Mthunzi-Kufa, Patience2012-04-202012-04-202010-02Mthunzi, P. 2010. Optical sorting and photo-transfection of mammalian cells. University of St Andrewshttp://research-repository.st-andrews.ac.uk/handle/10023/1254http://hdl.handle.net/10204/5810Copyright: 2010 The author. A Thesis submitted for the Degree of PhD at the University of St. AndrewsRecently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.enOptical cell sortingIntracellular dielectric taggingAxial and scattering forcesPhoto-transfectionFemtosecond laser pulsesCell arrestingNeuroblastomasPluripotent stem cellsEmbryonic stem cell differentiationReportMthunzi, P. (2010). <i>Optical sorting and photo-transfection of mammalian cells</i> (Workflow;6401). University of St Andrews. Retrieved from http://hdl.handle.net/10204/5810Mthunzi, P <i>Optical sorting and photo-transfection of mammalian cells.</i> Workflow;6401. University of St Andrews, 2010. http://hdl.handle.net/10204/5810Mthunzi P. Optical sorting and photo-transfection of mammalian cells. 2010 [cited yyyy month dd]. Available from: http://hdl.handle.net/10204/5810TY - Report AU - Mthunzi, P AB - Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm. DA - 2010-02 DB - ResearchSpace DP - CSIR KW - Optical cell sorting KW - Intracellular dielectric tagging KW - Axial and scattering forces KW - Photo-transfection KW - Femtosecond laser pulses KW - Cell arresting KW - Neuroblastomas KW - Pluripotent stem cells KW - Embryonic stem cell differentiation LK - https://researchspace.csir.co.za PY - 2010 T1 - Optical sorting and photo-transfection of mammalian cells TI - Optical sorting and photo-transfection of mammalian cells UR - http://hdl.handle.net/10204/5810 ER -