Thesis

Optimizing wide-field illumination and single-molecule photoswitching for localization microscopy

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17518
Person Identifier (Local)
  • 201785705
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Department, School or Faculty
Abstract
  • Nanometre-scale molecules such as proteins and DNA constitute the building blocks of biological structures responsible for every function of life. Conventional light microscopy allows to study cells but it meets a lateral resolution limit of 250 nm as described by Ernst Abbe in 1873. Abbe’s limit remained unbroken until the end of the twentieth century when E. Betzig, W. E. Moerner and S. Hell developed super-resolution microscopy. Our work concentrates on direct STochastic Optical Reconstruction Microscopy (dSTORM), a single molecule localisation microscopy (SMLM) technique, that consists of the photoswitching of fluorescent molecules and their localisation with nanometre precision followed by the reconstruction of a super-resolution image from the localisation information. However, the quality of results using dSTORM depend strongly on the careful tuning of many parameters from the microscopy hardware to sample preparation and data analysis. This work addresses several avenues of optimisation of the dSTORM process through four themes. First, improvements to hardware and data analysis for optimizing 3D SMLM are described in the context of building a versatile dSTORM-focused microscopy platform. The careful characterisation of our setup allowed the identification and investigation of several improvement paths. Second, a Forster Resonance Energy Transfer (FRET) -based method was explored to enable multi-channel dSTORM without chromatic aberrations. While the concept could be demonstrated in ensemble spectroscopy, single molecule imaging did not show proper channel separation which lead us to explore two aspects of dSTORM imaging to improve the channel separation, illumination across the field of view and the influence of the buffer on photoswitching. Third, we evaluated how a single Micro Electro Mechanical System (MEMS) mirror could produce a uniform illumination across a large field of view and compared it to a recognized optical beam-shaping device, the piShaper. Our approach matched the pishaper performance with an improved tuneability to produce other illumination schemes. We also presented our comparison method that can be applied to other illumination schemes. Finally, we systematically studied the influence of pH and Thiol concentration on photoswitching and on the quality of dSTORM imaging with Alex Fluor 647. We concluded that the thiolate concentration rather than pH or the total thiol concentration alone is the relevant parameter influencing photoswitching and the quality of results and observed a degradation in resolution for sub-optimal buffer conditions when we imaged the glucose transporter GLUT-4 in the plasma membrane of adipocytes. We also proposed a framework to optimise the switching buffer for dSTORM. Overall, this work explored several approaches to improve the quality of dSTORM and proposes several methods to achieve better results in dSTORM experiments through hardware, sample preparation and data analysis optimisation.
Advisor / supervisor
  • Van de Linde, Sebastian
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DOI
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