10th January 2017
The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.
1 Edinburgh Instruments, 2 Bain Square, Livingston, EH54 7DQ, UK
2 Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 6, UK
3 Princess Alexandra Eye Pavilion, Edinburgh and Centre for Clinical Brain Sciences, School of Clinical Sciences, University of Edinburgh, UK
4 Lein Applied Diagnostics, Reading Enterprise Centre, Whiteknights Rd, Reading RG6 6BU, UK
18th May 2016
Cu–N codoped TiO2 anatase thin films with a nanocolumnar architecture have been synthesized by RF-magnetron sputtering and characterized by Raman, scanning electron spectroscopy, and X-photoelectron spectroscopy. Absorption, photoluminescence, and photoluminescence lifetimes of the prepared samples have been investigated to understand the dynamics of the photogenerated carriers in connection to both introduced defects and the modified TiO2 band structure. At low concentrations Cu is mainly present as Cu+, while at higher concentrations the Cu2+ oxidation state prevails. Nitrogen, at low concentration and without the presence of copper dopant, substitutionally replaces oxygen to form a O–Ti–N linkage. With increasing concentration, interstitial nitrogen and Ti–O–N and Ti–O–N–O linkages are observed. In all codoped samples nitrogen is present as both interstitial and substitutional dopant. From photoluminescence spectra it is observed that nitrogen, in cooperation with Cu, more heavily affects the oxide structure, through Ti–N linkages, in such a way to quench the TiO2 exciton luminescence through charge trapping or energy transfer mechanisms. Time-resolved PL analysis evidenced that Cu–N codoping hinders the exciton radiative recombination in the anatase network, giving rise to increase of both the mean lifetime and trapping rate on defects at the nanocolumn surface.
9th August 2012
Noncognate or self peptide-MHC (pMHC) ligands productively interact with T-cell receptor (TCR) and are always in a large access over the cognate pMHC on the surface of antigen presenting cells. We assembled soluble cognate and noncognate pMHC class I (pMHC-I) ligands at designated ratios on various scaffolds into oligomers that mimic pMHC clustering and examined how multivalency and density of the pMHCs in model clusters influences the binding to live CD8 T cells and the kinetics of TCR signaling. Our data demonstrate that the density of self pMHC-I proteins promotes their interaction with CD8 co-receptor, which plays a critical role in recognition of a small number of cognate pMHC-I ligands. This suggests that MHC clustering on live target cells could be utilized as a sensitive mechanism to regulate T cell responsiveness.
1 Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
2 Edinburgh Instruments Ltd., Livingston, United Kingdom
3 Immudex Inc., Copenhagen, Denmark
20th April 2011
A novel route for early cataract diagnostics is investigated based on the excitation of tryptophan fluorescence (TF) at the red edge of its absorption band at 317 nm. This allows penetration through the cornea and aqueous humour to provide excitation of the ocular lens. The steepness of the red edge gives the potential of depth control of the lens excitation. Such wavelength selection targets the population of tryptophan residues, side chains of which are exposed to the polar aqueous environment. The TF emissions around 350 nm of a series of UV-irradiated as well as control lenses were observed. TF spectra of the UV cases were red-shifted and the intensity decreased with the radiation dose. In contrast, intensity of non-tryptophan emission with maximum at 435 nm exhibited an increase suggesting photochemical conversion of the tryptophan population to 435 nm emitting molecules. We demonstrate that the ratio of intensities at 435 nm to that around 350 nm can be used as a measure of early structural changes caused by UV irradiation in the lens by comparison with images from a conventional slit-lamp, which can only detect defects of optical wavelength size. Such diagnostics at a molecular level could aid research on cataract risk investigation and possible pharmacological research as well as assisting surgical lens replacement decisions.
1 Royal Society Industry Fellow at Edinburgh Instruments, Livingston, EH54 7DQ, UK
2 NHS Princess Alexandra Eye Pavilion, Edinburgh, EH3 9HA, UK
3 Abertay University, School of Social & Health Sciences, Dundee, DD1 1HG, UK
4 Edinburgh Instruments, Livingston, EH54 7DQ, UK
5 Heriot-Watt University, Physics Department, Edinburgh, EH14 4AS, UK
1st February 2011
This article describes novel data analysis of fluorescence lifetime-based protein kinase assays to identify and correct for compound interference in several practical cases. This ability, together with inherent advantages of fluorescence lifetime technology (FLT) as a homogeneous, antibody-free format independent of sample concentration, volume, excitation intensity, and geometry, makes fluorescence lifetime a practical alternative to the established “gold standards” of radiometric and mobility shift (Caliper) assays. The analysis is based on a photochemical model that sets constraints on the values of fluorescence lifetimes in the time responses of the assay. The addition of an exponential component with free floating lifetime to the constrained model, in which the lifetimes are constants predetermined from control measurements and the preexponential coefficients are “floating” parameters, allows the relative concentration of phosphorylated and nonphosphorylated substrates to be calculated even in the presence of compound fluorescence. The method is exemplified using both simulated data and experimental results measured from mixtures of dye-labeled phosphorylated and nonphosphorylated kinase substrates. A change of the fluorescence lifetime is achieved by the phosphorylated substrate-specific interaction with a bifunctional ligand, where one binding site interacts with the phosphate group and the other interacts with the dye.
17th February 2010
Commercial Fluorescence Lifetime Spectrometers have long suffered from the lack of a simple, compact and relatively inexpensive broad spectral band light source that can be flexibly employed for both quasi-steady state and time resolved measurements (using Time Correlated Single Photon Counting [TCSPC]). This paper reports the integration of an optically pumped photonic crystal fibre, supercontinuum source1 (Fianium model SC400PP) as a light source in Fluorescence Lifetime Spectrometers (Edinburgh Instruments FLS920 and Lifespec II), with single photon counting detectors (micro-channel plate photomultiplier and a near-infrared photomultiplier) covering the UV to NIR range. An innovative method of spectral selection of the supercontinuum source involving wedge interference filters is also discussed.
1st October 2006
The method of Time-Correlated Single Photon Counting (TCSPC) requires high repetitive light sources (>100 kHz) with pulse widths of ideally less than approximately 20 ps. While these light sources have been available for some time now in the form of Ti:Sapphire lasers, picosecond pulsed diode lasers (<90 ps) and light emitting diodes (<700 ps), they all have the drawback of either having no spectral tunability, or tunability over a very narrow spectral range (10 nm – 100 nm). While this is often sufficient for specific laboratory setups for measurements of fluorescence lifetimes, commercial Fluorescence Lifetime Spectrometers have suffered for a long time from the lack of the availability of simple, compact and relatively inexpensive broad spectral band light sources that can be employed for TCSPC. A new light source as an integral part of a commercial fluorescence lifetime spectrometer will be discussed that allows tunability over a wide spectral band of more than 500 nm.