Femtosecond Fluorescence Up-conversion Research Articles

Femtosecond Fluorescence Dynamics of Flavoproteins: Comparative Studies on Flavodoxin, Its Site-Directed Mutants, and Riboflavin Binding Protein Regarding Ultrafast Electron Transfer in Protein Nanospaces

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.

The Role of the Exciplex State in Photoinduced Electron Transfer of Phytochlorin−[60]Fullerene Dyads

The photoinduced electron transfer (ET) in five structurally different phytochlorin-fullerene dyads was studied in polar and nonpolar solvents using femtosecond fluorescence up-conversion and pump−probe transient-absorption techniques. Small changes in the structures of the dyads result in considerable changes in the ET properties and allow the determination of reorganization energies of the photoinduced reactions and electronic couplings between the initial and final states. After the excitation of the phytochlorin moiety to the second excited singlet state, the dyads relax rapidly to the first excited singlet state of phytochlorin. The first excited singlet state of phytochlorin is in equilibrium with an intramolecular exciplex state. In polar benzonitrile, the exciplex undergoes an electron transfer, and a complete-charge-separated (CCS) state is formed with a quantum yield close to unity. In contrast to the previously studied phytochlorin−fullerene dyads, the dyads in the present study form the CCS state also in nonpolar toluene with a yield influenced by minor changes in the molecular structure. The new dyads have a weaker phytochlorin−fullerene interaction due to longer separation distances between the two moieties. Therefore, the energies of the exciplex states are increased, and thus, their formation rates are reduced. In addition, the rates and yields of the complete charge separations are increased both in polar and nonpolar solvents. In benzonitrile, the reorganization energies for the transitions from the exciplex to the CCS and from the CCS to the ground state are 0.38 and 1.05 eV, respectively. The electronic couplings between the corresponding initial and final states of the two transitions mentioned above are 22 and 15 cm-1.

Ultrafast fluorescence detection in tris(2,2'-bipyridine)ruthenium(II) complex in solution: relaxation dynamics involving higher excited states.

The excited-state dynamics of a transition metal complex, tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)(3)](2+), has been investigated using femtosecond fluorescence upconversion spectroscopy. The relaxation dynamics in these molecules is of great importance in understanding the various ultrafast processes related to interfacial electron transfer, especially in semiconductor nanoparticles. Despite several experimental and theoretical efforts, direct observation of a Franck-Condon singlet excited state in this molecule was missing. In this study, emission from the Franck-Condon excited singlet state of [Ru(bpy)(3)](2+) has been observed for the first time, and its lifetime has been estimated to be 40 +/- 15 fs. Biexponential decays with a fast rise component observed at longer wavelengths indicated the existence of more than one emitting state in the system. From a detailed data analysis, it has been proposed that, on excitation at 410 nm, crossover from higher excited (1)(MLCT) states to the vibrationally hot triplet manifold occurs with an intersystem crossing time constant of 40 +/- 15 fs. Mixing of the higher levels in the triplet state with the singlet state due to strong spin-orbit coupling is proposed. This enhances the radiative rate constant, k(r), of the vibrationally hot states within the triplet manifold, facilitating the upconversion of the emitted photons. The vibrationally excited triplet, which is emissive, undergoes vibrational cooling with a decay time in the range of 0.56-1.3 ps and relaxes to the long-lived triplet state. The results on the relaxation dynamics of the higher excited states in [Ru(bpy)(3)](2+) are valuable in explaining the role of nonequilibrated higher excited sensitizer states of transition metal complexes in the electron injection and other ultrafast processes.

Femtosecond Fluorescence Anisotropy Studies of Excited-State Intramolecular Double-Proton Transfer in [2,2‘-bipyridyl]-3,3‘-diol in Solution

For [2,2‘-bipyridyl]-3,3‘-diol (BP(OH)2), dissolved in aprotic solvents, the time dependence of the fluorescence anisotropy has been studied using the femtosecond fluorescence up-conversion technique. A fast fluorescence anisotropy decay, with a characteristic time of ∼350 fs, is observed when detection is at 460 nm, i.e., near the blue edge of the BP(OH)2 broad-band emission. It is discussed that the fast decay is typical of emission from the “di-enol” excited Franck−Condon state for which the lifetime is limited by the first steps of a branched excited-state double double-proton-transfer process. The branched reaction includes concerted and consecutive double-proton-transfer. The fast decay of the “di-enol” excited state into “mono-keto” and “di-keto” excited states is indicative of a (quasi-)barrierless reaction. To explain that the rapid (∼350 fs) initial decay is manifested in the fluorescence anisotropy, it is conjectured that the electronic wave function characteristic of the emissive state is a reaction-coordinate dependent admixture of diabatic wave functions, these functions being characteristic of the “enol”- and “keto”-tautomers. The progress of the double-proton-transfer reaction is accompanied by a change of the admixture of the excited-state tautomer wave functions and in this way gives rise to a rapid decay in the fluorescence anisotropy. The fluorescence anisotropy of BP(OH)2 furthermore includes two slower decay components. These components, with time constants of a few tens of picoseconds, are related to the second step of the consecutive double proton-transfer kinetics (∼10 ps) and the rotational diffusion motions of the solute in the liquid (20−40 ps).

