An automated fluorescence lifetime imaging multiwell plate reader: application to high content imaging of protein interactions and label free readouts of cellular metabolism

This thesis reports on work performed in the development and application of an automated plate reading microscope implementing wide field time gated fluorescence lifetime imaging technology. High content analysis (HCA) imaging assays enabled by automated microscopy platforms allow hundreds of conditions to be tested in a single experiment. Though fluorescence lifetime imaging (FLIM) is established in life sciences applications as a method whereby quantitative information may be extracted from time-resolved fluorescence signals, FLIM has not been widely adopted in an HCA context. The FLIM plate reader developed throughout this PhD has been designed to allow HCA-FLIM experiments to be performed and has been demonstrated to be capable of recording multispectral, FLIM and bright field data from 600 fields of view in less than four hours. FLIM is commonly used as a means of reading out Förster resonance energy transfer (FRET) between fluorescent fusion proteins in cells. Using the FLIM plate reader to investigate large populations of cells per experimental condition without significant user input has allowed statistically significant results to be obtained in FRET experiments that present relatively small changes in mean fluorescent lifetime. This capability has been applied to investigations of FOXM1 SUMOylation in response to anthracycline treatment, and to studies of the spatiotemporal activation profiles of small GTPases. Furthermore, the FLIM plate reader allows FLIM-FRET to be applied to protein-protein interaction screening. The application of the instrument to screening RASSF proteins for interaction with MST1 is discussed. The FLIM plate reader was also configured to utilise ultraviolet excitation radiation and optimised for the measurement of autofluorescence lifetime for label-free assays of biological samples. Experiments investigating the autofluorescence lifetime of live cells under the influence of metabolic modulators are presented alongside the design considerations necessary when using UV excitation for HCA-FLIM.Open Acces

Stem Cell Oncology

Stem cell research is one of the fascinating areas of contemporary biology, but, as with many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates discoveries. Research on stem cell treatment continues to advance knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. The most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies, especially oncology. The Faculty of Medicine, Universitas Sumatera Utara, collaborated with the center of excellence and innovation (Pusat Unggulan Inovasi /PUI). The Stem Cell center of the Universitas Sumatera Utara (USU) organized an International Conference. The International Stem Cell and Oncology Conference (ISCOC) 2017 was an comprehensive academic conference in the field of stem cell and oncology research and also tropical medicine and related scientific topics. We expect this conference will benefit academics and practitioners in the field of health sciences in Indonesia. During the two day conference, researchers presented the most recent discoveries in stem cell and oncology and also general medical topics, and served as well in establishing networking for joint researchers and collaboration among participants. The editors and the scientific committee would like to thank the participants and express their gratitude to Faculty of Medicine of the Universitas Sumatera Utara for their support to ISCOC 2017. Lastly, we are most indebted for the generous support given by Universitas Sumatera Utara, Medan, Sumatera Utara, Indonesia