Monitoring physiological changes in cells after ionizing radiation using fluorescence lifetime imaging by time resolved single photon counting

• Main Author: Abdollahi Mirzanagh, Elham
• Format: Others
• Language: en
• Published: 2017
• Online Access: https://tuprints.ulb.tu-darmstadt.de/6972/1/ElhamAbdollahiMirzanagh_PhDThesis.pdf; Abdollahi Mirzanagh, Elham <http://tuprints.ulb.tu-darmstadt.de/view/person/Abdollahi_Mirzanagh=3AElham=3A=3A.html> (2017): Monitoring physiological changes in cells after ionizing radiation using fluorescence lifetime imaging by time resolved single photon counting.Darmstadt, Technische Universität, [Ph.D. Thesis]

Abstract Eukaryotic DNA is packed with histone and non-histone proteins into chromatin fibers. The higher order structure of the chromatin comprises loosely packed euchromatin and more densely packed heterochromatin. Euchromatin mainly consists of active gene regions, whereas repressive DNA is mainly included in heterochromatin. Remodeling of the local chromatin structure is critical for the regulation of gene expression and replication of the DNA, but also for repair, if DNA lesions are induced into chromatin. Exposure of the DNA to chemicals or ionizing radiation as well as natural processes can lead to an interruption of its integrity resulting in the formation of double strand breaks, an especially critical type of DNA damage. The inappropriate repair of double strand breaks may lead to genomic instability and finally to the development of cancer. To get further insight into the maintenance of genomic stability, the understanding of cellular responses to double strand breaks is highly required. The chromatin structure and its dynamics are suspected of playing a significant role in the regulation and facilitation of DNA repair. The main aim of this thesis has been to establish a chromatin compaction assay which is working in living mammalian cells and can be easily applied to different cell lines without the necessity of using genetic modifications. The major biological goal was to provide additional and independent evidence of a radiation-induced chromatin decondensation exceeding the hitherto proof based on fluorescence depletion. In addition other potential biological applications of the compaction assay should be explored. To achieve these aims, time correlated single photon counting fluorescence lifetime imaging microscopy (FLIM) was inspected and characterized in combination with different organic DNA dyes or fluorescent proteins, which might serve as chromatin compaction sensors. To test the irradiation response, the FLIM setup could be coupled to a beamline microscope or, alternatively combined with a 35 kV X-rays tube. The findings demonstrate that some single organic DNA binding dyes revealed a high dynamic range of chromatin compaction with respect to fluorescent histone FRET pairs. These newly established chromatin compaction probes were capable of detecting structural changes of chromatin induced via enzymatic treatments as well as osmolality changes. Furthermore, using postirradiative fixation, the exposure of murine cells to ion irradiation revealed a significant local enhancement of the lifetime values of dyes like Hoechst 34580 at sites of heterochromatic ion traversals, implying a local radiation- chromatin relaxation. Additionally, a global chromatin decompaction after X-rays irradiation could be detected in living cells proving the applicability of this approach as a live cell assay. Besides radiation induced changes, using the small differences in chromatin densities in different cell cycle phases or in resting versus cycling cells could be shown, demonstrating that the established FLIM-based chromatin compaction assay can also successfully applied to other questions or fields in biological research.