Excited State Fluorescence Research Articles

3P-113 ホタルルシフェラーゼによる生物的ジオキセタノン分解過程 : アニオン化から複数の発光励起状態生成物まで(電子状態,第46回日本生物物理学会年会)

3P-113 ホタルルシフェラーゼによる生物的ジオキセタノン分解過程 : アニオン化から複数の発光励起状態生成物まで(電子状態,第46回日本生物物理学会年会)

Interactions between protein and porphyrin-containing cyclodextrin supramolecular system: a fluorescent sensing approach for albumin

The interactions of meso-tetraphenylporphyrin (TPP), meso-tetraphenylporphyrin cobalt(ii) (CoTPP) and protein in the presence of a cyclodextrin derivative, heptakis(2,6-di-O-n-octyl)-beta-cyclodextrin (Oc-beta-CD), have been investigated. In the presence of Oc-beta-CD, significant increase of TPP fluorescence was realized, but the increased fluorescence was quenched by CoTPP. To further investigate the fluorescence-quenched system and explore its potential application in bioanalysis, a strategy has been devised to restore the quenching fluorescence of TPP upon interacting with protein. The restoration of TPP fluorescence in the present system is fast and accomplished upon interaction with bovine serum albumin (BSA) or human serum albumin (HSA). On the basis of the spectroscopic measurement and excited state fluorescence lifetime, the mechanism of TPP fluorescence quenching is attributed to formation of a ground-state complex of TPP and CoTPP, and the fluorescence restoration is attributed to the binding of CoTPP with the protein molecule which destroys the aggregate, releasing the free base porphyrin. With optimized conditions, the calibration equations are linear from 0.80 to 75.4 microg mL(-1) BSA and from 3.20 to 93.2 microg mL(-1) HSA. The corresponding detection limits are 0.32 microg mL(-1) for BSA and 1.06 microg mL(-1) for HSA, respectively. The method was used for the direct assay of HSA content in human serum. The result is comparable to that obtained by another method. The recovery from BSA in synthetic sample is also satisfactory.

Photodynamics of Polyene−Polymethine Transformations and Spectral Fluorescent Properties of Merocyanine Dyes

Absorption and fluorescence spectrum band moments (center of gravity, width, asymmetry, excess, and fine structure) have been determined in a wide range of solvents with different polarities for inverse solvatochromic di-, tetra-, and hexamethinemerocyanines derived from 1,3-diphenyl-2,3-dihydro-1H-benzimidazole. Juxtaposition of the quantum-chemically calculated (by the semiempirical AM1 method) charges, bond orders, and dipole moments of the merocyanine molecules in the ground and excited singlet states with the experimentally observed spectral fluorescent characteristics suggests that the molecular electronic structure in the two states can vary from a nonpolar polyene via a polymethine to a charge-separated polyene, depending on the length of the polymethine chain and the medium polarity. As shown, solvatofluorochromism gives rise to smaller spectral band shifts than those of solvatochromism. This effect is attributable to weaker intermolecular solute-solvent interactions in the fluorescent excited state due to the more equalized charges as compared to those of the ground state. A lack of mirror symmetry of the absorption and fluorescence spectra has been revealed for di- and tetramethinemerocyanines (broadened fluorescence bands) as well as for hexamethinemerocyanines (narrowed fluorescence bands); the two cases are accounted for by the different behavior of vibronic and intermolecular interactions in the course of absorption and emission. As found for merocyanines, the electronic structure of their fluorescent state approaches the cyanine limit and the ground state becomes increasingly polyene-like with lengthening of the polymethine chain. A close vicinity of the excited state to the cyanine limit causes a dramatic increase in fluorescence quantum yields and a decrease in Stokes shifts observed for higher merocyanine vinylogues.

