Olaf J Rolinski

Scottish Universities Physics Alliance, Glasgow, Scotland, United Kingdom

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Publications (49)135.35 Total impact

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    ABSTRACT: We have monitored the formation of toxic β-amyloid oligomers leading to Alzheimer's disease by detecting changes in the fluorescence decay of intrinsic tyrosine. A new approach based on the non-Debye model of fluorescence kinetics resolves the complexity of the underlying photophysics. The gradual disappearance of nonmonotonic fluorescence decay rates, at the early stages of aggregation as larger, tighter-packed oligomers are formed, is interpreted in terms of tyrosine-peptide dielectric relaxation influencing the decay. The results demonstrate the potential for a new type of fluorescence lifetime sensing based on dual excited-state/dielectric relaxation, with application across a broad range of biological molecules. The results also reconcile previously conflicting models of protein intrinsic fluorescence decay based on rotamers or dielectric relaxation by illustrating conditions under which both are manifest.
    No preview · Article · Aug 2015 · Journal of Physical Chemistry Letters
  • David J. S. Birch · Yu Chen · Olaf J. Rolinski
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    ABSTRACT: This chapter surveys some of the main capabilities, techniques, and measurements that are enabled by fluorescence. It covers spectra, quantum yield, lifetime, quenching, anisotropy, and microscopy, in each case citing topical review articles, many of the original references, underlying theory and modern day applications. Measuring absorption and fluorescence spectra is usually the first place to start in any fluorescence study. In order to illustrate how absorption and fluorescence spectra often interplay in tandem, the chapter considers the example of the auto-oxidation of 3,4-dihydroxy-l-phenylalanine (l-DOPA) to produce melanin. Lifetime measurement has emerged in recent years as the most powerful and versatile technique in fluorescence spectroscopy. The basis of fluorescence anisotropy techniques is to use polarized excitation to create a spatially selected, non-random, distribution of excited fluorescent molecules which then randomize, most commonly by Brownian rotation, but also at times by energy migration depending on the system.
    No preview · Article · Feb 2015
  • Olaf J Rolinski · Vladislav Vyshemirsky
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    ABSTRACT: The potentially highly informative, but complex fluorescence decay of amino acids in protein is not fully understood and presents a barrier to understanding. Here we have tested a new and general approach to describing experimentally measured the fluorescence decay in a heterogeneous macroscopic sample. The decay parameters carry information on the features of the kinetics induced by the environment's heterogeneity. Bayesian interference demonstrated that the model fits well to the fluorescence decay of tryptophan in human serum albumin (HSA). The approach has the potential to accelerate photophysical research of heterogeneous media and, specifically, to solve a critical outstanding problem in interpreting protein fluorescence, paving the way to further progress in biomedical research.
    No preview · Article · Dec 2014
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    ABSTRACT: The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.
    Full-text · Article · Jan 2014 · Applied Physics Letters
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    ABSTRACT: The development of Alzheimer's disease is associated with the aggregation of the beta-amyloid peptides Aβ1−40 and Aβ1−42. It is believed that the small oligomers formed during the early stages of the aggregation are neurotoxic and involved in the process of neurodegeneration. In this paper we use fluorescence decay measurements of beta-amyloid intrinsic fluorophore tyrosine (Tyr) and molecular dynamics (MD) simulations to study the early stages of oligomer formation for the Aβ1−40 and Aβ1−42 peptides in vitro. We demonstrate that the lifetime distributions of the amyloid fluorescence decay efficiently describe changes in the complex Tyr photophysics during the peptide aggregation and highlight the differences in aggregation performance of the two amyloids. Tyr fluorescence decay is found to be a more sensitive sensor of Aβ1−40 aggregation than Aβ1−42 aggregation. The MD simulation of the peptide aggregation is compared with the experimental data and supports a four-rotamer model of Tyr.
