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Lifetime image reconstruction super resolution microscopy: principles, applications and underlying photophysics

Culley, S; (2015) Lifetime image reconstruction super resolution microscopy: principles, applications and underlying photophysics. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Many biological structures exist on a scale smaller than can be resolved by conventional fluorescence microscopy, which has limited the study of cellular processes. For this reason, there has been a large amount of research over the past decade dedicated to the development of super resolution microscopy techniques, which allow optical imaging of structures below the so-called resolution limit. In particular there has been much interest in developing super resolution microscopy techniques capable of non-destructive imaging of living samples. A novel super resolution microscopy technique, lifetime image reconstruction super resolution (LIR-SR), is presented here; this uses continuous wave stimulated emission depletion (CW STED) to shorten the fluorescence lifetimes of fluorophores within a labelled sample. Differential lifetime shortening across the area of a scanning laser beam pair in the microscope results in spatial variation in the distribution of detected fluorescence on a nanosecond timescale, which can be subsequently used to reconstruct a super resolution image. Detailed theory of LIR-SR is explained, as well as the microscope hardware and computational methods used for its implementation. The technique is then tested on structures of known size and shape to gauge performance, and future directions for the technique are discussed. Spectroscopic studies of CW STED are also undertaken to better understand the underlying photophysics of the process. The effects of solvent viscosity on CW STED are investigated and analysed, and a mathematical model of CW STED is presented. Further investigations are then undertaken which address a wide range of factors which could affect CW STED, including out-of-focus fluorescence and involvement of the triplet state, and possible refinements to the model of CW STED are suggested in light of the experimental results.

Type: Thesis (Doctoral)
Title: Lifetime image reconstruction super resolution microscopy: principles, applications and underlying photophysics
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Third party copyright material has been removed from ethesis.
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy
URI: http://discovery.ucl.ac.uk/id/eprint/1470544
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