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Final Copy 2020 09 29 Tann This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Tanner, Hugh R C Title: Development of Novel Probes for Correlative Light Electron Microscopy General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Declaration Development of Novel Probes for Correlative Light Electron Microscopy Hugh Robert Charles Tanner April 10, 2020 A dissertation submitted to the University of Bristol in accordance with the requirements for the award of the degree Doctor of Philosophy in the Faculty of Biochemistry School of Biochemistry Word count: 50175 I Declaration Abstract This thesis covers two separate projects linked by the common theme of correlative light electron microscopy (CLEM) probes. Many imaging modalities can potentially be utilised within a CLEM experiment but for the purposes of this thesis laser scanning confocal microscopy and transmission electron microscopy for the focus. CLEM seeks to gain the positive attributes of both constituent techniques in order to acquire information otherwise unattainable using the same techniques in isolation. Firstly the development of a previously studied protein ligand binding pair into an affinity based CLEM probe. A dissociation constant between a mutagenic variant of the FK506 binding protein (FKBP) and synthetic ligand of FKBP (SLF’) was reported to be 94pM (Clackson et al., 1998). Enhanced green fluorescent protein (EGFP) was endogenously expressed on the protein of interest along with the FKBP(F36V) to produce a chimera. The small molecule SLF’ was conjugated with gold nanoparticles in a stoichiometric ratio of 1:1. The tight binding between FKBP(F36V) and SLF’ conjugated to AuNP was used to co-localise AuNPs to the protein of interest. This probe was used both in vivo and to investigate the putative helical oligomeric structures formed by SNX1 around lipid tubules in vitro. The second project investigated the feasibility of platinum nanoclusters (PtNCs) to be used as CLEM probes. Several atoms in size, the electrons within the structure become discretised which gives rise to a size dependent fluorescence. We discovered PtNCs can be silver enhanced in order to produce more electron density in the electron micrograph. The quantum yield of the PtNCs was shown to be 4.8%. In vivo CLEM experiments in HeLa cells produced sufficient fluorescence to be readily detected. Once silver enhanced electron dense punctae were visible that displayed a high correlation with previously acquired fluorescent signals Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: .................................................................. DATE:....................................................................... II Table of Contents Table of Contents Development of Novel Probes for Correlative Light Electron Microscopy .............................................. I Abstract ................................................................................................................................................... II Declaration .............................................................................................................................................. II Chapter 1 ................................................................................................................................................. 1 A Brief Introduction to Light and Electron Microscopy Techniques for the Life Sciences ...................... 1 1.1 Green Fluorescent Protein ................................................................................................................ 2 1.2 Light Microscopy ............................................................................................................................... 3 1.2.1 Fluorescence Microscopy............................................................................................................... 4 1.2.1.1 Laser Scanning Confocal Microscopy .......................................................................................... 5 1.2.2 Super Resolution Fluorescence Microscopy .................................................................................. 5 1.2.2.1 Stimulated Emission Depletion Microscopy ............................................................................... 5 1.2.2.2 PALM/STORM ............................................................................................................................. 6 1.2.2.3 Light Sheet .................................................................................................................................. 7 1.3 Electron Microscopy ......................................................................................................................... 7 1.3.1 Lens Defects and Resolution ........................................................................................................ 10 1.3.2 Transmission Electron Microscopy .............................................................................................. 10 1.3.3 Electron Microscopy Sample Preparation ................................................................................... 11 1.3.3.1 Negative Stain ........................................................................................................................... 11 1.3.3.2 Chemical Fixation ...................................................................................................................... 12 1.3.3.3 High Pressure Freezing and Plunge Freezing ............................................................................ 13 1.3.3.4 Processing Vitreous Frozen Samples ........................................................................................ 14 1.3.3.5 Freeze Substitution ................................................................................................................... 15 1.3.4 Scanning Electron Microscopy and Analytical Electron Microscopy Techniques ........................ 15 1.3.4.1 Scanning Electron Microscopy .................................................................................................. 15 1.3.4.2 High Angle Annular Dark Field Scanning Transmission Electron Microscopy ........................... 16 1.3.4.3 Electron Energy Loss Spectroscopy ........................................................................................... 19 1.3.4.4 Energy Dispersive X-ray............................................................................................................. 19 III Table of Contents 1.4 Correlative Light Electron Microscopy ............................................................................................ 20 1.5 CLEM Probes ........................................................................................................................... 21 1.5.1 CLEM Probes with an Endogenously Expressed Element ............................................................ 21 1.5.1.1 Horseradish Peroxidase ............................................................................................................ 21 1.5.1.2 APEX .......................................................................................................................................... 22 1.5.1.3 Metallothionein ........................................................................................................................ 22 1.5.2 Affinity Based CLEM Probes ......................................................................................................... 23 1.5.2.1 Gold Nanoparticles ................................................................................................................... 24 1.5.2.2 Quantum Dots ........................................................................................................................... 25 1.6 The Endosomal Network ................................................................................................................
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