eprintid: 1470544
rev_number: 36
eprint_status: archive
userid: 608
dir: disk0/01/47/05/44
datestamp: 2015-10-23 11:57:32
lastmod: 2020-10-05 21:20:33
status_changed: 2015-10-23 11:57:32
type: thesis
metadata_visibility: show
creators_name: Culley, S
title: Lifetime image reconstruction super resolution microscopy: principles, applications and underlying photophysics
ispublished: unpub
divisions: UCL
divisions: A01
divisions: B02
divisions: C08
divisions: D77
note: Third party copyright material has been removed from ethesis.
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.
date: 2015-08-28
date_type: published
oa_status: green
full_text_type: other
thesis_class: doctoral_open
language: eng
thesis_view: UCL_Thesis
primo: open
primo_central: open_green
verified: verified_manual
elements_id: 1046832
language_elements: English
lyricists_name: Culley, Sian
lyricists_id: SCULL85
actors_name: Culley, Sian
actors_id: SCULL85
actors_role: owner
full_text_status: public
pages: 249
event_title: UCL (University College London)
institution: UCL (University College London)
department: Physics & Astronomy
thesis_type: Doctoral
citation:        Culley, S;      (2015)    Lifetime image reconstruction super resolution microscopy: principles, applications and underlying photophysics.                   Doctoral thesis , UCL (University College London).     Green open access   
 
document_url: https://discovery-pp.ucl.ac.uk/id/eprint/1470544/8/Culley_whole_thesis.pdf.%20redacted.pdf