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Title: Super-resolution imaging to investigate the nanoscale localisation of proteins
Author: Francis, J. E.
ISNI:       0000 0004 7964 1782
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Any optical microscopy technique that overcomes the diffraction limit of light (250 nm) is considered to be super-resolution. Super-resolution optical fluctuation imaging (SOFI) can resolve structures below 250 nm by correlating fluctuations from probes that repeatedly switch between a fluorescent and non-fluorescent state. SOFI exists as an open-source algorithm that can be applied to fluorescent images from any optical system without additional hardware. Previous publications demonstrated the resolving capability of SOFI on filamentous structures without addressing any specific biological question. Therefore, through interdisciplinary collaboration, this work aimed to use SOFI as a tool to investigate complex biological systems. Several different proteins including hypoxia inducible factor two alpha (HIF-2a), components of neutrophil extracellular traps (NETs), and extracellular matrix (ECM) proteins such as laminins were resolved with SOFI by labelling their structures with a suitable fluctuating probe. Quantum dots (Qdots) have advantageous photophysical properties for SOFI, but they cannot specifically label nuclear proteins, so alternative approaches were employed, including the use of reversibly switching fluorescent proteins (RSFPs). The localisation of HIF-2a into speckles and its interaction with other HIF-related proteins was investigated using SOFI. Potential co-localisation of laminin N-terminus alpha 31 (LaNt a31) and laminin alpha 3 (LM a3), a subunit of laminin-332 (LM-332), was also more accurately probed using two-colour SOFI. Moreover, other super-resolution techniques were explored, including super-resolution radial fluctuations (SRRF), which enabled the fastmoving protein HIF-2a to be super-resolved in live cells. To achieve optimal super-resolution images, acquisition and post-processing parameters were stringently tested, with the image quality and resolution determined using quantitative software, such as the ImageJ plugin NanoJ-Super-resolution quantitative image rating and reporting of error locations (SQUIRREL). As well as two-dimensional (2D) SOFI, the reconstruction of three-dimensional (3D) SOFI images was also explored, to obtain additional z information about the protein of interest. Overall, using total internal reflection fluorescence (TIRF), light-sheet, and epifluorescence microscopy, a resolution enhancement for different proteins was achieved with SOFI by exploiting the random blinking of Qdots and using the RSFP Skylan-S. Through out-of-focus light removal and enhanced signal-to-noise ratio (SNR), additional structural information was obtained about several proteins, which could help to better understand their involvement in biological mechanisms related to pathological disease, which subsequently could lead to the development of new therapeutic targets in the future.
Supervisor: Levy, Raphael ; See, Violaine ; Bennett, Daimark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral