Ultrafast relaxation dynamics of a biologically relevant probe dansyl at the micellar surface

We report picosecond-resolved measurement of the fluorescence of a well-known biologically relevant probe, dansyl chromophore at the surface of a cationic micelle (cetyltrimethylammonium bromide, CTAB). The dansyl chromophore has environmentally sensitive fluorescence quantum yields and emission maxima, along with large Stokes shift. In order to study the solvation dynamics of the micellar environment, we measured the fluorescence of dansyl chromophore attached to the micellar surface. The fluorescence transients were observed to decay (with time constant approximately 350 ps) in the blue end and rise with similar timescale in the red end, indicative of solvation dynamics of the environment. The solvation correlation function is measured to decay with time constant 338 ps, which is much slower than that of ordinary bulk water. Time-resolved anisotropy of the dansyl chromophore shows a bi-exponential decay with time constants 413 ps (23%) and 1.3 ns (77%), which is considerably slower than that in free solvents revealing the rigidity of the dansyl-micelle complex. Time-resolved area-normalized emission spectroscopic (TRANES) analysis of the time dependent emission spectra of the dansyl chromophore in the micellar environment shows an isoemissive point at 21066 cm-1. This indicates the fluorescence of the chromophore contains emission from two kinds of excited states namely locally excited state (prior to charge transfer) and charge transfer state. The nature of the solvation dynamics in the micellar environments is therefore explored from the time-resolved anisotropy measurement coupled with the TRANES analysis of the fluorescence transients. The time scale of the solvation is important for the mechanism of molecular recognition.     

ENERGY TRANSFER AMONG THE CHROMOPHORES OF C-PHYCOCYANIN FROM Anabaena variabilis USING STEADY STATE AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Abstract We have investigated the model of energy transfer between sensitizing (s) and fluorescing (f) chromophores for the αβ monomer and for the separated α and β subunits of C-phycocyanin from Anabaena variabilis using fluorescence emission spectroscopy, fluorescence excitation polarization, and picosecond-resolved fluorescence decay kinetics. The fluorescence emission maximum occurs at 640 nm for all samples. The fluorescence excitation polarization is constant ( P = 0.40) across the absorption hand for the α subunit, but it increases across the absorption band towards longer wavelength for the β subunit and the αβ monomer. The fluorescence decay kinetics exhibit two exponential lifetimes of 1.3-1.5 ns and 340-500 ps for the αβ monomer and for the α and β subunit preparations.
We attribute the change in polarization across the absorption band to energy transfer among the three chromophores in the αβ monomer and among the two chromophores in the separated β subunit. The constant, relatively high polarization in the separated a subunit, having only one chromophore, is consistent with the absence of both energy transfer and chromophore rotation. The concentration of the α subunit did not affect the decay kinetics, suggesting that the short lifetime component does not arise from aggregation of the α subunits. The biexponential decay kinetics of the α subunit cannot be explained using the sensitizing-fluorescing model. The possibility of conformational interactions is under investigation.     

Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.     

Photoinduced intramolecular charge transfer in 9-aminoacridinium derivatives assisted by intramolecular H-bond

Fluorochromic dyes derived from 9-aminoacridinium containing a vinylene function with electron withdrawing groups such as diethyl [(acridinium-9-ylamino)methylene]malonate (I), ethyl [(acridinium-9-ylamino)methylene]cyanoacetate (II), [(acridinium-9-ylamino)methylene]malononitrile (III), are prepared and studied in their monoprotonated form. Absorption spectra of the new dyes are red shifted compared to that of the precursor dye. The observed dual fluorescence and multiexponential decay are ascribed to normal emission from the acridinium chromophore in addition to excited-state intramolecular charge transfer (ESICT) process. However, biexponential decay character is observed only for the dicyano derivative (compound III), whereas for the two other systems, more complex kinetics and a three-component decay is recovered. The analysis of the fluorescence decays in different solvents for the first two compounds reveals two short-lived components in the range of 160-350 ps and 1.1-3.0 ns, related to formation and decay of the ESICT state, plus a third one with decay time of about 9 ns, which is ascribed to the normal emission from the acridinium chromophore as an enol tautomer or as an intramolecular H-bond conformer (closed form tautomer). For the dicyano derivative, in which the absence of carbonyl group precludes the H-bond interaction, the biexponential fitting reveals a slightly fast formation rate of the ESICT state with values on the order of 10(10) s(-1), whereas its decay time is between 0.6 and 3.2 ns, depending on the solvent used.     

