Optical transfection and injection techniques applied to mammalian and embryonic cells

Lasers in cytology;Transfection;Micrurgy;Mammals;Embryos

Torres, Maria Leilani ; Dholakia, Kishan ; Dholakia, Kishan

University:University of St Andrews

Department:Physics & Astronomy (School of)

关键词: Lasers in cytology;    Transfection;    Micrurgy;    Mammals;    Embryos;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/2547/MariaTorresPhDThesis.pdf?sequence=10&isAllowed=y

来源: DR-NTU

【 摘 要 】

The delivery of biomolecules into living cells is an important methodology in celland molecular biology. Optical methods using lasers are attractive tools for suchapplication. However, the interaction of the laser with the cell depends on thelaser type and the parameters used. Hence, in this thesis, optical transfection andinjection of both mammalian and embryonic cells is demonstrated using a varietyof laser sources. Furthermore, some key issues are addressed by demonstratingalternative configurations of optoinjection and transfection systems to develop arobust, user-friendly device with potential for commercialisation.Most optical methods for the delivery of molecules rely on complex and expensivelaser systems that occupy a large footprint. In order for the system to be accessibleto end-users, transient transfection of plasmid DNA into mammalian cells using aninexpensive continuous wave 405 nm diode laser is demonstrated. In this work,the laser parameters are varied in order to optimise the transfection efficiency. Bycalculating the temperature change upon irradiation of the focused violet light, themechanism of violet diode laser transfection is elucidated. Furthermore, the systemis used to deliver small interfering RNA molecules to specifically knock down aparticular protein within the cell. This work is a major step towards an inexpensiveand portable optical transfection system.The critical issue of accurate targeting of the cell membrane is also addressed inconventional near-infrared femtosecond optical transfection systems. A near-infraredfemtosecond holographic system is built utilising a spatial light modulator in orderto provide fast three dimensional beam translation. Computer control of dosageand 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 Pomatoceroslamarckii embryo. The holographic system can be employed to optoinject avariety of macromolecules into the embryo, as well as orient and position the embryoby switching to the continuous wave mode of the laser. Such development ofoptical techniques to deliver biomolecules and orient embryos will benefit the fieldof developmental biology.Lastly, to achieve controlled cavitation, limiting the mechanical effects of a nanosecondlaser source, an optically trapped microsphere undergoes laser induced breakdownin the presence of a cell monolayer. Laser induced breakdown of a trappedmicrosphere 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 suitedfor cell transfection. This technique is an excellent tool for plasmid-DNA transfection of multiples of cells with both reduced energy requirements and cell lysiscompared to previously reported approaches.Demonstrating optimised and successful delivery of macromolecules with the varietyof laser sources used in this thesis will advance the applicability of opticalinjection and transfection and allow more potential users to access the technique.This thesis advances optical injection and transfection for optimised delivery ofmacromolecules to both mammalian cells and a developing embryo.