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Title: Advances in super-resolution microscopy and their application to photosynthesis
Author: Barnett, Samuel Fardell Hastings
ISNI:       0000 0004 6493 956X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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In recent years, microscopy has undergone a revolution that has made it much more powerful and able to achieve sub-diffraction resolutions that have in the past been the absolute limit of the optical microscope. These novel techniques, collected under the umbrella term "Super-resolution" have advanced the resolving power of an optical imaging system to the nanometre level, allowing a much deeper insight into biological problems in near native conditions. These techniques have differing levels of enhancement but typically lower the temporal resolution in favour increasing the spatial resolution. This thesis concerns both the advancement, through the development of a novel camera architecture and testing of a previously unknown label, and application of super-resolution microscopy to studying photosynthetic systems. How accurately each molecule can be located limits localisation microscopy in building a coordinate map. Here is demonstrated a camera that has the potential to increase that accuracy by discarding the concept of traditional images in favour of a continuous exposure. The camera is shown to outperform a traditional imaging regime, boosting the signal-to-noise ratio by two. Photosynthesis is a system that has been tuned over billions of years to produce proteins capable of absorbing and transferring photonic energy to create extended light harvesting arrays. We investigate the capabilities of one of these light-harvesting proteins, CpcA from the phycobilisome of the cyanobacterium Synechocystis sp. PCC6803, as a super-resolution label. CpcA is tested both in vitro, in the form of a nanopatterned surface; and in vivo, expressed in Escherichia coli. Additionally, the work presented here focusses on the investigation of photosynthesis with advanced microscopy techniques, notably hyperspectral confocal microscopy, localisation microscopy and structured illumination microscopy. These techniques are applied to investigate the in vivo protein distributions of many proteins known to be functionally involved in photosynthesis such as photosystem I, YidC, CURT1A, FtsH1, FtsH2 and FtsH4. Finally, the investigation of chloroplast thylakoid structure with structured illumination microscopy is demonstrated, showing morphological changes to the ultrastructure under different light conditions. These changes are further examined in mutants of proteins involved in regulation and adaption of the system to different light conditions.
Supervisor: Hunter, C. Neil ; Cadby, Ashley J. ; Johnson, Matthew P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available