| dc.description.abstract | With recent advances in optical super-resolution microscopy, biological structures can be imaged with single-nanometre resolution using visible light. One implementation thereof, DNA-PAINT (Point Accumulation for Imaging in Nano-scale Topography), is based on the highly specific and transient binding of fluorescently labelled oligonucleotides, the "imager strands", to complementary strands with which the targets are labelled, the "docking strands". The imager-docking binding events are detected as fluorescence blinking and can be localised with single-nanometre precision. From the set of localised events a super-resolution image can be assembled. DNA-PAINT has multiple advantages over other imaging methods, e.g. high photon yields resulting in high resolution, a free choice of fluorophores while being effectively free from photobleaching, straightforward implementation on a conventional fluorescence microscope and the possibility of temporally multiplexed and quantitative imaging. In this thesis, a test sample based on functionalised microspheres is developed, which allows for optimisation of various DNA-PAINT imaging parameters and for the characterisation and testing of new variations and modifications of DNA-PAINT. One such method which was developed for this thesis, Quencher-Exchange-PAINT, facilitates temporally multiplexed imaging, which is based on the sequential exchange of imager strands targeting different docking strands. The exchange step is replaced by addition of competitive quencher-strands, allowing for rapid, low-crosstalk imager exchange even in biological samples with limited diffusion. Additionally, Proximity-Dependent PAINT is introduced, which enables the imaging of the nanoscale distribution of protein pairs by interaction of two proximity probes which activates DNA-PAINT type binding. The technique is demonstrated both on the microsphere assay as well as in biological samples. Finally, approaches for enhancing the signal-to-noise ratio are explored. | en_GB |
