Bento Carvalho Almada, Pedro Manuel;
(2018)
Developing Highly Multiplexed Technology for High-throughput Super-resolution Fluorescence Microscopy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
High-Throughput imaging can reconstruct complex signalling networks, reveal unknown interactions and capture rare cellular events. Simultaneously, the development of Single Molecule Localization Super Resolution Microscopy has enabled molecular-level structural information to be obtained in a single cell. But the increase in resolution comes at a trade-off for the amount of molecular species that can be imaged and the time it takes to acquire data, all of which limit the applicability of super-resolution to high-throughput work-flows. The present work details a framework to address this. It combines three independent approaches: a microscope hardware design approach to increase the amount of data that can be obtained in a Super-Resolution experiment; an optofluidics platform that can be wholly synchronized with most microscopes; and a sequential labelling framework to increase the number of species that can be imaged in Super-Resolution in a single cell. The hardware design is validated by performing Single Molecule Localization of cytoskeleton components and its throughput is shown to be up to an order of magnitude larger than a corresponding commercial system. We demonstrate a complete optofluidics platform to integrate microfluidics with a microscope, enabling live imaging, drug application, fixation, and staining in single cells synchronized with imaging protocols. Finally, we show an efficient sequential labelling protocol that is compatible with the optofluidics platform, enabling several molecular species to be imaged in the same cells. Overall, our approach increases the speed and amount of data that can be acquired in a single of Super-Resolution experiment, as well as, by performing on-line fixation, considerably improves our capacity for High-Throughput experiments in Super-Resolution imaging.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Developing Highly Multiplexed Technology for High-throughput Super-resolution Fluorescence Microscopy |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10041569 |
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