AbstractThis 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
|Date of Award||29 Sep 2020|
|Supervisor||Paul Verkade (Supervisor) & Henkjan Gersen (Supervisor)|