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New Light Sources for Time-Correlated Single Photon Counting in Commercially Available Spectrometers

Roger Fenske, Dirk U. Näther, Mark Goossens, S. Desmond Smith

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.

The Supercontinuum Laser as a Flexible Source for Quasi-Steady State and Time-Resolved Fluorescence Studies

Roger Fenske, Dirk U. Näther, Richard B. Dennis, S. Desmond Smith

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.

Use of fluorescence lifetime technology to provide efficient protection from false hits in screening applications

Dmitry M. Gakamsky, Richard B. Dennis, S. Desmond Smith

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.

Exploring the possibility of early cataract diagnostics based on tryptophan fluorescence

Dmitry M. Gakamsky1, Bal Dhillon2,5, John Babraj3, Matthew Shelton4, S. Desmond Smith4,5

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.

Evidence that the Density of Self Peptide-MHC Ligands Regulates T-Cell Receptor Signaling

Nadia Anikeeva1, Dimitry Gakamsky2, Jørgen Schøller3, Yuri Sykulev1

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.