Higher Triplet Excited State Research Articles

Analysis of Photobleaching in Single-Molecule Multicolor Excitation and Förster Resonance Energy Transfer Measurements

Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.

C−O Bond Cleavage of Benzophenone Substituted by 4-CH2OR (R= C6H5 and CH3) with Stepwise Two-Photon Excitation

The C-O bond cleavage from benzophenone substituted with 4-CH2OR (p-BPCH2OR, 1-3), such as p-phenoxymethylbenzophenone (1, R= C6H5) and p-methoxymethylbenzophenone (2, R= CH3), occurred by a stepwise two-photon excitation during two-color, two-laser flash photolysis. On the other hand, no C-O bond cleavage occurred from p-hydroxymethylbenzophenone (3, R = H). The first 355-nm laser excitation of 1-3 generates p-BPCH2OR in the lowest triplet excited state (T1) which has an absorption at 532 nm. When p-BPCH2OR(T1) is excited with the second 532-nm laser to p-BPCH2OR in the higher triplet excited state (T(n)), the C-O bond cleavage occurred within the laser flash duration of 5 ns. The quantum yields of the C-O bond cleavage during the second 532-nm laser irradiation were found to be 0.015 +/- 0.007 and 0.007 +/- 0.003 for 1 and 2, respectively. Although these values are low, the diminishing 1(T1) or 2(T1) was found to convert, in almost 100% yield, to phenoxyl (C6H5O*) and p-benzoylbenzyl (BPCH2*) radicals or methoxyl (CH3O*) and BPCH2* radicals, respectively. The T(n) excitation energy, the energy barrier along the potential surface between the T(n) states and product radicals, and delocalization of the T(n) state molecular orbital including BP and CH2OR (R = C6H5, CH3, H) moieties are important factors for the occurrence of the C-O bond cleavage. It is found that the C-O bond cleavage and production of free radicals, such as BPCH2*, C6H5O*, and CH3O*, can be performed by a stepwise two-photon excitation. The present study is an example in which the chemical reactions can be selectively initiated from the T(n) state but not from the S1 and T1 states.

Significant Effects of Substituents on Substituted Naphthalenes in the Higher Triplet Excited State

Substituent effect on the lifetimes of a series of substituted naphthalenes (Np) in the higher triplet excited state (Tn) was studied with transient absorption measurements using the two-color two-laser flash photolysis technique. Lifetimes of Np(Tn) in cyclohexane solution were determined from the triplet energy transfer quenching by carbon tetrachloride to be 0.98-63 ps. The different lifetimes of Np(Tn) were explained by the energy gap law for the internal conversion from Np(Tn) to Np(T1), indicating that Np(Tn) quenched by carbon tetrachloride is assigned to Np(T2) with the longest lifetime among Np(Tn). The lifetime of Np(Tn) was correlative with the Hammett sigmap constant. Electronic characters of substituents showed a more significant influence on the energy of the T2 state than that of the T1 state.

Photophysical Properties of Oligo(2,3-Thienyleneethynylene)s

Photophysical properties of oligo(2,3-thienyleneethynylene)s (nTE, n denotes the number of thiophene rings, n = 2, 3) in benzene were investigated using steady-state, time-resolved fluorescence, and transient absorption spectroscopies. For 2TE, generation of the radiative S2 and nonradiative S1 states was confirmed. Upon excitation, the S2 state was initially generated and deactivated to the S1 state within 10 ps. The S1 state exhibited the transient absorption band at 470 nm, of which the lifetime was estimated to be 5.3 ns. In the case of 3TE, on the other hand, it was revealed that the radiative S1 state with a transient absorption peak at 650 nm was generated upon excitation. The T1 states of nTE were generated from the S1 states. The quantum yields were estimated to be 0.52 and 0.54 for 2TE and 3TE, respectively. Extremely fast reactions in the higher triplet excited state were indicated for both 2TE and 3TE.