Adenine, deoxyadenosine and deoxyadenosine 5 ′-monophosphate studied by femtosecond fluorescence upconversion spectroscopy

Aqueous solutions of adenine (A), deoxyadenosine (dA) and deoxyadenosine 5 ′-monophosphate (dAMP) were studied in room temperature by femtosecond fluorescence upconversion. The fluorescence decays cannot be described by single exponentials. They consist of an ultrafast component (230 fs for A, <100 fs for dA and dAMP) and a slower one (8 ps for A, 0.5 ps for dA and dAMP). The slow component constitutes 95% of the total fluorescence (time-integrated) for the base while only 24% for the nucleoside or the nucleotide. The initial fluorescence anisotropy is 0.30±0.03 for A, 0.25±0.05 for dA and dAMP. The anisotropy of the A fluorescence partially decays during its lifetime due to rotational diffusion.

Spectral Sensitization and Supersensitization of AgBr Nanocrystals Studied by Ultrafast Fluorescence Spectroscopy

Spectral sensitization and supersensitization of the silver bromide nanocrystals were studied by the femtosecond fluorescence up-conversion technique. An annihilation-free fluorescence from J-aggregates of the Dye 1 (3,3‘-disulfopropyl-5,5‘-dichloro-9-ethylthiacarbocyanine) adsorbed on the silver bromide nanocrystals (edge-to-edge distance from 40 to 900 nm) of different shapes (cubic and octahedral) was measured. Very fast nonexponential fluorescence decays were observed with a fast component from 0.4 to 2.5 ps, and an average decay time from 1.1 to 5.5 ps, depending on the type and size of AgBr grains. The average fluorescence decay time is several times longer on the cubic grains than that on the octahedral grains. Upon addition of a supersensitizer (SS) (3,3‘-disulfopropyl-9-ethyl-4,5,4‘,5‘-dibenzothiacarbocyanine), which is coadsorbed on the surface of silver bromide grains, the fluorescence decay became several times faster with a mean decay time of 0.60−1.3 ps. Different models of supersensitization were examined. The results were analyzed in the framework of the “hole-trapping” supersensitization model. Octahedral grains with different sizes were examined. The effective hole-trapping (electron transfer from SS to the excited J-aggregate) rate constant was found to be independent of the grain size and equal to ∼0.60 ps-1. The increase of the electron injection rate (from the excited J-aggregate to the AgBr conduction band) with the increase of the grain size is observed and a possible mechanism is discussed.

Visualization of excitation energy transfer processes in plants and algae.

Development of the time-resolved fluorescence spectroscopy in the pico-second time range and its application to the energy transfer processes in many photosynthetic organisms is reviewed here. This method enabled visualization of energy transfer processes by three-dimensional expression of fluorescence spectra and discrimination of kinetic components and spectral components. The second generation of the ultrafast fluorescence spectroscopy is the femto-second (fs) fluorescence up-conversion, and this has enabled analyses of the transfer processes from carotenoids to chlorophylls with a resolution of less than 100 fs. For future progress, a further development of the spectroscopy is indispensable as well as structural data at atomic resolution.

Thymine, thymidine and thymidine 5 ′-monophosphate studied by femtosecond fluorescence upconversion spectroscopy

We report fluorescence measurements of DNA components performed on the femtosecond time-scale using the fluorescence upconversion technique. Aqueous solutions of thymine (T), thymidine (dT) and thymidine 5 ′-monophosphate (TMP) were studied in room temperature by excitation at 267 nm and detection at wavelengths between 310 and 380 nm. The fluorescence decays are complex and cannot be described by single exponentials. About 25% of the fluorescence disappears within 150 fs and the remaining part decays more slowly when going from the base through the nucleoside to the nucleotide. The initial fluorescence anisotropy was found to be 0.35±0.03 and did not show any drastic change on the examined time interval.

DNA/RNA nucleotides and nucleosides: direct measurement of excited-state lifetimes by femtosecond fluorescence up-conversion

Fluorescence decay times of the nucleosides: adenosine, guanosine, cytidine and thymidine, and of the corresponding nucleotides, were determined using the technique of fluorescence up-conversion with femtosecond time resolution. The excited-state lifetimes of these nucleic acid molecules all fall in the sub-picosecond time scale, confirming the presence of an ultrafast internal conversion channel for both the nucleotides and the nucleosides; the nucleotides lifetimes are longer than those of the nucleosides by up to 20%. The ultrafast internal conversion is biologically relevant to the stability of DNA, and our results support the sub-picosecond repopulation of the ground state, consistent with transient absorption studies on the femtosecond time scale.

