen; Kupfer(I), Silber(I).

Thesis (M. Sc.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on Jan. 27, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Physics, University of Alberta. Includes bibliographical references.

Mechanical unfolding of -1 frameshift signals such as RNA pseudoknots have aimed to test the hypothesis that the stability of the pseudoknot is directly correlated to the frameshifting efficiency. Here we report unfolding of the Beet Western Yellow Virus (BWYV) pseudoknot by optical tweezers experiments complemented by computer simulations using steered molecular dynamics (SMD). Seven pseudoknot scenarios were studied: the wild-type pseudoknot in the presence and absence of Mg<super>2+</super>, the wild-type pseudoknot at high pH (deprotonated C8), and C8U, C8A, A24G, G19U, and G19UC mutant constructs. The mutants were selected to probe three key structural features of the BWYV pseudoknot, a triple-stranded helix at the base of stem 1, the stem junction region of stem 1 and stem 2, and a unique quadruple base-pair interaction involving a protonated cytosine in position 8 (C8). These regions are thought to control ribosomal frameshifting by different strategies such as thermodynamic stability, kinetic influences, and dynamics involving contacts with the ribosome. In addition, the mutants have been shown to either abolish frameshifting ability of the pseudoknot (C8 mutant cases and A24G), or actually increase the frameshifting efficiency (as seen with G19U and G19UC). We find three major conclusions from the stretching of the pseudoknot constructs with optical tweezers. First, stretching in the absence of Mg<super>2+</super> results in no observed unfolding transitions. We interpret this to mean that magnesium is indispensible for the stable folding of the pseudoknot. Second, we found that frameshifting efficiency is not correlated with the force required to unfold the pseudoknots. However, we observe the unfolding of stem 1 in all of the pseudoknots stretched, where stem 2 unfolding is below our noise level. For this reason, we cannot rule out the possibility that an estimate of the thermodynamic stability of the entire pseudoknot would correlate with frameshifting efficiency. And third, we found that each pseudoknot mutant that resulted in reduced frameshifting efficiency also exhibited more off-equilibrium unfolding transitions that the wild-type pseudoknot under comparable loading rates. We conclude from these studies that the resistance of a pseudoknot to unfolding is controlled by both thermodynamic and kinetic parameters. We then suggest new technologies that would allow for greater resolution in order to correlate pseudoknot unfolding behavior with -1 programmed ribosomal frameshifting events.

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