[Show abstract][Hide abstract] ABSTRACT: A series of bright, europium (III) complexes has been prepared based on an achiral heptadentate triazacyclononane ligand bearing two strongly absorbing, coordinated aralkynyl pyridyl moieties. The binding of chiral carboxylates, including α-hydroxy acids such as lactate and mandelate, has been monitored by emission spectroscopy and is signalled by the switching on of strong circularly polarised emission. In each case, an R-chiral carboxylate gave rise to emission typical of a Δ complex, most clearly shown in the form of the ΔJ = 4 transition manifold around 700 nm. Variations in the sign and magnitude of the CPL allow the enantiomeric purity and absolute configuration of the acid to be assessed in a sample. Analysis of the relative energies of the parent aqua complexes and their stereoisomeric adducts has been aided by lifetime measurements and density functional theory calculations.
[Show abstract][Hide abstract] ABSTRACT: A new modular super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) has been developed in order to break the optical diffraction barrier in Confocal Laser Scanning Microscopy (LSCM). This technique is based on using spatially modulated illumination intensity, whilst harnessing the fluorophore's non-linear emission response. It allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. The work is in its initial phase, but by using a custom built Electro Optical Modulator (EOM) in conjunction with functionalised Ln(iii) complexes as probes, a sub-diffraction resolution of ∼60 nm was achieved of selected cellular organelles in long term live cell imaging experiments.
[Show abstract][Hide abstract] ABSTRACT: The development of the brightest luminescent europium(III) complexes is traced, including analysis of the C3-symmetric core complex based on a functionalized triazacyclononane and identification of the most suitable strongly absorbing chromophore. Strategies for the synthesis of the complexes, including enantiopure analogues, are outlined and opportunities for applications in time-resolved microscopy and spectral imaging emphasised. Practicable examples are introduced, including selective organelle staining for cellular optical imaging at 65 nm resolution and the development of new bioassays using time-resolved FRET methods.
[Show abstract][Hide abstract] ABSTRACT: A second generation of N-methyl-D-aspartate (NMDA) receptor-targeted MRI contrast agents has been synthesised and evaluated in cellulo, based on established bicyclic NMDA receptor antagonists. Their use as responsive MR imaging probes has been evaluated in suspensions of NSC-34 cells, and one agent exhibited significant enhancements in measured longitudinal and transverse water proton relaxation rates (19 and 38% respectively; 3T, 298K). A biotin derivative of the lead compound was prepared and the specificity and reversibility of binding to the NMDA cell surface receptors demonstrated using confocal laser scanning microscopy. Competitive and reversible binding of glutamate to the receptors was also visualised, suggesting that the receptor-targeted approach may allow MRI to be used to monitor neuronal events associated with modulation of local glutamate concentrations.
[Show abstract][Hide abstract] ABSTRACT: An inverted microscope has been adapted to allow time-gated imaging and spectroscopy to be carried out on samples containing responsive lanthanide probes. The adaptation employs readily available components, including a pulsed light source, time-gated camera, spectrometer and photon counting detector, allowing imaging, emission spectroscopy and lifetime measurements. Each component is controlled by a suite of software written in LabVIEW and is powered via conventional USB ports.
[Show abstract][Hide abstract] ABSTRACT: A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.
[Show abstract][Hide abstract] ABSTRACT: A circularly polarized luminescence (CPL) spectrometer has been built and used to study the binding interaction of lactate and four different proteins with racemic EuIII and TbIII complexes in aqueous solution. Lactate binding gives rise to strong induced CPL spectra, and the observed emission dissymmetry factors vary linearly with enantiomeric composition. Particularly strong induced TbIII CPL also characterizes the binding interaction of alpha-1-acid glycoprotein with a dissociation constant, Kd, of 2.5 μM.