    Full-text · Article · Mar 2013
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    Jens U Sutter · David J S Birch · Olaf J Rolinski
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    ABSTRACT: We report the use of CdSe/ZnS core/shell quantum dots QDot800 (Invitrogen) as luminescence lifetime sensors for copper ions Cu2+(H2O)8 in solution with a sensitivity of <1 ppb that is relevant to intracellular copper concentrations. Excitation of QDot800 at 485 nm was found to be optimum in that it did not cause any change in the level of luminescence intensity or luminescence lifetime in the absence of copper ions. When excited at 485 nm a bi-exponential luminescence decay of QDot800 was observed suggesting the presence of two distinct emitting states, both capable of undergoing metal ion quenching that facilitates Cu2+ detection. Selectivity for copper, as against other transition metal ions, as well as other evidence, suggests the primary origin of the quenching is luminescence resonance energy transfer to both free and bound copper ions. The luminescence kinetics of quantum dots and their optimization and applicability for resonance energy transfer-based lifetime sensing in general is discussed.
    Full-text · Article · Apr 2012 · Measurement Science and Technology
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    Mariana Amaro · David J S Birch · Olaf J Rolinski
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    ABSTRACT: Aggregation of the peptide beta-amyloid is known to be associated with Alzheimer's disease. According to recent findings the most neurotoxic aggregates are the oligomers formed in the initial stages of the aggregation process. Here we use beta-amyloid's (Aβ's) intrinsic fluorophore tyrosine to probe the earliest peptide-to-peptide stages of aggregation, a region often merely labelled as a time lag, because negligible changes are observed by the commonly used probe ThT. Using spectrally resolved fluorescence decay time techniques and analysis we demonstrate how the distribution of 3 rotamer conformations of the single tyrosine in Aβ tracks the aggregation across the time lag and beyond according to the initial peptide concentration. At low Aβ concentrations (≤5 μM), negligible aggregation is observed and this is mirrored by little change in the fluorescence decay parameters, providing a useful baseline for comparison. At higher concentrations (≈50 μM), and contrary to what is generally accepted from ThT studies the rate of aggregation can be described by an exponential growth to a plateau in terms of the relative contributions of two of the three rotamers, with a characteristic aggregation time of ≈33 h.
    Full-text · Article · Mar 2011 · Physical Chemistry Chemical Physics
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    Jens U. Sutter · David J. S. Birch · Olaf J. Rolinski
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    ABSTRACT: We report changes in the photophysical properties of core-shell type CdSe/ZnS quantum dots (QDs) under optical irradiation. QDs either in aqueous solution or immobilized in a silica sol gel matrix have been excited at different wavelengths and fluxes. Illumination of the sample with 140 fs 700 nm Ti:sapphire laser pulses of the peak power of the order of 4 GW/cm2 caused gradual increase in the luminescence lifetime from an initial value of 3.5 increasing to 4.5 ns and an increase in luminescence intensity by ∼ 8%. Using about 16 GW/cm2 peak power resulted in a shortening of the luminescence lifetime to 3 ns and a decrease in intensity by ∼ 75%. Both photobrightening and photodarkening were fully reversible. We discuss the kinetics of photobrightening and photodarkening and investigate the suitability of QDs as luminescence lifetime sensors with tunable parameters.
    Full-text · Article · Jan 2011 · Applied Physics Letters
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    Olaf J Rolinski · Mariana Amaro · David J.S. Birch
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    ABSTRACT: Beta-amyloid (Abeta) aggregation, believed to be responsible for Alzheimer's disease, is monitored using its intrinsic fluorescence decay. Alterations in the fluorescence decay of tyrosine correlate with the Abeta aggregation at a much earlier stage than the traditionally used fluorescence intensity of Thioflavin T (ThT). Potentially the finding may underpin progress towards an earlier diagnosis of the onset of Alzheimer's disease and an improved approach to developing intervention therapies.
    Full-text · Article · Mar 2010 · Biosensors & Bioelectronics
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    ABSTRACT: Star-shaped oligofluorene consists of highly-fluorescent macromolecules of considerable interest for organic electronics. Here, we demonstrate controlled micro-patterning of these organic nanostructured molecules by blending them with custom-synthesized photo-curable aliphatic polymer matrices to facilitate solventless inkjet printing. The printed microstructures are spherical with minimum dimensions of 12μm diameter and 1μm height when using a cartridge delivering ∼1pL droplets. We evaluate the physical characteristics of the printed structures. Photoluminescence studies indicate that the blend materials possess similar fluorescence properties to neat materials in solid films or toluene solution. The fluorescence lifetime consists of two components, respectively 0.68±0.01ns (τ 1) and 1.23±0.12ns (τ 2). This work demonstrates that inkjet printing of such blends provides an attractive method of handling fluorescent nano-scaled molecules for photonic and optoelectronic applications.
