Fluorescence Time-correlated Single Photon Counting Research Articles

Femtosecond dynamics of the S2 and S1 fluorescence of ionic styryl dyes in polar solvents

Femtosecond fluorescence upconversion and picosecond time-correlated single-photon counting fluorescence experiments for bridged and unbridged ionic styryl dye compounds in polar solvents are reported. The measured fluorescence transients reveal S2 → S1 internal conversion (IC) with a typical time of 300 fs, independent of bridged or unbridged structure. The lifetime of the relaxed emissive S1 state differs considerably for the bridged and unbridged structures: when the styryl-group single bonds are unbridged, the S1-state lifetime is only about 20 ps and the non-radiative decay to the ground state is very effective. When both single bonds of the styryl dye are chemically bridged and only the double bond is free to rotate, the fast decay is suppressed and the lifetime becomes about 2 ns. In addition, the fluorescence of the ionic styryl dyes shows picosecond transient behavior that is attributed to vibrational cooling in the excited S1 state. Finally, a qualitative discussion is given of the influence of the donor–acceptor strength of the styryl dye compounds on the polymethine and stilbenoid character of the S1 state and how this affects the effectiveness of the single- and double-bond twisting relaxation pathways for this state.

Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur.

Three novel nodulation (Nod) factors were synthesized from chitotetraose and three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspects were studied: whether these amphiphilic molecules insert in membranes, whether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers in PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the Nod factors are associated with these particles. With time-correlated single-photon counting fluorescence spectroscopy, it was shown that upon Nod factor insertion in the membrane, the rotation of the fluorescent acyl chain was markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or from vesicles to root hairs. Nod factors transfer rapidly between membranes or from vesicles to root hair cell walls. However, they do not flip-flop between membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobium-legume interaction.

Fluorescence Lifetime Measurements and Spectral Analysis of Adamantyldiazirine

We report the first confirmed fluorescence lifetime measurement for a diazirine. We have obtained time-correlated single-photon counting fluorescence decays for adamantyldiazirine in a variety of solvents, over a wide range of temperatures (77−320 K) and across the diazirine absorption band (330−371 nm). The fluorescence lifetime of the primary decay component is on the order of 240 ps at ambient temperature and increases at lower temperatures. Arrhenius treatment of the fluorescence lifetime data indicates that the rate-limiting barrier for activated processes in the diazirine excited state is between 2.7 and 2.9 kcal/mol. Adamantyldiazirine's fluorescence lifetime appears to be unaffected by deuteration of the solvent, solvent polarity, or excitation energy. We also report and discuss the steady-state absorption and fluorescence emission spectra of adamantyldiazirine in a variety of solvents, as well as the infrared spectrum (KBr). We interpret the spectra with the help of ab initio (RHF/6-31G* and CIS/6-31G*), density functional (B3LYP/6-31G*), and semiempirical (PM3) calculations. The fluorescence quantum yield of adamantyldiazirine at ambient temperature was calculated to be ∼0.0012. Analysis of our data in the light of previous research leads us to conclude that little or no intermolecular chemistry is attributable to photoexcited adamantyldiazirine in solution at ambient temperature. Rather, fluorescence competes with one or more intramolecular photochemical processes.

Time-resolved confocal fluorescence microscopy of porphyrins for phototherapy

The application of a novel time-resolved confocal fluorescence microspectrometer to studies of the distribution and speciation of porphyrin photosensitizers in rat C6 cerebral glioma cells is described. The instrument combines a mode-locked argon ion laser excitation source with time-correlated single photon counting fluorescence detection and has sub-micron spatial and sub-nanosecond temporal resolution. The porphyrins studied were haematoporphyrin derivative (HpD), haematoporphyrin IX (HP), porphyrinc (Pc) and the tetrakiscarborane carboxylate ester of 2,4-(α,β-dihydroxyethyl) deuteroporphyrin IX (BOPP). From the heterogeneous emission observed in vitro, assignments and spatial location of various porphyrin species are proposed.