Femtosecond studies of double proton transfer in photoexcited [2,2’-bipyridyl]-3,3’-diol in solution

Double proton transfer dynamics of [2,2’-bipyridyl]-3,3’-diol in its photoexcited state is studied by means of femtosecond fluorescence upconversion spectroscopy. The measurements reveal very fast fluorescence anisotropy decay components, with typical times of ∼200 fs and 10 ps. The short-living (∼200 fs) component is attributed to di-enol tautomer emission; the 10 ps component is characteristic of the mono-keto to di-keto tautomer conversion.

Charge carrier dynamics in CdSe nanocrystals: implications for the use of quantum dots in novel photovoltaics

The dynamics of photo-generated electrons and holes in CdSe quantum dots have been studied using the femtosecond fluorescence upconversion technique, permitting an unambiguous examination of the excited state. The band edge emission shows an expected size dependence on the decay rate. We find that the deep trap emission is coupled to the band edge fluorescence, implicating surface states as important factors in the excited state lifetime of the hole. As a factor of the overall efficiency of solar cells, the rate of charge separation and the fate of the exciton are important considerations in the design of nanocrystal-based photovoltaic devices.

Spectroscopy and dynamics of excited-state intramolecular proton-transfer reaction in 5-hydroxyflavone

Spectroscopy and dynamics of excited-state intramolecular proton-transfer (ESIPT) in 5-hydroxyflavone (5HF) have been investigated. In cyclohexane dual fluorescence maxima at ∼420 and 700 nm were observed. The time scale for both proton and deuterium transfer in the excited state cannot be resolved under the response time of ca. 160 fs of our femtosecond fluorescence upconversion system. Following ESIPT the excited keto-tautomer undergoes an unusually fast relaxation rate of ∼1.2 ps −1 . Fast internal conversion is concluded to play a major role to account for the dominant radiationless deactivation.

Excited-State Intramolecular Proton Transfer in 10-Hydroxybenzo[h]quinoline

The dynamics of excited-state intramolecular enol−keto proton-transfer tautomerism in 10-hydroxybenzoquinoline (HBQ) and its deuterated analogue (DBQ) have been investigated by steady-state absorption and fluorescence spectroscopy, femtosecond fluorescence upconversion in combination with pump−probe transient absorption experiments in nonpolar solvents. In cyclohexane, the time scale for both proton and deuterium transfer in the excited state cannot be resolved under the response limit of ca. 160 and 200 fs, respectively, of our current upconversion and transient absorption systems. The initially prepared keto tautomer is in a higher lying excited state, possibly the state (prime indicates the keto-tautomer form) which then undergoes a ∼330 fs → internal conversion, resulting in a highly vibrationally excited state. Subsequently, a solvent-induced vibrational relaxation takes place in a time scale of 8−10 ps followed by a relatively much longer, thermally cooled → decay rate of 3.3 × 109 s-1 (τf ∼ 300 ps-1). The results in combination with extremely weak enol fluorescence resolved from the steady-state measurement lead us to conclude that excited-state intramolecular proton transfer (ESIPT) is essentially barrierless. The rate of ESIPT upon 385−405 nm excitation may be determined within the period of low-frequency, large-amplitude vibrations incorporating the motion of atoms associated with the hydrogen bond.

Femtosecond Fluorescence Anisotropy Studies of Solvation-Induced Intraligand Charge Transfer in Photoexcited Aluminum(III) Tris(8-hydroxyquinoline)

For the organic light emitting diode compound aluminum(III) tris(8-hydroxyquinoline) (Alq3), dissolved in dimethylformamide (DMF), the time dependence of the fluorescence anisotropy has been studied using the femtosecond fluorescence upconversion technique. Upon excitation within the first absorption band, near 364 nm, with polarized laser pulses of duration less than 150 fs, an initial fluorescence anisotropy of about 0.2 is found to rapidly decay with a time constant of 2.0 ± 0.2 ps. The observed fast anisotropy decay component is concomitant with the solvation-induced dynamic Stokes shift of about 1000 cm-1. When excitation is at wavelengths below 330 nm, the fluorescence of Alq3 in liquid solution does not show any anisotropy effect. It is discussed that the emissive lowest excited electronic state of Alq3 is ligand localized and that its electronic wave function is affected by solvation. More specifically, the electronic wave function is considered to be an admixture of wave functions belonging to se...