Fluorescence decay of α-chymotrypsin studied by the picosecond-resolved single photon-counting technique

The fluorescence decay of the multi-tryptophan-containing enzyme α-chymotrypsin in Tris buffer (pH 7.8) at room temperature was studied using a frequency-doubled, synchronously-pumped picosecond rhodamine-6G laser excitation source with time-correlated single photon-counting detection. The fluorescence decay parameters were computed with a non-linear least-squares iterative reconvolution program. The goodness-of-fit was tested with well-known graphical methods such as residuals plots and the autocorrealtion function. Numerical tests (reduced chi-square, ordinary runs test and the Durbin—Watson statistic) were included to improve the reliability of the residuals analysis. Normal distribution of the weighted residuals was checked with the normal probability plot, and with computation of the mean and standard deviation of the weighted residuals. α-Chymotrypsin exhibited triple-exponential fluorescence decay kinetics with decay times of 615 ± 76 ps, 1.7 ± 0.2 ns, and 4.3 ± 0.3 ns. The fractional fluorescence contributions depended on the emission wavelength. The fluorescence spectra of the components contributing to the total fluorescence were calculated from the steady-state fluorescence spectrum and fluorescence decays at different emission wavelengths, and from convoluted time-resolved emission spectra and a fluorescence decay measurement.     

Intramolecular energy hopping and energy trapping in polyphenylene dendrimers with multiple peryleneimide donor chromophores and a terryleneimide acceptor trap chromophore

Intramolecular F?rster-type excitation energy transfer (FRET) processes in a series of first-generation polyphenylene dendrimers substituted with spatially well-separated peryleneimide chromophores and a terryleneimide energy-trapping chromophore at the rim were investigated by steady-state and time-resolved fluorescence spectroscopy. Energy-hopping processes among the peryleneimide chromophores are revealed by anisotropy decay times of 50--80 ps consistent with a FRET rate constant of k(hopp) = 4.6 ns(-1). If a terryleneimide chromophore is present at the rim of the dendrimer together with three peryleneimide chromophores, more than 95% of the energy harvested by the peryleneimide chromophores is transferred and trapped in the terryleneimide. The two decay times (tau(1) = 52 ps and tau(2) = 175 ps) found for the peryleneimide emission band are recovered as rise times at the terryleneimide emission band proving that the energy trapping of peryleneimide excitation energy by the terryleneimide acceptor occurs via two different, efficient pathways. Molecular- modeling-based structures tentatively indicate that the rotation of the terryleneimide acceptor group can lead to a much smaller distance to a single donor chromophore, which could explain the occurrence of two energy-trapping rate constants. All energy-transfer processes are quantitatively describable with F?rster energy transfer theory, and the influence of the dipole orientation factor in the F?rster equation is discussed.     

Distinguishing chromophore structures of photocycle intermediates of the photoreceptor PYP by transient fluorescence and energy transfer

The cinnamoyl chromophore is the light-activated switch of the photoreceptor photoactive yellow protein (PYP) and isomerizes during the functional cycle. The fluorescence of W119, the only tryptophan of PYP, is quenched by energy transfer to the chromophore. This depends on the chromophore's transition dipole moment orientation and spectrum, both of which change during the photocycle. The transient fluorescence of W119 thus serves as a sensitive kinetic monitor of the chromophore's structure and orientation and was used for the first time to investigate the photocycle kinetics. From these data and measurements of the ps-fluorescence decay with background illumination (470 nm) we determined the fluorescence lifetimes of W119 in the I(1) and I (1') intermediates. Two coexisting distinct chromophore structures were proposed for the I(1) photointermediate from time-resolved X-ray diffraction ( Ihee, H., et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 7145 ): one with two hydrogen bonds to E46 and Y42, and a second with only one H-bond to Y42 and a different orientation. Only for the first of these is the calculated fluorescence lifetime of 0.22 ns in good agreement with the observed one of 0.26 ns. The second structure has a predicted lifetime of 0.71 ns. Thus, we conclude that in solution only the first I(1) structure occurs. The high resolution structure of the I(1') intermediate, the decay product of I(1) at alkaline pH, is still unknown. We predict from the observed lifetime of 1.3 ns that the chromophore structure of I(1') is quite similar to that of the I(2) intermediate, and I(1') should thus be considered as the alkaline (deprotonated) form of I(2).     

