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    Interfacial colloidal assembly guided by optical tweezers and tuned via surface charge
    (Elsevier Inc, 2022-09) Pradhan S; Whitby CP; Williams MAK; Chen JLY; Avci E
    HYPOTHESIS: The size, shape and dynamics of assemblies of colloidal particles optically-trapped at an air-water interface can be tuned by controlling the optical potential, particle concentration, surface charge density and wettability of the particles and the surface tension of the solution. EXPERIMENTS: The assembly dynamics of different colloidal particle types (silica, polystyrene and carboxyl coated polystyrene particles) at an air-water interface in an optical potential were systematically explored allowing the effect of surface charge on assembly dynamics to be investigated. Additionally, the pH of the solutions were varied in order to modulate surface charge in a controllable fashion. The effect of surface tension on these assemblies was also explored by reducing the surface tension of the supporting solution by mixing ethanol with water. FINDINGS: Silica, polystyrene and carboxyl coated polystyrene particles showed distinct assembly behaviours at the air-water interface that could be rationalised taking into account changes in surface charge (which in addition to being different between the particles could be modified systematically by changing the solution pH). Additionally, this is the first report showing that wettability of the colloidal particles and the surface tension of the solution are critical in determining the resulting assembly at the solution surface.
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    The development of optical nanomachines for studying molecules : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Mechatronics Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Andrew, Philippa-Kate
    Optical tweezers have been used for a number of applications since their invention by Arthur Ashkin in 1986, and are particularly useful for biological and biophysical studies due to their exceptionally high spatial and force-based resolution. The same intense laser focus that allows light to be used as a tool for micro-nanoscale manipulation also has the potential to damage the objects being studied, and the extremely high force resolution is coupled with the limitation of very low forces. There is potential to overcome these drawbacks of optical manipulation through making use of another laser based technique: two-photon absorption polymerisation (TPAP). This thesis has brought these together to demonstrate the uses of optical nanomachines as helpful tools for optical tweezer studies. The project was highly interdisciplinary, concerning the intersection of optical trapping, 3D micromachine design and development, and DNA stretching. The thesis was based around the strategy of first developing microrobots and demonstrating their manipulation using optical tweezers, then adjusting the design for specific applications. Microlevers were developed for lever-assisted DNA stretching and amplification of optical forces. The influence of design features and TPAP parameters on microlever functionality was investigated; particularly the influence of overlapping area and presence of supports, and the effects of differently shaped "trapping handles". These features were important as lever functionality was tested in solutions of different ionic strength, and stable trapping of the levers was required for force amplification. DNA stretching was chosen as a target application for distanced-application of optical forces due to its status as a well-known and characterised example of single-molecule studies with optical tweezers. Amplification of optical forces was also seen as an application that could demonstrate the utility of optical micromachines, and microlevers with a 2:1 lever arm ratio were developed to produce consistent, two-fold amplification of optical forces, in a first for unsupported, pin-jointed optical microrobotics. It is hoped that in the future fully-remote, micromachine-assisted studies will extend optical tweezer studies of laser-sensitive subjects, as well as increasing the forces that can be applied, and the results obtained in this thesis are encouraging. All in all, the thesis confirms the potential of optical micromachines for aiding studies using optical tweezers, and demonstrates concrete progress in both design and application.
