Optical transfection and injection techniques applied to mammalian and embryonic cells

Abstract

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.