Homolytic cleavage of C–Si bond of p-trimethylsilylmethylacetophenone upon stepwise two-photon excitation using two-color two-laser flash photolysis

C–Si bond cleavage of p-trimethylsilylmethylacetophenone( 1) occurred in a higher triplet excited state (T n ), giving mainly p-acetylbenzyl radical with the transient absorption in the region of 295–360 nm, with a quantum yield of 0.046 ± 0.008 using the two-color two-laser photolysis techniques. In contrast, the C–Si bond cleavage of p-trimethylsilylmethylbenzophenone( 2) was absent in the T n state whose energy is larger than the C–Si bond cleavage energy. The results can explain existence of a bond cleavage crossing between potential surfaces of the T n state and a dissociative state of the C–Si bond for 1, but not for 2.

Stepwise Photocleavage of C−O Bonds of Bis(substituted-methyl)naphthalenes with Stepwise Excitation by Two-Color Two-Laser and Three-Color Three-Laser Irradiations

Stepwise photocleavage of naphthylmethyl-oxygen (C-O) bonds of mono(substituted-methyl)naphthalenes [1- and 2-ROCH2Np, R = 4-benzoylphenyl (BP), phenyl (Ph), and methyl (CH3)] and bis(substituted-methyl)naphthalenes [1,8-(ROCH2)2Np and 1,4-(ROCH2)2Np, R = BP and Ph] was observed to give the naphthylmethyl radicals (NpCH2* or ROCH2NpCH2*) in almost 100% yield with two-step or three-step excitation by the two-color two-laser or three-color three-laser irradiation, respectively, at room temperature. The C-O bond cleavage quantum yields of 1-PhOCH2Np, 2-PhOCH2Np, 1,8-(PhOCH2)2Np, and 1,4-(PhOCH2)2Np were higher than those of 1-BPOCH2Np, 2-BPOCH2Np, 1,8-(BPOCH2)2Np, and 1,4-(BPOCH2)2Np. No C-O bond cleavage occurred from 1,8-(HOCH2)2Np and 2-CH3OCH2Np in the higher triplet excited state (T(n)). The experimental results show that the C-O bond cleavage was determined not only by the position of the substituents on Np but also by the type of the substituents. The C-O bond cleavage of 1-ROCH2Np was more efficient than that of 2-ROCH2Np. In the case of 1,8-(ROCH2)2Np and 1,4-(ROCH2)2Np (R = BP and Ph), the first C-O bond cleavage from the T(n) states occurred to give ROCH2-substituted naphthylmethyl radicals (1,8- and 1,4-ROCH2NpCH2*) when the T1 states, generated with the 308-nm first laser irradiation, were excited using the 430-nm second laser. The second C-O bond cleavage occurred when 1,8- and 1,4-ROCH2NpCH2* in the ground state [1,8- and 1,4-ROCH2NpCH2*(D0)] were excited to the excited states [1,8- and 1,4-ROCH2NpCH2*(D(n))] using the third 355-nm laser during the three-color three-laser flash photolysis at room temperature. It was revealed that acenaphthene was produced as the final product during the stepwise C-O bond cleavages of 1,8-(BPOCH2)2Np and 1,8-(PhOCH2)2Np. This is a successful example of stepwise cleavage of two equivalent C-O bonds in a molecule using the three-color three-laser photolysis method.

Theoretical Study of Singlet and Triplet Excitation Energies in Oligothiophenes

We have analyzed singlet and triplet excitation energies in oligothiophenes (up to five rings) using time-dependent density-functional theory (TD-DFT) with different exchange-correlation functionals and compared them with results from the approximate coupled-cluster singles and doubles model (CC2) and experimental data. The excitation energies have been calculated in geometries obtained by TD-DFT optimization of the lowest excited singlet state and in the ground-state geometries of the neutral and anionic systems. TD-DFT methods underestimate photoluminescence energies but the energy difference between singlet and triplet states shows trends with the chain-length similar to CC2. We find that the second triplet excited state is below the first singlet excited state for long oligomers in contrast with the previous assignment of Rentsch et al. (Phys.Chem. Chem. Phys. 1999, 1, 1707). Their photodetachment photoelectron spectroscopy measurements are better described by considering higher triplet excited states.