[Show abstract][Hide abstract] ABSTRACT: The synthetic utility of europium complexes with three coordinated 4-bromopyridyl groups for chromophore elaboration has been assessed in palladium-catalysed Sonogashira coupling reactions, and in copper(i) mediated click reactions of the triazide derivative, generated in situ. The crystal structure of the Eu complex of a p-OMe-phenyl substituted triazole at 100 K is reported in which the pendant triazole sensitising moieties interdigitate in the solid-state lattice. The triazole complex can be separated into Δ and Λ enantiomers by chiral HPLC but is weakly emissive in methanol (ε 5.5 mM(-1) cm(-1); λexc 320 nm; ϕ 0.2%), contrasting with a set of four alkynyl-aryl derivatives which are one thousand times brighter and absorb strongly with broad absorption maxima in the range 332 to 360 nm. An enantiopure europium complex gives an intense CPL signal in solution that is the strongest yet reported.
[Show abstract][Hide abstract] ABSTRACT: Nine very bright europium(iii) complexes with different macrocyclic ligands have been prepared that exhibit excellent cell uptake behaviour and distinctive sub-cellular localisation profiles, allowing the use of fluorescence microscopy and time-gated spectral imaging to track their fate in cellulo. Their use as cellular imaging stains is described for the selective illumination of mitochondria, lysosomes or the endoplasmic reticulum of various mammalian cell types.
Chemical Science 01/2014; 5(5). DOI:10.1039/C3SC53056F · 8.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cell-staining behaviour of a set of five emissive europium complexes has been studied in Nicotiana tabacum BY-2 cells and pollen tubes, Nicotiana benthamiana plant leaves and in the root hairs of the wild-type plant Arabidopsis thaliana (Columbia). The cell walls were stained selectively, notably in the tobacco BY-2 cells, by the complex [EuL1] that contains one azathiaxanthone chromophore. Internalisation only occurred in cells that had been deliberately permeabilised or were dying. No uptake was observed within healthy plant leaves. In root hairs, the cell wall was strongly stained as well as the mitochondria, revealed by time-lapsed microscopy that showed the tumbling of the mitochondria in the living tissue, confirmed by co-localisation studies. In pollen tubes, the cell wall was also stained; rapid bursting of the pollen tip occurred following incubation with [Eu.L1], triggered by excitation with 405 nm laser light. Such behaviour is consistent with local perturbation of cell wall permeability and integrity, associated with the reactivity of the chromophore triplet excited state.
[Show abstract][Hide abstract] ABSTRACT: A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualise mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualisation by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalisation observed. Another approach, examining the location of a terbium analog via sensitised emission also exhibited non-specific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labelled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.
ACS Chemical Neuroscience 11/2013; 5(2). DOI:10.1021/cn400175m · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mono-C-substitution of the 1,4,7-triazacyclononane ring induces formation of single enantiomers of Eu(iii) complexes with nonadentate N6O3 ligands. The absolute configuration of each complex is determined by the stereogenicity of the C-substituent, revealed by comparison of the sign and sequence of CPL transitions for a series of complexes.
[Show abstract][Hide abstract] ABSTRACT: A series of five europium(III) complexes has been prepared from heptadentate N5 O2 ligands that possess a brightness of more than 10 mM(-1) cm(-1) in water, following excitation over the range λ=330-355 nm. Binding of several oxy anions has been assessed by emission spectral titrimetric analysis, with the binding of simple carboxylates, lactate and citrate involving a common ligation mode following displacement of the coordinated water. Selectivity for bicarbonate allows the rapid determination of this anion in human serum, with Kd =37 mM (295 K). The complexes are internalised quickly into mammalian cells and exhibit a mitochondrial localisation at early time points, migrating after a few hours to reveal a predominant lysosomal distribution. Herein, we report the synthesis and complexation behaviour of strongly emissive europium (III) complexes that bind oxy-anions in aqueous media with an affinity and selectivity profile that is distinctively different from previously studied systems.
