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Armes, Helen Elizabeth Harcourt.Pdf A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details IMPLEMENTING SUPER-RESOLUTION PALM MICROSCOPY IN FISSION YEAST By Helen Elizabeth Harcourt Armes SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY The University of Sussex September 2016 1 Declaration I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature:........................................... Date:................................................... 2 This thesis is dedicated to my family and friends 3 Acknowledgements This has been a very cross-disciplinary project with contributions from a number of different labs and I have had support from a lot of different people. I would like to start by thanking my supervisor Professor Tony Carr for having me in his group and suggesting these projects. He has been a very supportive, always showing interest in what I have done and making suggestions for next steps. I would like to thank Professor David Klenerman (University of Cambridge) for letting me spend a year in his lab learning the ropes of super-resolution microscopy. I would also like to thank Dr Steve Lee and Dr Matthieu Palayret for teaching me to use the microscope and Dr David Lando (Prof. Ernest Laue’s Group, University of Cambridge) for assisting with learning to handle S. pombe. I’d also like to acknowledge all the members of the Klenerman lab at that time who made me feel very welcome and the other members of the Laue lab who were involved in the PALM collaboration. At the University of Sussex I would like to thank Dr Adam Watson who has given a lot of his time to help me with biological work and to provide me with the strains I needed. I would like to thank Dr Manisankar Maiti for tireless work on aligning, calibrating and maintaining the instruments I have used, I never would have got the FCS data without him. Also Dr Mark Osborne for his input into setting up the FCS system and interpreting my results. I would like to thank Dr Alex Herbert, the writer of PeakFit and diverse other plugins I have used for my data analysis, not only did he devote a lot of time to developing these tools but also a lot of time to explaining them to me. I would like to thank Dr Thomas Etheridge who always had at least ten suggestions of different things to try whenever I got stuck. I would like to thank my co-supervisor Dr Eva Hoffman for patiently and persistently 4 encouraging me to start writing, and for reading and correcting first drafts. I would also like to thank Dr Jo Murray for offering extra support in proof reading this thesis I would like to thank Dr Dorota Dziadkowiec and Anna Barg for sharing strains, data and encouragement with me for the Rrp1/2 collaboration. I would like to thank all other members of both the Carr and Murray lab groups for being generally friendly, knowledgeable and always ready to provide advice. I would also like to thank the friends and family who have kept me sane for the last four years. In particular my partner Simon who has provided much support and many cups of tea and my college wives who have been there for me almost constantly via Facebook, encouraging me when I felt like giving up. 5 UNIVERSITY OF SUSSEX Helen Elizabeth Harcourt Armes A thesis submitted for the degree of Doctor of Philosophy IMPLEMENTING SUPER-RESOLUTION PALM MICROSCOPY IN FISSION YEAST SUMMARY Fluorescence microscopy is a popular biological technique because it allows the study of cells in great detail. However, the resolution achievable is limited by the diffraction properties of light, meaning that fine detail cannot be resolved. Various super-resolution microscopy methods have been developed to break this resolution limit. This thesis focuses on the single molecule localisation microscopy techniques. My host laboratory focuses on DNA replication and repair pathways using the model organism Schizosaccharomyces pombe (fission yeast). The aim of this thesis is thus to apply the technique of photo-activatable localisation microscopy (PALM) to specific biological questions in order to establish its benefits and limitations. In theory, in PALM every molecule will be imaged once and, as such, could be counted. So far this has been largely limited to membrane proteins. Using a combination of artificially created fluorescent oligomers, endogenous ribonucleotide reductase proteins tagged with mEos and computer simulations I studied the feasibility of counting highly expressed cytoplasmic proteins and assigning them to complexes of known or unknown stoichiometry. I established that density of expression is a significant limiting factor when using PALM to resolve complex stoichiometry. I thus went on to develop a variation of fluorescence correlation spectrometry to study the same protein complexes to see if we could determine their stoichiometry by diffusion speed. I established that the technique could differentiate between quite small changes in size. However the endogenous complex did not respond well to the fluorophore used so I was not able to establish its size. Using the PALM system I also studied a biological molecule, Rrp2, which was expressed at such low levels it was not possible to observe with conventional fluorescence microscopy. I established that we were able to observe this protein at endogenous levels and characterised its behaviour in response to stress. 6 Table of Contents Declaration ......................................................................................................................................... 1 Acknowledgements ........................................................................................................................... 3 SUMMARY ........................................................................................................................................ 5 Table of Contents ............................................................................................................................. 6 Table of Figures .............................................................................................................................. 12 List of Tables ................................................................................................................................... 15 List of Abbreviations ..................................................................................................................... 16 1. Introduction ............................................................................................................................. 18 General introduction and thesis aims ................................................................................ 18 Super-resolution microscopy .............................................................................................. 19 Light microscopy ............................................................................................................. 19 Fluorescence microscopy ............................................................................................... 20 Labelling strategies for fluorescence microscopy ................................................. 23 The limit of resolution ................................................................................................... 27 Illumination schemes to improve the signal and resolution in fluorescence microscopy ....................................................................................................................... 33 Breaking the diffraction limit - Super-resolution microscopy .................................. 38 Photo-switchable and -activatable fluorophores ................................................... 42 Localising fluorophores from their point spread functions ................................ 46 Applications of SMLM in biological systems ........................................................ 51 Background to the biological questions ............................................................................ 54 The yeast Schizosaccharomyces pombe as a model organism ........................................... 55 The class Ia ribonucleotide reductase complex .......................................................... 58 DNA damage and genomic instability – sensing and responding ........................... 62 Fork stalling and collapse .......................................................................................... 63 Double strand breaks ................................................................................................ 65 Cell cycle checkpoints ............................................................................................... 65 HR repair of double strand breaks ......................................................................... 71 2. Materials and Methods ........................................................................................................... 75 Growth media ......................................................................................................................
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