Raman spectroscopy of phthalocyanines and their sulfonated derivatives

The aggregation and photochemistry of the copper (II) 3, 4″, 4″, 4‴-tetrasulfonated phthalocyanine, free base phthalocyanine and copper (II) phthalocyanine have been studied by UV–VIS absorption spectroscopy and resonance Raman spectroscopy (RRS). The vibrational mode ν 3 of (Cu(tsPc) 4− has been used as a probe in RRS measurements. The photochemistry of monomers and dimers of (Cu(tsPc) 4− has been studied in liquid solutions of H 2O and DMSO as well as in frozen matrices. Low temperature Raman measurements in a broad temperature range have been carried out for free base phthalocyanine and copper (II) phthalocyanine in DMSO to identify the nature of emissive bands observed in the Raman spectra. It has been shown that the dimerization equilibrium constant K for tetrasulfonated phthalocyanine Cu(tsPc) 4− is strongly shifted towards monomeric form in DMSO solutions and in human blood compared to aqueous systems. The emission band at around 682 nm in DMSO and aqueous solutions observed at 77 K for tetrasulfonated salt of copper(II) phthalocyanine in concentrated solutions has been assigned to the radical transient species generated during the photoinduced dissociation with the electron transfer between the molecules of phthalocyanines. The emission at 527 nm in DMSO and at 556 nm in water has been preliminarily assigned to the fluorescence from the higher excited triplet state T n →T 1.

Competitive Marcus-Type Electron Transfer and Energy Transfer from the Higher Triplet Excited State

Electron transfer (ELT) and energy transfer (ENT) from the higher excited triplet state (Tn) (n > 1) of various naphthalene derivatives (NpDs) to some polychlorobenzenes (CBs) such as chlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene were examined. It is confirmed that the ELT process from the NpDs (Tn) to CBs obeys Marcus's theory from the driving force dependence of the ELT yield. This is the first example of the Marcus-type ELT from the Tn state. As a competitive process of the ELT, the ENT from NpDs (Tn) to CBs giving the higher excited state of CBs (Tn) (NpD (Tn) + CB (S0) → NpD (S0) + CB (Tn)) was observed although a part of CBs (Tn) decomposed by the C−Cl bond dissociation giving chlorocyclohexadienyl radical and chloride radical. This is also the first example of intermolecular ENT from molecules in the Tn state to quenchers giving the quenchers in the Tn state.

Transient Phenomena of Polyphenyls in the Higher Triplet Excited State

Transient phenomena of polyphenyls (PP), such as biphenyl (BP), p-terphenyl (TP), and 1,3,5-triphenylbenzene (TPB) in the higher triplet excited states (Tn, n ≥ 2), were investigated with the nanosecond−nanosecond (ns−ns) two-color two-laser excitation method. PP(Tn) was generated by the excitation of PP in the lowest triplet excited state (PP(T1)) at the wavelength tuned to the absorption of PP(T1). Bleaching of the transient absorption of BP(T1) and TP(T1) was observed during the second laser irradiation in the presence of chloroalkanes (RCl) such as carbon tetrachloride (CCl4), dichloromethane (CH2Cl2), or 1,2-dichloroethane (DCE). In the case of TPB, clear bleaching of TPB(T1) was observed in the presence of CCl4, while no bleaching was observed in the presence of CH2Cl2 and DCE. No formation of PP•+ was detected during the quenching of PP(Tn) by RCl. Therefore, the bleaching of PP(T1) is attributed to triplet energy transfer quenching of PP(Tn) by RCl followed by the C−Cl bond cleavage. The lifetimes (τTn) of PP(Tn) were estimated from the dependence of the quenching efficiency on the RCl concentration. It was found that the τTn value decreased with the increasing number of benzene rings, BP(Tn) (950 ps) > TP(Tn) (620 ps) > TPB(Tn) (360 ps). The quenching mechanism of PP(Tn) by RCl was discussed.