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    Biophysical investigations of cells focusing on the utility of optical tweezers : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Biophysics and Soft Matter Group, School of Fundamental Science, Massey University, New Zealand
    (Massey University, 2021) Pradhan, Susav
    The aim of this thesis was to explore the utility of different biophysical techniques, particularly optical tweezers (OT), in the investigation of the mechanical properties and interactions of biological samples. Specifically, MCF7 cells and their extracted nuclei were investigated mechanically, while the adhesion property of selected bacteria to the milk fat globule was also used as an exemplar. Biological cells have the ability to actively respond to external mechanical forces exerted by the microenvironment. The cellular response can be viscous, elastic, or viscoelastic in nature depending on the nature of the applied forces and the mechanical stresses applied. Changes in the mechanical properties of cells and nuclei have emerged as a prominent hallmark of many human diseases, particularly in neurodegenerative and metastatic diseases. In this thesis, to understand the application of these techniques to biological systems better, bulk rheology and microrheology studies were first performed on a model viscoelatic fluid (PEO). Particularly, the passive and active microrheology of this model viscoelastic material was characterized using optical tweezers and video particle tracking to develop the prerequisite experimental and analytical methods. Using the experimental knowledge gained from applying optical tweezers to standard materials, a mechanistic approach was developed in order to better understand how the mechanical properties of MCF7 cells change when the amount of heterochromatin protein (HP1a) present inside the nuclei was reduced. (HP1a) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. While the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin modifying enzymes have been studied previously, here we measure how the material properties of the cells are modified following the knockdown HP1a. Indentation experiments using optical tweezers revealed that the knockdown cells have apparent Young’s modului significantly lower than control cells. Similarly, tether experiments performed using optical tweezers revealed that the membrane tensions of knockdown cells were lower than those of control cells. This assay led to further work on studying the mechanical properties of nuclei extracted from MCF7 cells. A combination of atomic force microscopy, optical tweezers, and techniques based on micropipette aspiration was used to characterize the mechanical properties of nuclei extracted from HP1a knockdown or matched control cells. Similar to the previous finding on cells, local indentation performed using atomic force microscopy and optical tweezers found that the knockdown nuclei have apparent Young’s modului significantly lower than control nuclei. In contrast, results from pipette-based techniques in the spirit of microaspiration, where the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1a knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organisation observed by various microscopy techniques between the MCF7 control and HP1a knock-down nuclei correlated well with the results of our measured mechanical responses and our hypotheses regarding their origin. Finally, not just the mechanical properties of the cells but also their interactions (an interaction between the milk fat globule membrane and two bacterial strains - Lactobacillius fermentum strains - 1487 and 1485) was explored as a side project by probing with optical tweezers. The difference in bacterial cell surface properties of these two strains and its effects on intestinal epithelial barrier integrity has already been studied. This study focuses on measuring the adhesion force between membrane and bacteria using optical tweezers. The results suggested that L. fermentus AGR1487 strongly interacts with MFGM compared to AGR1485. All in all, this thesis demonstrates how biophysical techniques can provide valuable insights into understanding biological systems.
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    Setup and calibration of a suite of state-of-the-art microrheology techniques : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physics at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Mansel, Bradley William; Mansel, Bradley William
    Microrheology is the study of the flow and deformation of fluids on the micrometre scale. It has many benefits over the use of traditional rheomoeters to measure the mechanical properties of fluids. Microrheology has small sample sizes, can extract information about the underlying heterogeneities, often has a lower setup cost, can measure to higher frequencies and can measure the viscoelasticity of in-vivo samples. Work has been carried out to setup and calibrate four different microrheology techniques, namely: diffusing wave spectroscopy, dynamic light scattering, multiple particle tracking and probe laser tracking with a quadrant photodiode and optical traps. This resulted in the ability to measure the viscoelastic properties of a material over approximately eight orders of magnitude, with nanometre resolution on the most sensitive technique; diffusing wave spectroscopy. The link between free Brownian motion and a particle diffusing in a harmonic potential was used to calibrate the trap strength of the optical tweezers, enabling a comparison of three different trap calibration techniques. Calibration of the trap strength in optical tweezers resulted in a good agreement between different methods, although, the power spectral density method proved easier to implement and more accurate over the range of laser powers, making it the superior method to use. To illustrate the power of microrheology techniques, the mechanical properties of standard viscous and viscoelastic fluids were first compared. Also organelles in pollen tubes were tracked to simply and accurately measure properties of a complex biological system in-vivo.