Graham Hungerford

Near‐infrared spark source excitation for fluorescence lifetime measurements

We have extended the range of excitation wavelengths from spark sources used in single photon timing fluorometry into the near infrared by means of the all‐metal coaxial flashlamp filled with an argon‐hydrogen gas mixture. At 750 nm this mixture gives ∼15 times the intensity available from pure hydrogen for a comparable pulse duration. Measurements are demonstrated by using the laser dye IR‐140 in acetone, for which a fluorescence lifetime of 1.20 ns is recorded.

Time-correlated single-photon counting fluorescence decay studies at 930 nm using spark source excitation

The authors report extension of the spectral range of fluorescence decay time measurements up to 930 nm using spark source excitation, time-correlated single-photon counting and a low-noise detection system incorporating the Philips XP2257B photomultiplier.

Thermal control over the extent of photoinduced electron transfer in helical oligopeptides

Time-resolved fluorescence studies of an electron donor–acceptor modified helical oligopeptide, in acetonitrile, show a biexponential decay with a short lifetime component attributed to rapid photoinduced electron transfer (PET), which increases from 15% of the total fluorescence at –40 °C to 58% on warming to 0 °C and then more gradually to 67% at 60 °C.

The fluorescence properties of DCM

Abstract The steady-state and time-resolved fluorescence properties of 4-dicyanomethylene-2-methyl-6- p -dimethylaminostyryl-4 H-pyran (DCM) in polar solvents as a function of temperature and concentration are shown to be consistent with thermal equilibrium between the trans and cis isomers. The high degree of spectral overlap of the two isomers and their distinct fluorescence decay times are dominant features in the photophysics of DCM. The presence of cis-isomer fluorescence in dimethylsulphoxide, in addition to that of the trans isomer, is identified for the first time. Further evidence, as shown by the fluorescence kinetics, is presented for ground-state concentration quenching of the cis isomer to give the trans isomer in DMSO. No evidence is found for significant DCM aggregation in solution, but such effects are observed in lipid bilayers wherein the use of DCM as a membrane probe is proposed.

Instrumentation for Red/Near-Infrared Fluorescence

As we mentioned at the outset, the study and application of near-IR fluorescence is only at the beginning. The success or failure of this promising approach will depend largely on the synthesis of suitable probe molecules and the performance of the appropriate instrumentation. We can only guess which, if either, will prove the limiting factor, but if the history of UV/visible fluorescence is anything to go by, IR fluorescence will evolve stepwise through the interplay between the different disciplines. The greatest promise in fluorescence instrumentation undoubtedly lies in the development of new semiconductor sources and detectors, combining wide-band spectral properties and fast time-resolution with low cost, reliability, and ease of use. It is very possible that the semiconductor devices discussed in this chapter only give us a snippet of what ultimately might be achieved in terms of device performance and fluorescence applications.

Influence of secondary structure on the decay kinetics of fluorescent donor-acceptor labelled peptides☆

Time-resolved fluorescence decays from a series of methoxynaphthalene labelled peptides in ethyl acetate were monitored over the temperature range -40 to 60 degrees C. The quenching effect of a piperidone acceptor group placed at various positions along the peptide chain relative to the fluorescent methoxynaphthalene donor was studied. In this moderately polar solvent the mechanism of quenching is most likely electron transfer, although a Dexter exchange mechanism cannot be ruled out. Both donor and acceptor moieties were covalently attached to the side-chains of glutamic acid residues. These were either placed adjacently, in the case of a dipeptide, or separated by three and six amino acids within a 12 and 15 amino-acid oligopeptide, respectively. The presence of the piperidone group resulted in a reduction in the fluorescence lifetime and a change from a simple monoexponential decay to more complex behaviour. This was found to vary reversibly with temperature and not to be caused by impurities. Modelling of the fluorescence decays was carried out using either the sum of two exponentials or a distribution of decays. For the dipeptide the best fit was a distribution while in the case of the 12-mer two clearly distinguishable populations could be observed. The results for the 15-mer were equivocal. Importantly, regardless of the fitting method used the quenching rate was found to be fastest for the 12-mer. The slower quenching rates observed for the dipeptide compared to the oligopeptides provide strong evidence that secondary structure promotes better electronic coupling between the donor and acceptor. The biexponential fluorescence behaviour for the 12 amino-acid oligopeptide is ascribed to two slowly (> 10 ns) interconverting conformational states. Comparison with circular dichroism and infrared obtained in acetonitrile indicates these two conformers are likely to be an alpha-helix and a 3(10)-helix with electronic coupling strongest in the latter case.

Spark source infrared fluorometry

The application of our development of 750 nm excitation from an argon-hydrogen filled spark source to time-resolved fluorescence probe studies of lipid membranes and inverse micelles is reported. The laser dye IR-140 was studied using the single-photon counting technique both in a lipid membrane of L-(alpha) dipalmitoylphosphatidylcholine (DPPC) and in sodium sulfosuccinic acid bis (2-ethylhexyl) ester (AOT) in iso-octane. In DPPC a dramatic change in the fluorescence behavior of IR-140 is observed between the gel and liquid crystalline phases. In inverse micelles of AOT an increase in the intensity of the peak fluorescence emission and a decrease in fluorescence lifetime is noticed on increasing the water content.

Fluorescence studies of a labeled model peptide in membrane and micellar media

Both micellar and lipid membrane systems have been used as models to provide further information regarding peptide properties in biological systems. Peptides are basic architectural units in nature and the study of their properties in membranes and other non-homogeneous media is of fundamental importance. We present the results for a time-resolved and steady state fluorescence study of two 4-methoxy-naphthalene labelled twelve amino acid residue model peptides that we have synthesised. One peptide, with a N-tert-Butoxycarbonyl (BOC) modified N-terminal, was incorporated into small unilamellar vesicles of L-(alpha) dipalmitoylphosphatidycholine (DPPC) and the other, with a free amino group, into inverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in 2,2,4-trimethylpentane. Steady state fluorescence and time-resolved fluorescence lifetime and anisotropy measurements show the first peptide to be situated in the lipid bilayer. In the inverse micelle system there is evidence for the peptide being situated at the surfactant waterpool interface.

Time-resolved fluorescence study of electron transfer in a model peptide system

At present there is a great deal of interest in the study of the transference of energy in biological systems. For example, electron transfer is of major importance in many synthetic and biological processes and in nature is mediated by proteins. Information regarding this process is therefore useful in leading to a greater understanding of phenomena such as photosynthesis and respiration. Previous work on protein systems has shown the electron transfer process to be complex to analyze because of the presence of competing pathways. This has led to the use of model systems to simplify the kinetics. We have synthesized novel model systems using peptides containing both a fluorescent methoxy- naphthalene donor and a dicyanoethylene group as a potential electron acceptor and observed fluorescence quenching for both dipeptide and oligopeptide systems. Biexponential fluorescence decay behavior was observed for all donor acceptor systems, with an increase in the amount of the shorter fluorescence decay component on increasing temperature.