Rate Constant of Bimolecular Triplet Energy Transfer from Chrysene in the Higher Triplet Excited States

The rate constant of the bimolecular triplet energy transfer from chrysene in the second higher triplet excited state (T2) to triplet quenchers (Q) such as biphenyl, naphthalene, and carbon tetrachloride was determined based on the lifetime of chrysene(T2) (45 ± 7 ps) measured directly with ns−ps two-color two-laser flash photolysis method. The rate constant was found to be larger than the diffusion-controlled rate constant in the solvents. The occurrence of the fast bimolecular triplet energy transfer is considered clear evidence of the Ware theoretical model on the energy transfer quenching of the excited species with short lifetimes by the quencher.

Intermolecular electron transfer from naphthalene derivatives in the higher triplet excited states.

Intermolecular electron transfer (ELT) from a series of naphthalene derivatives (NpD) in the higher triplet excited states (T(n)) to carbon tetrachloride (CCl(4)) in Ar-saturated acetonitrile was observed using the two-color two-laser flash photolysis method. The ELT efficiency depended on the driving force of ELT. Since the ELT from the T(n) state occurred competitively with the internal conversion (IC, T(n) --> T(1)) and the triplet energy transfer (ENT), the ELT became apparent only when sufficient free energy change of ELT was attained. On the other hand, ELT from the T(1) state was not observed, although ELT from the T(1) state with sufficiently long lifetime has a slightly exothermic driving force. The fast ELT from the T(n) state and lack of the reactivity of the T(1) state were explained well by the "sticky" dissociative electron-transfer model based on one-electron reductive attachment to CCl(4) leading to the C-Cl bond cleavage.

Quenching processes of aromatic hydrocarbons in the higher triplet excited states-energy transfer vs. electron transferElectronic supplementary information (ESI) available: The quenching of DBA(Tn) by CCl4, CHR(Tn) by NAP, the evidences of no DBA and CHR ions produced during two-color two-laser flash photolysis, and the evidence of formation of benzene/Cl complex. See http://www.rsc.org/suppdata/cp/b4/b400128a/

Quenching processes of several aromatic hydrocarbons (AH) such as naphthalene (NAP), dibenz[a,h]anthracene (DBA), and chrysene (CHR) in the higher triplet excited states (T2) by different quenchers (Q) such as p-dichlorobenzene, o-dicyanobenzene aromatic compounds, and chloroalkanes (RCl), have been investigated by the two-color two-laser excitation method. AH in the higher triplet excited states (AH(Tn, n ≥ 2)) initially generated by the excitation of AH(T1) at the wavelength tuned to the absorption of AH(T1). AH(Tn) decays to a AH(T2) with the longest lifetime among AH(Tn) through the fast internal conversion. In the presence of Q, the competition of triplet energy transfer (TENT) and electron transfer (ELT) reactions between AH(T2) and Q are expected. However, no AH radical cation was observed, especially when the quenchers were chloroalkanes such as carbon tetrachloride (CCl4), methylene dichloride (CH2Cl2), 1,2-dichloroethane, which are good electron acceptors. It is suggested that the TENT is important during the quenching of AH(Tn) by Q. The lifetimes of NAP(T2), DBA(T2), and CHR(T2) were calculated from the TENT quenching experiments. It was found that the lifetimes of AH(T2) increase in the order of NAP(T2) (4.5 ps) < DBA(T2) (16 ps) < CHR(T2) (60 ps), which is consistent very well with the energy gap law for the transition from AH(T2) to AH(T1).

Benzophenones in the higher triplet excited states.

Transient phenomena of benzophenone (BP) in the higher triplet excited state (Tn) have been investigated by the two-colour two-laser excitation method. Triplet energy transfer from BP(Tn) to quenchers (Q) occurred within the duration of a laser pulse (5 ns) to give Q(T1) with higher triplet energy than that of BP(T1). The quantum yield of the triplet energy-transfer quenching of BP(Tn) by CCl4 was found to be 0.0023 +/- 0.0002 from the bleaching of the transient absorption of BP(T1) and the absorbed photon number. It appears that internal conversion from BP(Tn) to BP(T1) is the predominant process. The lifetimes (tauTn) of BP(Tn) and several substituted benzophenones (BPs) in the higher triplet excited state [BPs(Tn)] were estimated from the dependence of the Q concentration on the efficiency of the triplet energy-transfer quenching of BP(Tn) by Q, and found to be 110-450 ps, depending on the nature of the substituents on the BPs. The effect of the substituents on tauTn may be explained by the energy gap between the Tn and T1 states, because the main deactivation pathway for BPs(Tn) is the internal conversion process. In contrast, the substituent effect on the lifetimes of BPs(T1) cannot be explained by the energy gap law. The transient behaviour of Q(T1) depends on the properties of the quencher. Sequential triplet energy transfer from Q(T1) to BP occurred for p-dichlorobenzene and tert-butylbenzene as quenchers, while Q(T1) reacted partly with Q to form triplet excimers (3Q2*) for benzene, chlorobenzene, and o-dichlorobenzene as quenchers. When CCl4 was used as the quencher, the homolytic cleavage of a C-Cl bond of CCl4(T1) occurred to give Cl* and Cl3C* radicals.

