Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free-space bulk optical setup. Here in this thesis, a few novel fabrication methods are devised to obtain easy and inexpensive fabrication of microlensed optical fibers, which can be used to replace traditional optical setup and perform femtosecond optical transfection. These fabrication methods offer the flexibility to fabricate a microlens which can focus femtosecond laser pulses at 800 nm to a small focal spot whilst keeping a relatively large working distance. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. This thesis reports the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining lensed fiber based optical transfection system with a micro-capillary based microfluidic system. Finally, based on an imaging fiber (coherent optical fiber bundle), the first endoscope-like integrated system for optical transfection with subcellular resolution epifluorescence imaging was built. The transfection efficiency of these fiber based systems is comparable to that of a standard free-space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of genetic material for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, such as the dosing of tissue slices to study neuron activities, targeted drug delivery, and in particular for using endoscope-like integrated systems for targeted in vivo optical microsurgery.

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.

Optical cell manipulation allows precise and non-invasive exploration of mammalian cell function and physiology for medical applications. Plants, however, represent a vital component of the Earth's ecosystem and the knowledge gained from using optical tools to study plant cells can help to understand and manipulate useful agricultural and ecological traits. This thesis explores the potential of several biophotonic techniques in plant cells and tissue. Laser-mediated introduction of nucleic acids and other membrane impermeable molecules into mammalian cells is an important biophotonic technique. Optical injection presents a tool to deliver dyes and drugs for diagnostics and therapy of single cells in a sterile and interactive manner. Using femtosecond laser pulses increases the tunability of multiphoton effects and confines the damage volume, providing sub-cellular precision and high viability. Extending current femtosecond photoporation knowledge to plant cells could have sociological and environmental benefits, but presents different challenges to mammalian cells. The effects of varying optical and biological parameters on optical injection of a model plant cell line were investigated. A reconfigurable optical system was designed to allow easy switching between different spatial modes and pulse durations. Varying the medium osmolarity and optoinjectant size and type affected optoinjection efficacy, allowing optimisation of optical delivery of relevant biomolecules into plant cells. Advanced optical microscopy techniques that allow imaging beyond the diffraction limit have transformed biological studies. An ultimate goal is to merge several biophotonic techniques, creating a plant cell workstation. A step towards this was demonstrated by incorporating a fibre-based optical trap into a commercial super-resolution microscope for manipulation of cells and organelles under super-resolution. As proof-of-concept, the system was used to optically induce and quantify an immunosynapse. The capacity of the super-resolution microscope to resolve structure in plant organelles in aberrating plant tissue was critically evaluated.

Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

Broadband spectral content is required to support ultrashort pulses. However this broadband content is subject to dispersion and hence the pulse duration of corresponding ultrashort pulses may be stretched accordingly. I used a commercially-available adaptive ultrashort pulse shaper featuring multiphoton intrapulse interference phase scan technology to characterise and compensate for the dispersion of the optical system in situ and conducted experimental and theoretical studies in various inter-linked topics relating to the light-matter interaction. Firstly, I examined the role of broadband ultrashort pulses in novel light-matter interacting systems involving optically co-trapped particle systems in which inter-particle light scattering occurs between optically-bound particles. Secondly, I delivered dispersion-compensated broadband ultrashort pulses in a dispersive microscope system to investigate the role of pulse duration in a biological light-matter interaction involving laser-induced cell membrane permeabilisation through linear and nonlinear optical absorption. Finally, I examined some of the propagation characteristics of broadband ultrashort pulse propagation using a computer-controlled spatial light modulator. The propagation characteristics of ultrashort pulses is of paramount importance for defining the light-matter interaction in systems. The ability to control ultrashort pulse propagation by using adaptive dispersion compensation enables chirp-free ultrashort pulses to be used in experiments requiring the shortest possible pulses for a specified spectral bandwidth. Ultrashort pulsed beams may be configured to provide high peak intensities over long propagation lengths, for example, using novel beam shapes such as Bessel-type beams, which has applications in biological light-matter interactions including phototransfection based on laser-induced cell membrane permeabilisation. The need for precise positioning of the beam focus on the cell membrane becomes less strenuous by virtue of the spatial properties of the Bessel beam. Dispersion compensation can be used to control the temporal properties of ultrashort pulses thus permitting, for example, a high peak intensity to be maintained along the length of a Bessel beam, thereby reducing the pulse energy required to permeabilise the cell membrane and potentially reduce damage therein.