Noninvasive probing of aqueous Triton X-100 with steady-state and frequency-domain fluorometry

Most fluorescence spectroscopic methods that investigate the properties of surfactant-based systems are invasive in nature as they rely on information afforded by an externally-introduced probe. External probes may adversely affect the colloidal property of interest. Information from chromophoric surfactants as endogenous probes of micellization process is presented. Fluorescence from phenyl moiety of a common nonionic surfactant Triton X-100 in water is demonstrated to be rich in information on surfactant aggregation. While Triton X-100 concentration-dependent fluorescence emission and excitation can be effectively used to monitor the presence of micelles, frequency-domain excited-state fluorescence intensity decay data affords key dynamic parameters characterizing Triton X-100 micelles.

Influence of detergent concentration on aggregation and spectroscopic properties of light-harvesting complex II

Aggregation of photosynthetic light-harvesting complexes strongly influences their spectroscopic properties. Fluorescence yield and excited state lifetimes of the main light-harvesting complex (LHC II) of higher plants strongly depend on its aggregation state. Detergents are commonly used to solubilize membrane proteins and/or to circumvent their aggregation in aqueous environments. Nonlinear polarization spectroscopy in the frequency domain (NLPF) was performed with LHC II over a wide concentration range of the mild detergent n-dodecyl beta-D: -maltoside (beta-DM). Additionally, conventional absorption-, fluorescence- and circular dichroism-spectra were measured.The results indicate that: (i) conventional spectroscopic techniques are not well suited to investigate aggregation effects. NLPF provides a novel approach to overcome this problem: NLPF spectra display dramatic alterations upon even minor beta-DM concentration changes. (ii) Commonly used detergent concentrations (around or slightly above the critical micellar concentration) apparently do not lead to complete trimerization of LHC II. A long-wavelength species in the NLPF spectra (peaking at about 685 nm), indicative of residual aggregation, persists up to DM-concentrations of 0.06%. (iii) High-resolution NLPF spectra indicate the existence of a species with a considerably shortened excited state lifetime. (iv) No indication of denaturation was found even at the highest beta-DM concentrations used. (v) A specific change in interaction between certain chlorophyll(s) b and a xanthophyll molecule, probably neoxanthin, was detected upon aggregation as well as at higher beta-DM concentrations. The results are discussed with respect to the still elusive mechanism of nonradiative dissipation of excess excitation energy in the antenna system.

Spectroscopic properties of Yb3+ in NaYbP2O7 diphosphate single crystals

Abstract Yb3+ absorption and fluorescent emissions were investigated for NaYbP2O7 diphosphate single crystals. The interpretation of electronic energy level positions has been done by using the comparison of absorption and emission spectra with those of vibronic sideband energy positions from Raman and IR absorption spectroscopies. The Yb3+ energy levels scheme in this host was drawn. The decay time for infrared Yb3+ (5F5/2 excited state) fluorescence was in the range of 1–2 ms. The interest feature is leading to broad emission band at room temperature from 940 nm to 1100 nm, which seems of high interest for ultra-short laser pulse production.

Functional Promiscuity Correlates with Conformational Heterogeneity in A-class Glutathione S-Transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.

Laser spectroscopy and dynamics of the jet-cooled AsH2 free radical

The A (2)A(1)-X (2)B(1) electronic transition of the jet-cooled AsH(2) free radical has been studied by laser-induced fluorescence (LIF), wavelength-resolved emission, and fluorescence lifetime measurements. The radical was produced by a pulsed electric discharge through a mixture of arsine (AsH(3)) and high pressure argon at the exit of a pulsed valve. Nine vibronic bands were identified by LIF spectroscopy in the 505-400 nm region, including a long progression in the bending mode and two bands (1(0) (1) and 1(0) (1)2(0) (1)) involving the excited state As-H symmetric stretch. Single vibronic level emission spectra showed similar activity in the bending and symmetric stretching frequencies of the ground state. High-resolution spectra of the 0(0) (0) band exhibited large spin splittings and small, resolved arsenic hyperfine splittings, due to a substantial Fermi contact interaction in the excited state. The rotational constants obtained in the analysis gave effective molecular structures of r"(0)=1.5183(1) A, theta"(0)=90.75(1) degrees and r'(0)=1.4830(1) A, theta'(0)=123.10(2) degrees . The excited state fluorescence lifetimes vary dramatically with rovibronic state, from a single value of 1.4 micros to many with lifetimes less than 10 ns, behavior which the authors interpret as signaling the onset of a predissociative process near the zero-point level of the ground state.

