Synthetic studies on enhancers for europium luminescence

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Lanthanide luminescence and in particular sensitised emission from both europium (III) and terbium (III) is reviewed. The use of both europium and terbium as biological markers is covered, concentrating on time-resolved fluoroimmunoassays (TR-FIA), and more recently nucleic acid hybridisation assays. A new approach is described for the design of cooperative ligands that enhance europium luminescence in aqueous solution. This involves a three-component system comprised of the europium. ion, a sensitising ligand, 1,10-Phenanthroline-2,9- biscarboxylic acid, and a separate non-sensitising ligand, N-(butylacetamido)- ethyl enediamine-N, N, N'-tri acetate. The synthesis of a novel class of europium sensitiser is described 5-(Hexanoylphenanthridinium)-2,9-biscarboxylic acid- 1,1 O-phenanthroline. A homogeneous method for identifying the presence of a single strand of DNA is described which employs 5-(Hexanoylphenanthridinium)-2,9-biscarboxylic acid- ,10- phenanthroline as a cooperative sensitiser of europium (M) luminescence

Molecular rheometry: direct determination of viscosity in L-o and L-d lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol–sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing