부산대학교 도서관

This PhD thesis reports the development and application of fluorescence imaging technologies for studying biological processes on spatial scales below the diffraction limit. Two strategies were addressed: firstly fluorescence lifetime imaging (FLIM) to study molecular processes, e.g. using Förster resonance energy transfer (FRET) to read out protein interactions, and secondly direct imaging of nanostructure using super-resolution microscopy (SRM). For quantitative FRET readouts, the development and characterisation of an automated multiwell plate FLIM microscope for high content analysis (HCA) is described. Open source software was developed for the data acquisition and analysis, and approaches to improve the performance of time-gated imaging for FLIM were evaluated including different methods to despeckle the laser illumination and testing of an enhanced detector. This instrument was evaluated using standard fluorescent dye samples and cells expressing fluorescent protein-based FRET constructs. It was applied to an assay of live cells expressing a FRET biosensor and to FRET readouts of aggregation of a membrane receptor (DDR1) in fixed cells. A novel instrument, combining structured illumination microscopy (SIM) with FLIM, was developed to explore the combination of SRM and FLIM-FRET readouts. This enabled the simultaneous mapping of molecular readouts with FLIM and super-resolved imaging. The SIM+FLIM system was applied to image collagen-stimulated DDR1 aggregation in cells, to image DNA structures during the cell cycle and to explore interactions between cell organelles. A novel SRM approach based on a stimulated emission of depletion (STED) microscope incorporating a spatial light modulator (SLM) was developed to provide straightforward robust alignment with collinear excitation/depletion beams, aberration correction, an extended field of view and multiple beam scanning for faster STED image acquisition. The performance of easySLM-STED was evaluated by imaging bead samples, labelled vimentin in Vero cells and the synaptonemal complex in homologs of C. elegans germlines.