Excited state properties, fluorescence energies, and lifetime of a poly(fluorene‐pyridine) copolymer, based on TD‐DFT investigation

The structural and electronic properties of the fluorene-pyridine copolymer (FPy)(n), (n = 1-4) were investigated theoretically by means of quantum mechanical calculations based on density functional theory (DFT) and time-dependent DFT (TD-DFT) using the B3LYP functional. Geometry optimizations of these oligomers were performed for the ground state and the lowest excited state. It was found that (FPy)(n) is nonplanar in its ground state, whereas a more pronounced trend toward planarity is observed in the S(1) state. Absorption and fluorescence energies have been extrapolated to infinite chain length making use of their good linearity with respect to 1/n. An extrapolated value of 2.64 eV is obtained for vertical excitation energy. The S(1)<--S(0) electronic excitation is characterized as a highest occupied molecular orbital to lowest unoccupied molecular orbital transition and is dominating in terms of oscillator strength. Fluorescence energies and radiative lifetime were calculated as well. The obtained results indicate that the fluorescence energy and radiative lifetime of (FPy)(n) are 2.16 eV and 0.38 ns, respectively. The decrease of fluorescence energy and radiative lifetime with the increase in the chain length is discussed.

Synthesis, photophysical and photochemical properties of substituted zinc phthalocyanines

The synthesis, photophysical and photochemical properties of the 4-({3,4,5-tris-[2-(2-ethoxyethoxy)ethyloxy]benzyl}oxy) and 4-({3,4,5-tris-[2-(2-ethoxyethoxy)ethyloxy]benzyl}thio) zinc(ii) phthalocyanines are reported for the first time. The new compounds have been characterized by elemental analysis, IR, (1)H and (13)C NMR spectroscopy, electronic spectroscopy and mass spectra. General trends are described for photodegradation, singlet oxygen, fluorescence and triplet excited state quantum yields, and triplet state and fluorescence lifetimes of these compounds in dimethylsulfoxide (DMSO). The fluorescence of the complexes was quenched by benzoquinone (BQ). The effects of the substitution on the photophysical and photochemical parameters of the zinc(II) phthalocyanines (6, 7 and 8) are also reported. Photophysical and photochemical properties of phthalocyanine complexes are very useful for PDT applications. The substituted Zn(II) phthalocyanines showed high triplet and singlet oxygen quantum yields. High singlet oxygen quantum yields are very important for Type II mechanism. Thus, these complexes show potential as Type II photosensitizers.

Quenching and Blinking of Fluorescence of a Single Dye Molecule Bound to Gold Nanoparticles

A fluorescein derivative (SAMSA) bound to gold nanoparticles of different diameters is investigated by time-resolved fluorescence at the single molecule level in a wide dynamic range, from nanosecond to second time scale. The significant decrease of both SAMSA excited state lifetime and fluorescence quantum yield observed upon binding to gold nanoparticles can be essentially traced back to an increase of the nonradiative deactivation rate, probably due to energy transfer, that depends on the nanoparticle size. A slow single molecule fluorescence blinking, in the ms time scale, has a marked dependence on the excitation intensity both under single and under two photon excitation. The blinking dynamics is limited by a low probability nonlinear excitation to a high energy state from which a transition to a dark state occurs. The results point out a strong coupling between the vibro-electronic configuration of the dye and the plasmonic features of the metal nanoparticles that provide dye radiationless deactivation channels on a wide dynamic range.

Luminescence techniques and characterization of the morphology of polymer latices: 2. Fluorescence lifetime, phosphorescence and fluorescence anisotropy studies

Five poly(n-butyl methacrylate), PBMA, latex dispersions have been prepared, each incorporating a different fluorescent label, via a two-stage seeded emulsion polymerization. The resultant latices contain ca. 35% by weight total solids and are of 80 (±10) nm diameter as determined by photon correlation spectrometry. Luminescence spectroscopic techniques, namely fluorescence (and phosphorescence) excited state lifetime measurements in addition to time-resolved anisotropy experiments have provided useful information regarding the morphology, microviscosity and water permeability of the resultant particles. A picture of the PBMA colloid emerges of an interior which is highly viscous and water impermeable in nature. Indeed, the environment is protective enough to sustain room temperature stabilized phosphorescence from both an acenaphthylene and 9-phenanthrylmethyl methacrylate labeled dispersion through simple nitrogen purging of the solutions. However, the current spectroscopic measurements should be viewed with the knowledge that each luminescent label may fashion its own distinctive microenvironment within the latex during polymerization.

Amide‐Based Molecular Knots as Platforms for Fluorescent Switches

A series of amide-based molecular knots equipped selectively with fluorescent dansyl and/or pyrenesulfonyl moieties were synthesized from the readily available tris(allyloxy)knotane. UV/Vis absorption spectra, emission spectra, and the emission lifetimes of the fluorescent knotanes were investigated in chloroform at 298 K. The absorption spectra of the knotanes correspond to those of mixtures of their UV-active constituents. The fluorescence quantum yields and lifetimes of the dansyl and pyrenesulfonyl moieties are partly quenched by the knotane platform. In the KN(Da)(2)(Py) species, the fluorescent excited state of the dansyl units (lambda(max)=510 nm) lies at lower energy than the fluorescent excited state of the pyrenesulfonyl unit (lambda(max)=385 nm), the emission of which is accordingly quenched with sensitization of the dansyl fluorescence. In the KN(Ao)(2)(Da), KN(Ao)(Da)(2), and KN(Da)(3) species, the addition of acids causes the protonation of their dansyl units with a consequent decrease in the intensity of the dansyl band at 510 nm and appearance of the emission band of the protonated dansyl unit (lambda(max)=340 nm). Each dansyl unit of KN(Ao)(Da)(2) and KN(Da)(3) undergoes the independent protonation. In these incompletely protonated knotanes the fluorescence of the protonated dansyl units is partly quenched by nonprotonated ones. These processes can be quantitatively reversed upon addition of a base. In KN(Da)(2)(Py), an increase of the fluorescence of its pyrenesulfonyl group is observed when the dansyl groups are protonated. The results obtained show that the readily available and easily functionalizable amide-knotanes can be used as an interesting scaffold to obtain fluorescent switches.

Refined analysis of the luminescent centers in the Yb 3+:CaF 2 laser crystal

A detailed spectroscopic study of various Yb 3+:CaF 2 crystals with dopant concentrations ranging from 0.03 to 2 at% Yb 3+ has been performed. By using site-selective laser excitation, the low-temperature (7 K) excitation and emission spectra recorded in the case of the lower doped samples (0.03% and 0.05%) are found to be dominated by three types of sites with excited state fluorescence lifetimes of about 2, 8 and 10 ms. Confronted with crystal field calculations, these sites have been identified as Yb 3+ ions in trigonal (C 3v), tetragonal (C 4v) and cubic environments, respectively. At higher dopant concentrations (>0.1 at%), evidence is found of a third kind of site, whose contribution increases up to be dominant as dopant concentration is increased above about 1 at%Yb 3+ and which gives rise to the lasing center which has been recently discovered and operated.

Photophysics and spectroscopy of the higher electronic states of zinc metalloporphyrins: a theoretical and experimental study

The photophysics of the S2 and S1 excited states of zinc porphyrin (ZnP) and five of its derivatives (ZnOEP, ZnTBP, ZnTPP, ZnTFPP, ZnTCl8PP) have been investigated by measuring their steady-state absorption and fluorescence spectra, quantum yields and excited state lifetimes at room temperature in several solvents. The radiative and radiationless decay constants of the fluorescent excited states accessible in the visible and near UV regions of the spectrum have been obtained. Despite the similarities in the Soret spectra of these compounds, their S2 excited state radiationless decay rates differ markedly. Although the S2-S1 electronic energies of a given zinc porphyrin vary linearly with the Lippert (refractive index) function of the solvent, the S2 radiationless decay rates of the set of compounds do not follow the energy gap law of radiationless transition theory. Calculations, using time-dependent density functional theory (TDDFT), of the energies and symmetries of the complete set of excited states accessible by 1- or 2-photon absorption in the near UV-visible have also been carried out. Substitution on the porphyrin macrocycle framework affects the ground state geometry and alters the electron density distributions, the orbital energies and the relative order of the excited electronic states accessible in the near UV-blue regions of the spectrum. The results are used to help interpret both the nature of the electronic transitions in the Soret region, and the relative magnitudes of the radiationless transition rates of the excited states involved.

Metabolic sensing using fluorescence

With a view to assessing the current boundaries of non-invasive sensing of metabolites we compare the photophysical properties of melanin nanoparticles in vivo, size selected in natural form from Sepia officinalis extracted from cuttlefish, and synthesised from l-dihydoxyphenylalanine (DOPA) in water pools of reverse micelles. The fluorescence emission spectra and excited state decay components correlate well with the particle size. Recent developments in optical technology are discussed in the context of the formidable challenges and opportunities for metabolic sensing presented by endogenous fluorophores.

Absorption and fluorescence spectral properties of donor-acceptor ethenes bearing indole and p-nitrophenyl substituents

Synthesis, absorption and fluorescence properties (λf, λex, Stokes shifts, Φf, τf, correlation plots of Δf and ET(30) with Φf and Stokes shifts, μf, low temperature fluorescence properties and excited state geometry) of 3-(4-nitrophenyl ethenyl-E)-NH-indole (1) and 3-(4-nitrophenyl ethenyl-E)-N-acetyl indole (2) are described. Occurrence of singlet excited state torsional motions and presence of more than one excited species, which are significantly influenced by the solvent polarity, have been observed. It is suggested that while 1 fluoresces predominantly from the conformationally relaxed intramolecular charge transfer (CRICT) state, 2 fluoresces from three different types of excited species, including the locally excited state, intramolecular charge transfer excited state and the CRICT state.

Fluorescence spectroscopic studies to characterize the internal environment of tetraethyl-orthosilicate derived sol–gel bulk and thin films with aging

Characterization of the internal environment of a sol–gel matrix is an important area of investigation in optical biosensors. In the present study, different sol–gel compositions were prepared by varying the water (H 2O) to tetraethyl-orthosilicate (TEOS) ratio ( R) from 1 to 16 and the changes in the internal environment of the sol–gel both in bulk and thin films as a function of aging (storage) were investigated using fluorescence spectroscopy. We focussed on the fluorescence characteristics , viz. emission and excited state lifetime of Hoechst 33258 (H258), a bisbenzimidazole derivative, which was used as fluorescence probe entrapped in the TEOS derived sol–gel bulk and thin films. These sols were prepared at a low pH (∼2.0) and the thin films were coated by dip coating technique at withdrawal speeds of 1 cm/min and 0.1 cm/min. Usually, uniform thin films were obtained at a high speed (1 cm/min) and partially cracked film at a low speed (0.1 cm/min) as observed by fluorescence microscope. These observations did not change during aging. On the contrary, three months long observations on steady-state fluorescence emission measurements on H258 depicted a blue shift from 535 nm to 508 nm at R = 1 in the sol–gel bulk, whereas at higher ratios this was not prominent. At all ratios, dual emission bands were observed in thin films. This may be due to faster sol–gel to xerogel transition during aging depending on the ratio ( R). Analysis of the excited state decay profiles of H258 revealed a double exponential fitting having a short ( τ 1) and a long ( τ 2) component in both fresh and during aging, in the sol–gel bulk and thin films, indicating heterogeneity in the internal environment. The value of τ 1 increased from 0.4 ns to 1.2 ns whereas τ 2 attained a value from 3.0 ns to 3.6 ns at R = 1 upon aging in the sol–gel bulk. The corresponding values of τ 1 and τ 2 in thin films were 0.3 ns and 3.5 ns, respectively. The values of these decay components in thin films did not alter much due to storage, but their relative contributions showed more systematic changes in the thin films. The observed changes could be correlated to rigidification in the bulk depending on the ratio ( R). This process was very slow at R ≥ 4. The heterogeneity in the internal environment of bulk and thin films upon aging appeared to be different as revealed from analysis of excited-state lifetime. Thus, the bisbenzimidazole derivative H258 appears to be very useful probe for characterizing the internal environment of both the sol–gel bulk and thin films.

Fluorescence Photoconversion Kinetics in Novel Green Fluorescent Protein pH Sensors (pHluorins)

Genetically encoded pH-sensitive green fluorescent proteins (GFPs) offer significant control over localization, spectral properties, and environmental sensitivity for cell biology and neuroscience applications. Quantitative analysis of biological applications and full exploitation of novel GFP properties rely on a fundamental understanding of their molecular photophysics. In this contribution, the ground- and excited-state fluorescence properties of Ecliptic and Ratiometric pHluorins, two pH-sensitive GFP probes of presynaptic activity, are presented and compared with Sapphire, a neutral phenol GFP mutant. Molecular dynamics have been characterized using fluorescence correlation spectroscopy (FCS) and time-correlated single-photon counting (TCSPC) as a function of pH, excitation wavelength (including 405 nm), detection wavelength, and illumination intensity. As a cellular pH indicator, Ecliptic (EcGFP) is particularly well-suited to the physiological pH range (pKa = 7.2 ± 0.2) compared with Sapphire (H9; ...

Symmetry switching of the fluorescent excited state in alpha,omega-diphenylpolyynes.

By using MO calculations based on DFT, absorption, and fluorescence spectroscopy, we have comprehensively studied the low-lying excited singlet states of alpha,omega-diphenylpolyynes (DPY) having 1-6 triple bonds. The a(g) vibrational modes of the C(triple bond)C stretching and of the phenyl ring motion were observed in the fluorescence spectra of diphenylacetylene and 1,4-diphenylbutadiyne. On the other hand, in the fluorescence spectra of the long DPY with the triple-bond number (N) more than two, the phenyl ring motion with a(g) symmetry disappeared and the b(1g) modes of the phenyl ring twisting (approximately 400 cm(-1)) and of the C-H bending (approximately 900 cm(-1)) were detected. The observed fluorescent states of DPY with N < or = 2 and N > or = 3 are assigned to the 1(1)B(1u) (pi(x)pi(x*)) and 1(1)A(u) (pi(x)pi(y*) and/or pi(y)pi(x*)) states, respectively, based on the vibronic structures, the relatively short lifetimes, and the solvatochromic shifts of the fluorescence spectra. Not only the allowed transition of 1(1)B(1u) <-- S(0) but also the forbidden transition of 1(1)A(u) <-- S(0) was detected in the fluorescence excitation spectra of the long DPY with N > or = 3. The low-lying excited state with A(u) symmetry is characteristic in polyyne, which does not exist in polyene. The oscillator strength (f) of the first absorption band in DPY decreases with an increase in N, which is the opposite behavior of the all-trans-alpha,omega-diphenylpolyenes. The N-dependence of the f value is understood by the configuration interaction between the 1(1)B(1u) and 2(1)B(1u) (pi(y)pi(y*)) states, which is consistent with the reduction of the nonlinear optical response of polyyne.