Gustafsson, Nils S;
(2017)
Enabling live-cell super-resolution microscopy by computational analysis and fluorescent probe design.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Super-resolution fluorescence microscopy has become a well-established tool for structural cell biology, achieving lateral resolutions on molecular scales. Limitations to the application of super-resolution in live-cell imaging remain however. Particularly, the requirement for intense phototoxic illumination, prolonged acquisition times, non-physiological media and extensive computational analysis. This thesis aims to address these issues, through both analytical and biochemical approaches. Three analytical methods are presented and evaluated, including a method for nano-meter precision microtubule end tracking applicable to live-cells, and a Hidden Markov model of fluorophore photoswitching, for use in live-cell experimental design. The third, Super-Resolution Radial Fluctuations (SRRF), is a novel image processing algorithm that forms the basis of a new approach to live-cell super-resolution. Low-illumination, live-cell imaging of actin dynamics in T-cell synapses at resolutions better than 100 nm are demonstrated in physiologically relevant media using SRRF. In addition, SRRF enables super-resolution in modern widefield or confocal microscopes using conventional fluorophores with illumination orders of magnitude lower than methods such as single molecule localisation or stimulated emission depletion. The computational framework developed for SRRF, NanoJ, is an open-source java library of high performance algorithms that has enabled the development of a number of GPU accelerated ImageJ/FIJI plugins for super-resolution. While SRRF allows super-resolution in live-cells, achieving resolutions on the order of 10s of nm remains challenging. To this end, Super-Beacons (SBs), novel DNA-based super-resolution probes that photoswitch spontaneously under environments relevant for live-cell imaging, are presented. The design of SBs allows the engineering of photoswitching within the physical environment of the cell, mediated through structural, chemical or thermal control. Single molecule imaging and characterisation of the photoswitching of the probes is performed. The use of SBs is verified in fixed-cell imaging of benchmark structures and live-cell super-resolution of interferon inducible transmembrane proteins is demonstrated resolving the endosomal membrane with 65 nm resolution.
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