    Full-text · Article · Oct 2009 · Applied Physics A
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    ABSTRACT: We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carls-berg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm -1 to 1632 cm-1, indicating a shift from α-helical structure to β-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.
    Full-text · Article · Jul 2009 · ChemPhysChem
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    ABSTRACT: We aimed to develop microsensors for eventual glucose monitoring in diabetes, based on fluorescence lifetime changes in glucose/galactose-binding protein (GBP) labelled with the environmentally sensitive fluorophore dye, badan. A mutant of GBP was labelled with badan near the binding site, the protein adsorbed to microparticles of CaCO3 as templates and encapsulated in alternating nano-layers of poly-l-lysine and heparin. We used fluorescence lifetime imaging (FLIM) with two-photon excitation and time-correlated single-photon counting to visualize the lifetime changes in the capsules. Addition of glucose increased the mean lifetime of GBP-badan by a maximum of ∼2 ns. Analysis of fluorescence decay curves was consistent with two GBP states, a short-lifetime component (∼0.8 ns), likely representing the open form of the protein with no bound glucose, and a long-lifetime component (∼3.1 ns) representing the closed form with bound glucose and where the lobes of GBP have closed round the dye creating a more hydrophobic environment. FLIM demonstrated that increasing glucose increased the fractional proportion of the long-lifetime component. We conclude that fluorescence lifetime-based glucose sensing using GBP encapsulated with nano-engineered layer-by-layer films is a glucose monitoring technology suitable for development in diabetes management.
    No preview · Article · Jul 2009 · Biosensors & Bioelectronics
  • Olaf J Rolinski · Katherine Scobie · David J S Birch
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    ABSTRACT: We present a description of fluorescence decay kinetics in complex environments based on gamma functions rather than the conventional approach using exponentials. The gamma function description is tested in measurements on the temperature dependence of the protein human serum albumin (HSA), N-acetyl tryptophanamide (NATA), and 2, 5-dipenyl oxazole (PPO). The monitoring of macromolecular structure and dynamics is demonstrated by means of distinct tryptophan (Trp) rotamer populations and their interconversion in HSA.
    No preview · Article · Jun 2009 · Physical Review E
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    ABSTRACT: Hydrated nanopores offer a unique environment for studying biological molecules under controlled conditions and fabricating sensors using fluorescence. Silica nanopores for example are non-toxic, biologically and optically compatible with protein, and can be easily synthesized to entrap protein and exclude potentially interfering macromolecules, while transmitting analytes of interest. A well known problem when polymerizing orthosilicates to fabricate silica sol-gel nanopores is the release of alcohol, which denatures proteins. We will describe how using the fluorescence of PRODAN (6-propionyl-2-(N,N-dimethylamino) naphthalene) to monitor methanol generated during polymerization has helped define a protocol with enhanced biocompatibility. The improved biocompatibility of sol-gel nanopores synthesized using tetramethyl orthosilicate (TMOS) has been demonstrated by preserving the unstable native trimer form of allophycocyanin (APC) for up to 500 Hrs without the need to covalently binding the subunits together. This has enabled the observation of native APC trimer by means of its fluorescence in a pore down to the single molecule level. In this paper we demonstrate how PRODAN and another polarity sensitive dye, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, Nile red (NR) report on pore polarity and successfully extend protein encapsulation to nano-channels of alumina (Al2O3). Improved biocompatibility of nanopores has potential impact in nanomedicine where the ability to study single biomolecules is a primary goal as it underpins our understanding of disease pathology and therapeutics at the most fundamental level. In sensing also the advantages of nanopore isolation of metabolite-specific protein for detecting non-fluorescent metabolites has been demonstrated. Similar approaches can in principle be developed for both single-molecules and lab-on-a-chip sensors.
    No preview · Article · Jan 2009 · Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
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    Olaf J Rolinski · David J S Birch
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    ABSTRACT: Some fluorescence dyes in complex media, such as those found in biology, demonstrate nonextensive kinetics, which implies representing their fluorescence decays in terms of lifetime distributions rather than simple exponentials. Complex kinetics usually discourage application to lifetime sensors, as it is believed, that additional molecular mechanisms employed for detection of an analyte will make the resulting kinetics ambiguous and the sensor response inconclusive. In this paper we investigate theoretically the applicability of complex dye kinetics as a fluorescence resonance energy transfer based lifetime sensor and demonstrate that the nonextensive nature of its kinetics does not decrease the sensing performance, and indeed even provides richer structural information than a simple exponential behavior.