Chemistry - A European Journal 07/2013; 19(29). DOI:10.1002/chem.201301273 · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A ratiometric Eu(III) complex has been developed that localises selectively within the endoplasmic reticulum of living cells. Careful calibration, using a time-gated spectral imaging microscope, allows the intensity ratio of emission bands and the variation of excited state lifetime to be used for pH determination, with a pKa of 7.15.
Chemical Communications 05/2013; 49(47). DOI:10.1039/c3cc42308e · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis, structure and photophysical properties of a series of highly emissive europium complexes is reported. Certain complexes enter mammalian cells by macropinocytosis and stain the mitochondria selectively, allowing observation of the Eu emission in cellulo by time-gated spectral imaging.
Chemical Communications 01/2013; 49(16). DOI:10.1039/c2cc35247h · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of Eu and Tb complexes of four different chiral ligands incorporating an azaxanthone sensitiser has been evaluated as probes for the bicarbonate anion. Their binding affinities were assessed at ambient pH with bicarbonate, lactate, citrate, phosphate and serum albumin. Binding was signalled by modulation of circularly polarised luminescence and apparent affinity constants were measured by examining changes in emission intensity ratios. Competition experiments show that with these species and ATP present at normal physiological values, bicarbonate can be determined selectively over the concentration range 10 to 35 mM. Bicarbonate levels are also reported by using a mixture of Eu and Tb complexes of a common ligand, examining the ratio of red/green emitted light. These methods have been adapted for the determination of bicarbonate in human serum and used for the assessment of mitochondrial levels of bicarbonate in several different cell types with confocal microscopy.
Chemistry - A European Journal 09/2012; 18(37):11604-13. DOI:10.1002/chem.201201738 · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Europium and terbium complexes of two structurally related ligands have been evaluated as optical probes to monitor changes in lysosomal pH; calibration using ionophores and fluorescent probes allows monitoring of the time dependence of lysosomal pH change, examining the green/red intensity ratio from internalised Tb-Eu complexes.
Chemical Communications 07/2012; 48(68):8520-2. DOI:10.1039/c2cc34267g · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Image and spectral intensity from bicarbonate-selective europium(III) probes localised in the mitochondria of cells is modulated reversibly by variation of external pCO(2), and is suppressed by addition of the carbonic anhydrase inhibitor, acetazolomide.
Chemical Communications 07/2011; 47(26):7347-9. DOI:10.1039/c1cc11853f · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This work aims to develop and validate a rapid analytical method that enables the measurement of citrate in seminal fluid samples. Samples were obtained from men who were within 9 weeks of a vasectomy operation. Two age ranges were examined, between 40-43 and 50-53 years old, with nearly 100 samples in each case. No patient clinical history was available for this anonymous study, simulating a random screening cohort. The concentration of citrate in 0.5 μL seminal fluid samples was assessed, using a europium emission luminescence method. This involves the ratiometric analysis of two well-separated europium(III) emission bands. Spectral data were obtained using a time-gated spectrometer whose construction and modification is described. Citrate values were confirmed by independent measurements using a citrate lyase enzymatic assay and by 700 MHz ¹H NMR analysis of the seminal fluid. Citrate concentrations were not statistically different between age groups and averaged 35.0(±14.6) mM for the 40-43 group, and 28.2(±12.7) mM for the 50-53 cohort; in each case a polymodal distribution was observed.
Journal of pharmaceutical and biomedical analysis 05/2011; 56(2):352-8. DOI:10.1016/j.jpba.2011.05.023 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A terbium complex has been designed that may be used to sensitise formation of singlet oxygen (phi(em) 12%), allowing the creation of a ratiometric sensory system for pO(2) (versus the insensitive Eu analogue) and the definition of a reactive probe that can be used to cause local damage to cells loaded with the complex and exposed to laser excitation.
Chemical Communications 12/2009; DOI:10.1039/b920222f · 6.83 Impact Factor