Sensitized reactions by benzophenones in the higher triplet excited state

Transient behaviors of benzophenone (BP) and several substituted benzophenones in the higher triplet excited state (T n , n>2) were investigated by two-color two-laser excitation technique. Energy transfer from BP(T n ) to quenchers (Q) having higher E T1 than that of BP and the second energy transfer from Q(T 1) to BP(S 0) were observed. The lifetime of BP(T n ) is estimated to be 450 ps which is long enough for a bimolecular reaction to occur. The decay of Q(T 1) depended on the concentration and structure of the Q. BP(T n )-sensitized cleavages of C–Cl bond of CCl 4 and C–Br bond of bromobenzene occurred.

Properties of chrysene in the higher triplet excited state

Properties of chrysene in the higher triplet excited state were studied by the two-color two-laser flash photolysis method. Triplet energy transfers from chrysene in the higher triplet excited state to quenchers such as biphenyl and naphthalene, and from the quenchers in the triplet excited state back to chrysene in the ground state were observed to proceed at the diffusion-controlled rate. From dependence of the quenching efficiency on the quencher concentration, the lifetime of chrysene in the higher triplet excited state was estimated to be 60 ps with considering the time-dependent quenching.

Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis

Irreversible photobleaching imparts the unavoidable limitation on single-molecule spectroscopy: when the fluorescent probe molecule becomes nonfluorescent, the experiment is over. A detailed study of the photobleaching rate for rhodamine 6G under vacuum reveals that low excitation rates yield long photochemical survival times and unprecedented numbers of emitted photons. A four-level system is used to model the photobleaching rate, reproducing the experimental bleaching rate and photon yield from single molecule and ensemble experiments. A higher triplet excited state T n is found to be the predominant reactive state for photobleaching. This model produces a calculated quantum yield for photobleaching on the order of 7×10 −7 bleaching events per excitation to T n .

Ab initio calculations on excited states of VCH

The ground state and various excited states of triplet and singlet VCH have been calculated using ab initio multiple reference configuration interaction (MRCI) methods. The ground state is calculated to be a 3 Δ state arising predominantly from the single electron configuration ··8σ 2 3π 4 9σ 1 1δ 1 with a calculated V–C bond length of 1.7061 Å and a V–C stretching frequency of 837 cm −1, which are in excellent agreement with experiment. The first excited state is the corresponding isoconfigurational low spin state, 1 Δ , which lies 4670 cm −1 above the ground state. Higher excited triplet states are found to correlate well with experimentally known states.

Photoinduced Intramolecular Proton Transfer and Intermolecular Hydrogen-Atom Abstraction of 1-Piperidinoantraquinone (PAQ)

Abstract The lowest excited singlet and triplet states of PAQ give rise to formation of the N-ylide compound at 77 K and the reduced compound at room temperature, respectively. In the latter case, however, indirect population of the lowest excited triplet state from a higher excited singlet state (through a higher excited triplet state) is very important.

Femtosecond Double-Pulse Excitation Study of α-Sexithienyl Film

Relaxation dynamics from a higher triplet excited state in α-sexithienyl (hereafter abbreviated as 6T) film was investigated by double-pulse excitation spectroscopy. Very rapid formation (within 1 ps) of the lowest singlet excited state and an intermolecular ion-pair state were observed. The mechanism of this rapid conversion process from a higher electronic excited state was discussed.