Excited-State Proton Transfers in 9-Aminoacridine Carboxamides

Excited-state proton transfers have been investigated for several isomeric and homologous N-((dimethylamino)-alkyl)-9-aminoacridine carboxamides in a time-correlated single photon counting fluorescence study. The pH behavior of these DNA intercalators` steady-state absorption and fluorescence spectra has also been investigated. While the excited-state pK{sub a}* for removal of a proton from the singly protonated species is close to the ground-state pK{sub a} in the parent molecule 9-aminoacridine, the presence of the carboxamide substituent renders pK{sub a}* much lower than pK{sub a} for the corresponding deprotonation in the present compounds. This fact, along with the presence of an additional deprotonation site at the distal N atom of the side chain, endows these molecules with complex pH dependence in their excited-state kinetics. Triexponential model functions are generally required to fit the experimental fluorescence decays. The pH dependence of the component lifetimes and preexponential factors can be simulated with a kinetic model for sequential deprotonations involving four aminoacridine species in both the ground and fluorescing states. These results provide a baseline study for comparisons with these intercalators` excited-state kinetics in DNA. 37 refs., 12 figs., 2 tabs.

10] Maximum likelihood analysis of fluorescence data

Publisher Summary In principle, fluorescence spectroscopy provides a powerful tool for the investigation of structure and function in biological macromolecules. There are two fundamental considerations in the use of fluorescence data for such purposes. First, the fluorescence may be used simply as a signal of an event. Second, fluorescence data may be interpreted in terms of the physicochemical character, the structure, and the dynamics of the environs of the fluorophore. This chapter focuses on the analysis of fluorescence intensity decay data gathered with time-correlated single photon counting (TCSPC) instrumentation. The method of analysis considered obviously can be applied generally, but the principal interest is in its use for the study of protein fluorescence. The chapter describes the use of the maximum likelihood method for the analysis of TCSPC fluorescence intensity decay data. The results of analyses is presented in the chapter as an illustration obtained by the computer program FDA (written in FORTRAN 77), which have developed in the past few years. The program is interactive, but it can also run in batch mode.

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.

Charge separation in carotenoporphyrin-quinone triads: synthetic, conformational, and fluorescence lifetime studies

Carotenoid-porphyrin-quinone triad molecules undergo a photodriven two-step electron-transfer reaction which results in the generation of a high-energy charge-separated state with a lifetime on the microsecond time scale at ambient temperatures in fluid solution. These systems mimic the initial charge separation steps of photosynthesis. A series of these tripartite molecules which differ systematically in the nature of the linkages joining the porphyrin to the quinone and carotenoid moieties has been synthesized in order to investigate the effect of structure on the yield and lifetime of the charge-separated state. The time-averaged solution conformations of these molecules have been determined from porphyrin ring current induced shifts in the /sup 1/H NMR resonances of the carotenoid and quinone moieties. Studies of the triads and related molecules in dichloromethane solution using time-correlated single photon counting fluorescence lifetime techniques have yielded the rate constant for the first of the photoinitiated electron-transfer steps as a function of the linkage joining the porphyrin and the quinone. The rate constants range from 1.5 x 10/sup 8/ to 9.7 x 10/sup 9/ s/sup -1/. For most members of the series, the results are consistent with an exponential dependence of the electron-transfer rate on the experimentally determined donor-acceptor separation, with themore » exponential factor ..cap alpha.. = 0.6 A/sup -1/.« less

The binding of 1, N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1, N 6-ethenoNAD (εNAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of εADP and carefully purified εNAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20°C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for εNAD) and 23 ns (for εADP), in good agreement with literature values obtained under similar conditions. εNAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48–57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free εNAD and 21 ns for bound εNAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of εNAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter γ, which represents the molar fraction of εNAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Possibilities and limitations of the time-correlated single photon counting technique: a comparative study of correction methods for the wavelength dependence of the instrument response function

One of the principal reasons for poor fits in time-correlated single photon counting fluorescence decay measurements is the wavelength dependence of the instrument response function. Over the years several correction methods have been developed to account for or correct for this effect. The most recent and widely used procedures are critically compared in this paper. A comparison of the channel shift technique, the pseudoscatterer technique, the excitation pulse-shape mimic technique and the delta function convolution method demonstrates that the latter method is the superior one. This method requires the measurement of the fluorescence decay of a reference compound with single exponential decay kinetics under identical conditions as used for the sample. A modified functional form is used to describe the sample decay law. Since no experimental data are altered in any way, a correct residual analysis is possible. Simulations show that fluorescence lifetimes can be recovered accurately, as long as the reference decay time is sufficiently different from that of the sample. Biexponential decays can be resolved successfully when the two decay times are well separated and different from that of the reference. The difference between the standard normal variates of chi-square for single and double exponential fits can be used to distinguish between mono- and bi-exponential decays. The usefulness of the delta function convolution method is demonstrated by fluorescence decay measurements of a series of different samples with very short decay times. Fluorescence lifetimes as short as 10 ps could be resolved accurately and reliably.