Excited-state intramolecular charge transfer in 9-aminoacridine derivative

A new fluorochromic dye was obtained from the reaction of 9-aminoacridine with ethyl-2-cyano-3-ethoxyacrylate. It displays complex fluorescence that is ascribed to normal emission from the acridine chromophore in addition to excited-state intramolecular charge transfer (ESICT) formed upon light excitation. The analysis of the fluorescence decays in different solvents reveals two short-lived components in the range of 80-450 ps and 0.7-3.2 ns, ascribed to the formation and decay of the intramolecular charge transfer (ICT) state, in addition to a third component of about 9.0 ns, which is related to the normal emission from the acridine singlet excited state, probably in an enol-imine tautomeric form. The ICT emission is readily quenched by water addition to polar solvents, and this effect is ascribed to changes in the keto-amine/enol-imine equilibrium of this fluorochromic dye.     

Lifetime determination of fluorescence and phosphorescence of a series of oligofluorenes

The photoluminescence (PL) properties of oligofluorenes with 2-ethylhexyl group in 9, 9' position in solution and as thin films were investigated by time-resolved techniques at both room temperature and 77 K. The fluorescence lifetimes of the oligomers decrease with chain length. The lifetimes tau follow the relation tau=386+808(1/n) (ps) where n is the number of fluorene units in the oligomer. Concentration and laser excitation energy dependences of PL spectra of the oligofluorenes are also given. Phosphorescence was observed for oligofluorenes in the frozen matrix of MTHF at 77 K. The lifetime of phosphorescence increases with increasing molecular length. Similar emission bands were observed for oligofluorenes with a central ketogroup. A lifetime analysis clearly reveals that the "green emission" of the oligomers free of ketogroups results from a phosphorescence with lifetime tau of 3 ms while the green emission from the keto-oligomer is a fluorescence from a charge transfer pi-pi* level of tau=8 ns.     

RESOLUTION OF SPECTRAL AND LIFETIME HETEROGENEITY OF TRYPTOPHAN FLUORESCENCE IN BACTERIORHODOPSIN

Abstract— The picosecond time-resolved fluorescence decay of bacteriorhodopsin (BR) was analyzed by the maximum entropy method. Results showed five distributions of lifetimes indicating at least five decay components. A wavelength-dependent study of emission decay of BR was carried out in the wavelength region from 310 to 390 nm. The decay at each wavelength was resolvable into four decay components by the discrete exponential analysis. The three short lifetime components (100 ± 20 ps, 400 ± 50 ps and 1.0 ± 0.1 ns) were independent of wavelength, whereas the longest lifetime component was wavelength dependent (varying from 4.1 ns at 310 nm to 5.7 ns at 390 nm). These results are inconsistent with the existing model of associating the fluorescence of bacteriorhodopsin with two or four lifetime components. An attempt is made to associate the five decay components with the emitting tryptophans of BR.     

Ultraviolet laser induced fluorescence of human aorta

Laser induced fluorescence from normal human aorta is studied with u.v. excitations of 305 to 310 nm, observing emission from 320 to 500 nm. In this region LIF lineshapes are strongly dependent on the excitation wavelength, suggesting that at least two fluorophores are being observed. The short wavelength fluorophore, peaking at 34Onm, is identified as tryptophan, while the longer wavelength fluorophore, peaking at 387 nm, is associated with collagen and elastin. In addition, fluorescence time decays of each component are measured with a time correlated photon counting system. A four-exponential fit of each decay is necessary to extract fluorescence lifetimes, which range from 33 ps to 8.6 ns.     

