Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.552494
Title: Optical sorting and photo-transfection of mammalian cells
Author: Mthunzi, Patience
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 2010
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Abstract:
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.
Supervisor: Dholakia, Kishan; Council for Scientific and Industrial Research, South Africa Sponsor: University of St Andrews
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.552494  DOI: Not available
Keywords: Optical cell sorting ; Intracellular dielectric tagging ; Axial and scattering forces ; Photo-transfection ; Femtosecond laser pulses ; Cell arresting ; Neuroblastomas ; Pluripotent stem cells ; Embryonic stem cell differentiation ; QH585.5F56M8 ; Flow cytometry ; Femtosecond lasers ; Transfection ; Mammals--Cytology
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