    Preview · Article · Nov 2008 · The Journal of Chemical Physics
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    ABSTRACT: Here we describe progress toward our objective of detecting single nonfluorescent hydrated metal ions. Single-ion detection represents detection and spectroscopy at the ultimate sensitivity level of approximately 1.6 x 10(-24) M. Achieving this goal would provide a breakthrough in analytical science and allow much more detailed insight into sensor-ion interaction than that available with conventional bulk detection methods. We combine recent advances in confocal microscopy with the sensitivity and the noninvasive nature of fluorescence by analyzing Förster resonance energy transfer between sensor fluorophores and transition metal ions.
    Full-text · Article · Feb 2008 · Annals of the New York Academy of Sciences
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    Olaf J Rolinski · Andrew Martin · David J S Birch
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    ABSTRACT: A nonextensive model of decay kinetics has been used to describe fluorescence behavior of tryptophan in human serum albumin on binding two flavonoids, quercetin and morin. We demonstrate that this approach, alternative to multiexponential representation of usually complex decays of tryptophan, is more adequate and can be beneficial in noninvasive lifetime sensing based on intrinsic fluorescence.
    Preview · Article · Feb 2008 · Annals of the New York Academy of Sciences
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    Olaf J Rolinski · Andrew Martin · David J S Birch
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    ABSTRACT: Human serum albumin (HSA) complexation with quercetin, a flavonoid commonly present in human diet, was monitored by means of fluorescence decays of the single HSA tryptophan - Trp214. Data analysis based on fitting to multiexponential functions and determining the lifetime distributions revealed a high sensitivity of tryptophan fluorescence to binding quercetin. Results are discussed in terms of the rotamer model for tryptophan, HSA-quercetin complexation and potential HSA to quercetin energy transfer. Evidence for quercetin stabilising tryptophan rotamers in HSA is presented.
    Preview · Article · May 2007 · Journal of Biomedical Optics
  • Olaf J. Rolinski · Aaron Hernandez-Santana · Duncan Graham
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    ABSTRACT: We demonstrate a new fluorescence resonance energy transfer (FRET) based approach to determine the donor-acceptor distributions and apply it to two model molecular systems: double stranded DNA labeled with Hoechst 33258 and FAM, and perylene randomly surrounded by cobalt ions in a bulk solution. The approach makes some generic assumptions regarding the FRET kinetics, but no a priori assumptions regarding the distribution function.
    No preview · Conference Paper · Sep 2006
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    N.D. Evans · L Gnudi · O.J. Rolinski · D.J.S. Birch · J.C. Pickup
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    ABSTRACT: The aim of this study was to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence lifetime of cells in an in vitro cell culture model. Autofluorescence decay functions were measured in 3T3-L1 adipocytes by time-correlated single-photon counting (excitation 370nm, emission 420-480nm). Free NADH had a two-exponential decay but cell autofluorescence fitted best to a three-exponential decay. Addition of 30mM glucose caused a 29% increase in autofluorescence intensity, a significantly shortened mean lifetime (from 7.23 to 6.73ns), and an increase in the relative amplitude and fractional intensity of the short-lifetime component at the expense of the two longer-lifetime components. Similar effects were seen with rotenone, an agent that maximizes mitochondrial NADH. 3T3-L1 fibroblasts stained with the fluorescent mitochondrial marker, rhodamine 123 showed a 16% quenching of fluorescence intensity when exposed to 30mM glucose, and an increase in the relative amplitude and fractional intensity of the short lifetime at the expense of the longer lifetime component. We conclude that, though the effect size is relatively small, glucose can be measured non-invasively in cells by monitoring changes in the lifetimes of cell autofluorescence or of a dye marker of mitochondrial metabolism. Further investigation and development of fluorescence intensity and lifetime sensing is therefore indicated for possible non-invasive metabolic monitoring in human diabetes.
    Full-text · Article · Sep 2005 · Journal of Photochemistry and Photobiology B Biology