Energy transfer in calixarene-based cofacial-positioned perylene bisimide arrays

The synthesis of multichromophoric perylene bisimide-calix[4]arene arrays with up to five perylene units (containing orange, violet, and green perylene bisimide chromophores) and of monochromophoric model compounds was achieved by subsequent imidization of mono-Boc functionalized calix[4]arene linkers with three different types of perylene bisimide dye units. The optical properties of all compounds were studied with UV/vis absorption and steady state and time-resolved fluorescence spectroscopy. Upon excitation of the inner orange dye at 490 nm of array 3, strong fluorescence emission of the outer green perylene bisimide (PBI) chromophore at 744 nm is observed. The fluorescence excitation spectra of compounds 3 and 4 (lambdadet = 850 nm) show all absorption bands of the parent chromophores (e.g., all perylene units contribute to the emission from S1 state of the green PBI). Thus, the fluorescence emission and excitation spectra as well as time-resolved data of fluorescence lifetimes in the absence (tauD = 5.1 ns) and in the presence of an acceptor (tauDA = 0.8 ns) suggest efficient energy transfer processes between the perylene bisimide dye units. For the bichromophoric array 4, the energy transfer rate is calculated to a value of 1.05 x 109 s-1. These results demonstrate highly efficient energy transfer in cofacially assembled dye arrays.     

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.     

FLUORESCENCE DECAY KINETICS and CHARACTERISTICS OF BIMOLECULAR EXCITON ANNIHILATION IN CHLOROPLASTS

Abstract— The decay profiles of the fluorescence of dark-adapted spinach chloroplasts (0^A°C) excited with single 30 ps 532 nm laser pulses of varying intensities were measured with a low-jitter streak camera system. By comparing the decay profiles of the fluorescence at low and high laser fluences, i.e. in the absence and presence, respectively, of dynamic bimolecular exciton-exciton annihilation effects, the duration of such dynamic annihilation events can be estimated. A simple model suggests that the influence of bimolecular annihilation events on the fluorescence decay kinetics should disappear within a time interval corresponding to the low intensity, unimolecular lifetime of the exciton population which is subject to exciton-exciton annihilation. The low intensity fluorescence decay profiles are characterized by three to four lifetimes (Reviewed by A. R. Holzwarth, Photochem. Photobiol. 43,707–725, 1986); it is shown here that only the shortest fluorescence components are subject to exciton annihilation, since the kinetics of the fluorescence decay are influenced by annihilations only within the initial 150–200 ps time interval after the excitation pulse. The amplitudes (but not the decay kinetics) of the longer-lived fluorescence components are decreased at high levels of laser pulse excitations, suggesting that these components are derived from the shorter-lived fluorescence decay components. The implications of these results are*discussed within the contexts of current models of the fluorescence in chloroplasts.     

Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.     

Time-resolved and steady-state fluorescence spectroscopy of eumelanin and indolic polymers

Eumelanin plays a variety of important physiological roles in human skin. However, its structure and fundamental properties still remain poorly understood. Although the absorbance of eumelanin is broad and reveals little about its structure, a variety of techniques have revealed the presence of a disordered array of chromophores within the melanin compound. In order to examine the fluorescence decay dynamics of these chromophores, time-resolved spectroscopy was applied to solutions of synthetic eumelanin and a melanin-like polymer of N-methyl,5-hydroxy,6-methoxyindole (N-Me-5H6MI). Solutions were excited with 80 fs laser pulses at 355, 370, 390 and 400 nm, and decay time courses were acquired at 20 nm intervals between 400 and 600 nm for each excitation wavelength. Decay profiles for both eumelanin and the polymer exhibited a characteristic multiexponential behavior with decay times between 0.5 and 15 ns, although steady-state spectra for the polymer exhibited only two peaks. The long-decay component in the polymer showed a significant decrease in both amplitude (30-5%) and decay time (14-6 ns) with increasing emission wavelength. In contrast, the amplitude and decay time in melanin increased slightly (10-15% and 7-10 ns, respectively) from 400 to 520 nm emission, at which point they leveled off. These trends were consistent for all excitation wavelengths. These results suggest that the multiexponential behavior of melanin fluorescence is characteristic of each oligomer within the eumelanin compound, and is consistent with the assertion that the diversity of constituents within eumelanin provides it with a robustness in spectral properties.     