Ultrafast Carrier Dynamics in CdSe Nanocrystals Determined by Femtosecond Fluorescence Upconversion Spectroscopy

Femtosecond fluorescence upconversion has been utilized to study the band edge and deep trap emission dynamics of cadmium selenide (CdSe) nanocrystals (NC's) ranging in size from 27 to 72 Å in diameter. Both the band edge rise time and decay show a direct correlation to NC size, and a rise time that depends on excitation energy. Surface-oxidized and non-oxidized NC's display the same band edge fluorescence decay kinetics, but the relative amplitudes of the short and long components differ. The deep trap emission that appears within 2 ps is attributed to ultrafast relaxation of a surface selenium dangling bond electron to the valence band where it combines radiatively with the initial photogenerated hole. By this process, the large amplitude of the band edge emission that is attributed to direct electron/hole recombination is attenuated within the initial 2−6 ps. The long lifetime of the band edge emission originates from a triplet state, with an energy lying just below the lowest electronic level consistent with the “Dark Exciton”. The extended deep trap emission arises from the relaxation of the excited-state conduction band electron to a surface-localized hole or vice-versa. A new model is presented which describes these mechanisms for exciton relaxation in CdSe quantum dots.

Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Reactions of Flavin Chromophores in Protein Nanospace

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp.NH) or tyrosine (Tyr.OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp.NH.....F.....Tyr.OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC → Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr.OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr.OH to excited F.

Exciton Annihilation in J-Aggregates Probed by Femtosecond Fluorescence Upconversion

Femtosecond fluorescence upconversion was used to study the excited-state dynamics of two-dimensional J-aggregates of a thiacyanine dye. At high intensities the excited-state population decay is faster than exponential and the decay is intensity dependent. A singlet−singlet exciton annihilation decay mechanism is consistent with the observed behavior. A value of 4 × 1012 molecule s-1 is calculated for the exciton annihilation constant γ. Exciton annihilation measurements reported in the literature for J-aggregates of several cyanine dyes are recast into the same units for comparison. The physical state of these J-aggregates is elucidated to explore the relationship between the magnitude of the exciton annihilation constants and the liquid-crystalline nature of these aqueous colloidal systems. Attempts to time-resolve spectral features related to intraband relaxation in the excited excitonic states of the thiacyanine J-aggregate prior to annihilation indicate that these relaxation processes are faster than 100 fs at room temperature.

Femtosecond Studies of Barrierless Rotamers in Solution

Femtosecond fluorescence upconversion experiments have been performed for photoexcited auramine (a diphenylaminemethane dye) in liquid solution. Following pulsed laser excitation, the fluorescence shows a multiexponential decay with an initial fast decay of 2 ps. The lifetime of the relaxed excited state is 30 ps or longer, depending on the viscosity of the solvent. Concomitant with the initial fast decay a dynamic Stokes shift is observed. From a study of the excited state dynamics as a function of solvent viscosity it is concluded that auramine undergoes a picosecond (quasi)-barrierless relaxation from an emissive to a non-emissive excited state. The relaxation within the excited state arises from torsional diffusion motions of the phenyl rings of the auramine molecule.

Vibrational Motion of Electron Donor-Acceptor Complexes Time-Resolved by Femtosecond Fluorescence Spectroscopy

The excited-state dynamics of several electron donor-acceptor complexes were studied by a femtosecond fluorescence up-conversion technique. The spontaneous fluorescence decay of the complexes contains an oscillatory component superimposed with an ultrafast decay component. The oscillatory component was assigned to the out-of-plane vibration of the acceptors (tetracyanoethylene (TCNE), chloranil, fluoranil) having b3u symmetry. The time-dependent fluorescence spectrum for the TCNE - hexamethylbenzene (HMB) complex was reconstructed from the fluorescence dynamics at different wavelengths and the fluorescence peak-shift correlation function was obtained. The ultrafast relaxation of the fluorescence peak position is superimposed with the oscillatory component. The main component of the spectral relaxation of 115 fs is attributed mainly to the intramolecular vibrational energy redistribution process in both donor and acceptor parts of the complex. The oscillatory behavior of the fluorescence peak position demonstrates the modulation of the transition frequency during the vibrations. The time dependence of the mean transition moment was constructed. It oscillates with the same frequency (155 cm−1), which is an indication of the non-Condon transition. Thus, two mechanisms are observed to be responsible for the oscillations: the modulation of the transition frequency and the modulation of the mean transition moment by the vibrational motion.

Femtosecond studies of twisting dynamics of auramine in solution

Femtosecond fluorescence upconversion experiments as performed for photoexcited auramine (a diphenylaminemethane dye) in liquid solution are discussed. Upon pulsed laser excitation the solute shows a dynamic Stokes shift as well as a drop in fluorescence intensity in only a few picoseconds. From a study of the excited state dynamics as a function of solvent viscosity it is concluded that auramine undergoes a picosecond (quasi)-barrierless relaxation from an emissive state to a non-emissive excited state that decays to the ground state in about 30 ps. The relaxation is controlled by the torsional motions of the phenyl rings of the auramine molecule.