Abstract not available

Thesis (Ph.D.) - University of St Andrews, May 2007.

Thesis (Ph.D.) - University of St Andrews, October 2007.

Thesis (M.S.)--University of North Texas, Dec., 2008. / Title from title page display. Includes bibliographical references.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2005. / Dr. Cheng Zhu, Committee Chair ; Dr. Periasamy Selvaraj, Committee Co-Chair ; Dr. Timothy Wick, Committee Member ; Dr. Lyle Sinor, Committee Member ; Dr. Raymond Vito, Committee Member ; Dr. Robert Nerem, Committee Member.

Thesis (Ph.D.) - University of St Andrews, May 2009.

Thesis (M.Phil.) - University of St Andrews, June 2009.

The delivery of biomolecules into living cells is an important methodology in cell and molecular biology. Optical methods using lasers are attractive tools for such application. However, the interaction of the laser with the cell depends on the laser type and the parameters used. Hence, in this thesis, optical transfection and injection of both mammalian and embryonic cells is demonstrated using a variety of laser sources. Furthermore, some key issues are addressed by demonstrating alternative configurations of optoinjection and transfection systems to develop a robust, user-friendly device with potential for commercialisation. Most optical methods for the delivery of molecules rely on complex and expensive laser systems that occupy a large footprint. In order for the system to be accessible to end-users, transient transfection of plasmid DNA into mammalian cells using an inexpensive continuous wave 405 nm diode laser is demonstrated. In this work, the laser parameters are varied in order to optimise the transfection efficiency. By calculating the temperature change upon irradiation of the focused violet light, the mechanism of violet diode laser transfection is elucidated. Furthermore, the system is used to deliver small interfering RNA molecules to specifically knock down a particular protein within the cell. This work is a major step towards an inexpensive and portable optical transfection system. The critical issue of accurate targeting of the cell membrane is also addressed in conventional near-infrared femtosecond optical transfection systems. A near-infrared femtosecond holographic system is built utilising a spatial light modulator in order to provide fast three dimensional beam translation. Computer control of dosage and targeting allows us to explore the potential of different targeting modalities. An enhanced optoinjection and transfection on mammalian cells is demonstrated. Furthermore, the system is applied to optically manipulate a developing Pomatoceros lamarckii embryo. The holographic system can be employed to optoinject a variety of macromolecules into the embryo, as well as orient and position the embryo by switching to the continuous wave mode of the laser. Such development of optical techniques to deliver biomolecules and orient embryos will benefit the field of developmental biology. Lastly, to achieve controlled cavitation, limiting the mechanical effects of a nanosecond laser source, an optically trapped microsphere undergoes laser induced breakdown in the presence of a cell monolayer. Laser induced breakdown of a trapped microsphere allows control over several parameters, such as the microsphere material, position of the breakdown from the monolayer and the size of the microsphere. Optimising these parameters provide limited mechanical effects, particularly suited for cell transfection. This technique is an excellent tool for plasmid-DNA transfection of multiples of cells with both reduced energy requirements and cell lysis compared to previously reported approaches. Demonstrating optimised and successful delivery of macromolecules with the variety of laser sources used in this thesis will advance the applicability of optical injection and transfection and allow more potential users to access the technique. This thesis advances optical injection and transfection for optimised delivery of macromolecules to both mammalian cells and a developing embryo.

Over the last few decades, the use of light to control and manipulate microscopic particles has become widespread. These methods are enabling new areas of research to flourish across the physical and biological sciences. This thesis describes investigations into both optical trapping and the closely related field of optical sorting. It documents the development of a variety of new techniques. The thesis begins with a short review of optical trapping and existing methods for sorting mixtures of microscopic particles. The first half of this chapter highlights some of the reasons behind optical trapping's rapid growth in popularity. By reviewing an array of methods for sorting particles and discussing the relative merits of each, the case for optical sorting is established. The second chapter describes research into using a spatial light modulator to create three-dimensional optically trapped colloidal structures using the time-sharing technique. Limiting factors inherent in the technology are discussed in detail. The third chapter reviews a sophisticated particle-tracking software package that has proved to be a considerable success. It was developed explicitly with colloidal microscopy in mind and experimental plots produced by the software are used throughout the thesis. Experimental studies have been performed into the behaviour of microscopic particles moving under the influence of two classes of propagation-invariant beams: Mathieu beams and Bessel beams. The Bessel beam studies have been complimented by a theoretical model and have led ultimately to a new method for the static optical sorting of both solid particles and biological cells, with particular emphasis on human blood. The fifth and final chapter describes how re-configurable optical devices can be implemented to spatially separate different colloidal species. A new method for creating arbitrary optical landscapes using an acousto-optic modulator is reported. This new technique is then used to optically sort four particle species simultaneously - the first experimental demonstration of polydisperse optical fractionation. Additionally, experiments are reported that demonstrate controlled, static optical sorting using a spatial light modulator.

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.