A new instrument for time-resolved reduction of scattered radiation in fluorescence measurements

An opto-electronic cross-correlation system was employed to reduce the scattering influence in fluorescence measurements. A stable optical delay line incorporated into the instrument was positioned to yield detection at a fixed time after excitation; the optimal delay time was determined simply from the ratio of the fluorescence decay curve to a similar curve portraying scattering response. Signal-to-scattering background enhancements greater than two were measured for the very short-lived (τ = 0.7 ns) fluorophores whereas a six-fold increase was measured for fluorophores with longer lifetimes. The shortest lifetime which would benefit from time-resolution in this system is limited by the time-response of the photomultiplier tube (1.1 ns FWHM); the excitation pulses are on the order of 6 ps.     

The thiourea group modulates the fluorescence emission decay of fluorescein-labeled molecules

We have investigated the spectral properties and emission characteristics of fluorescein-5-thiocarbamoyl-N,N'-caproate (FITC-ACA) to examine the origin of the complex emission decay often observed in fluorescein-labeled molecules. The covalent attachment of fluorescein to epsilon-amino-n-caproic acid does not perturb the prototropic transitions of the chromophore or the general fluorescence characteristics of the various prototropic forms. However, both the monoanion and dianion forms of FITC-ACA are quenched relative to free fluorescein and exhibit a complex emission decay that is described by two discrete lifetimes. The thiourea group that links the chromophore to the caproic acid is shown to modulate the emission properties of the FITC-ACA. We show that the emission decay can also be analyzed using the asymmetric distribution model of Alcala et al. In this analysis, the tauL and tauu parameters that represent the lower and upper lifetime limits of the distribution reflect the quenched (0 ns) and unquenched lifetimes, respectively. The beta parameter that describes the distribution of lifetimes between the two limiting states can be related to the quenching efficiency of the thiourea group and to the structure and dynamics of the FITC-ACA molecule.     

Radiative and Nonradiative Lifetimes of Band Edge States and Deep Trap States of CdS Nanoparticles Determined by Time-correlated Single Photon Counting

Emission lifetimes of band edge and deep trap states of CdS nanoparticles with different surface capping were measured using time-resolved single-photon-counting^[1]. For unpassivated nanoparticles with low fluorescence yield, the emission is dominated by deep trap states and the decay can be fit to a single exponential with a time constant of 5 ns that is independent of excitation intensity. For surface passivated nanoparticles with strong luminescence, the emission is dominated by band edge states and the decay is fit to a double exponential with time constants of a few ns and 50 ns. While the 50 ns decay is independent of excitation intensity, the fast component is strongly dependent on intensity. For the fast decay component, the amplitude decreases non-linearly and the time constant becomes longer (from 2.5 to 7.9 ns) as the intensity decrease. The results support the model of exciton-exciton annihilation^[2] upon trap state saturation at high excitation intensities.     

Examination of chlorin-bacteriochlorin energy-transfer dyads as prototypes for near-infrared molecular imaging probes

Abstract New classes of synthetic chlorin and bacteriochlorin macrocycles are characterized by narrow spectral widths, tunable absorption and fluorescence features across the red and near-infrared (NIR) regions, tunable excited-state lifetimes (<1 to >10 ns) and chemical stability. Such properties make dyad constructs based on synthetic chlorin and bacteriochlorin units intriguing candidates for the development of NIR molecular imaging probes. In this study, two such dyads (FbC-FbB and ZnC-FbB) were investigated. The dyads contain either a free base (Fb) or zinc (Zn) chlorin (C) as the energy donor and a free base bacteriochlorin (B) as the energy acceptor. In both constructs, energy transfer from the chlorin to bacteriochlorin occurs with a rate constant of approximately (5 ps)(-1) and a yield of >99%. Thus, each dyad effectively behaves as a single chromophore with an exceptionally large Stokes shift (85 nm for FbC-FbB and 110 nm for ZnC-FbB) between the red-region absorption of the chlorin and the NIR fluorescence of the bacteriochlorin (lambda(f) = 760 nm, Phi(f) = 0.19, tau approximately 5.5 ns in toluene). The long-wavelength transitions (absorption, emission) of each constituent of each dyad exhibit narrow (相似文献