Fluorescence Line Narrowing Research Articles

The Power of Line-Narrowing Techniques: Applications to Photosynthetic Chromoprotein Complexes and Autofluorescent Proteins

Laser spectroscopic techniques at low temperature, such as fluorescence line-narrowing and hole burning, enable an increase of spectral resolution by a factor of 103–105 compared to conventional spectroscopy at room temperature. With these methods, it is possible to retrieve a fingerprint of the species involved and to measure the rates of dynamic processes that normally remain hidden in the broad absorption bands. A few applications carried out in our laboratory will be discussed: (1) the determination of energy transfer rates in the peripheral LH2 complex of purple bacteria; (2) the study of spectral diffusion and its implications in three types of systems: (a) the B820 and B777 subunits of the LH1 complex of purple bacteria, (b) the photosystem II reaction center (PSII RC) and CP47 antenna complex of green plants, and (c) an organic glass doped with bacteriochlorophyll a; (3) the unraveling of 0-0 transitions and the pathways of photoconversion between a number of conformations of the green fluorescent protein mutant S65T; (4) the measuring of electron-phonon coupling strengths in PSII RC and the red fluorescent protein DsRed; and (5) the determination and comparison of the homogeneous linewidths and optical dephasing in photosynthetic chromoprotein complexes and autofluorescent proteins.

Investigation of site-selective symmetries of Eu3+ ions in KPb2Cl5 by using optical spectroscopy

Site-selective time-resolved spectroscopy of Eu3+ in KPb2Cl5 has been investigated by using fluorescence line narrowing technique. A crystal field analysis and simulation of the experimental results has been performed in order to parametrize the crystal field at the Eu3+ sites. Three symmetry independent crystal field sites for the rare-earth ion in this crystal were found. A plausible argument about the crystallographic nature of these sites is given.

Role of Fluorescence Line-Narrowing Spectroscopy and Related Luminescence-Based Techniques in the Elucidation of Mechanisms of Tumor Initiation by Polycyclic Aromatic Hydrocarbons and Estrogens

Formation of DNA adducts by various carcinogens represents the first critical event in the mechanism of tumor initiation. The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are biologically activated by two major mechanisms: one-electron oxidation to produce radical cations and monooxygenation to form bay-region diol epoxides. The PAH−DNA adducts formed by these mechanisms are stable adducts that remain in DNA unless removed by repair and depurinating adducts that are lost from DNA by cleavage of the glycosyl bond. Identification of PAH−DNA adducts has relied heavily on low-temperature, laser-based fluorescence spectroscopy under non-line-narrowing (NLN) and line-narrowing (FLN) conditions. These spectroscopies can be used for chemical identification, conformational analysis, and/or probing the microenvironment of DNA (or protein) adducts. Small and co-workers have pioneered the use of FLN spectroscopy in this research. For example, the structures of the depurinating adducts formed by the PAHs benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and dibenzo[a,l]pyrene have been elucidated. Understanding of the mechanism of tumor initiation by PAHs has relied on identifying and quantifying the DNA adducts formed. The insights gained from the study of PAH−DNA adducts enabled us to discover the estrogen metabolites that form depurinating DNA adducts and can be potential endogenous initiators of human cancer. Small and co-workers have also studied the estrogen−DNA adducts and estrogen−thioether conjugates by using FLNS and related luminescence-based techniques and have demonstrated that the level of the 4-hydroxyestrone-1-N3-adenine depurinating adduct in breast tissue from a woman with breast carcinoma was significantly higher than that in breast tissue from women without breast cancer. The fluorescence- and phosphorescence-based techniques they are developing will be applied to analyzing estrogen adducts and conjugates as biomarkers of susceptibility to breast and other types of human cancer.

Emitting Excitonic Polaron States in Core LH1 and Peripheral LH2 Bacterial Light-Harvesting Complexes

Hole-burned absorption and line-narrowed fluorescence spectra along with nanosecond fluorescence decay kinetics have been studied at 5 K in core LH1 and peripheral LH2 antenna complexes isolated from the photosynthetic purple bacterium Rhodobacter sphaeroides. A dual nature for the respective emission bands has been confirmed in both complexes and has been assigned to the nearly free excitons weakly coupled to lattice vibrations and to the strongly coupled self-trapped excitons. The apparent phonon structure of quasi-free excitons has been analyzed resulting in a total Huang-Rhys factor, a characteristic of the electron-phonon coupling strength, equal to S = 0.85 ′ 0.10 in LH1 and to S = 1.05 ′ 0.10 in LH2. An estimate for self-trapped excitons is a few times larger. Excitonic polarons are thus proper excitations in LH 1 and LH2 complexes, as the electron-phonon coupling cannot be ignored.

Time-resolved fluorescence line narrowing spectroscopy and fluorescence quenching in Nd3+-doped fluoroarsenate glasses

Abstract The spectral migration of energy among the Nd 3+ ions in fluoroarsenate glasses of the Na 4 As 2 O 7 , BaF 2 , YF 3 system with different Nd 3+ concentrations (0.5, 2, 3, 4, and 5 mol%) has been investigated by using time-resolved fluorescence line narrowing (TRFLN) spectroscopy. The features of the time-resolved fluorescence line narrowing 4 F 3/2 → 4 I 9/2 emission spectra obtained under resonant excitation at different wavelengths along the 4 I 9/2 → 4 F 3/2 transition as a function of Nd 3+ concentration reveal the existence of energy migration between discrete regions of the inhomogeneously broadened spectral profile. The analysis of the time evolution of the 4 F 3/2 → 4 I 9/2 narrowed emission shows that the electronic mechanism responsible for the ion–ion interaction can be identified as a dipole–dipole energy transfer process. From the thermal behavior of lifetimes of the 4 F 3/2 state a T 3 dependence for the non-radiative Nd–Nd relaxation processes has been found in the 10–80 K temperature range for Nd 3+ concentrations higher than 0.5 mol% which is in agreement with a two-site non-resonant process.

Probing the interaction of benzo[a]pyrene adducts and metabolites with monoclonal antibodies using fluorescence line-narrowing spectroscopy.

A new approach for studying antibody-antigen interactions of DNA adducts and metabolites of polycyclic aromatic hydrocarbons (PAHs) is demonstrated in which fluorescence line-narrowing spectroscopy (FLNS) is used. It is based on the fact that in an FLN spectrum the relative intensities of the line-narrowed bands (that correspond to the excited-state vibrations) are, in general, strongly dependent on the local environment of the fluorophore. Information on the nature of the interactions can be obtained by comparing the FLN spectra of the antigen-antibody complexes to the spectra of the antigen in different types of solvents (H-bonding, aprotic, and pi-electron-containing solvent molecules) recorded under the same conditions. The antigens used were the DNA adduct 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and the metabolite (+)-trans-anti-7,8,9,10-benzo[a]pyrenetetrol (BP-tetrol) of benzo[a]pyrene; two monoclonal antibodies (MAbs) have been developed to selectively bind these compounds. It is shown that, for BP-tetrol, H-bonding solvents have a pronounced effect on the FLN spectra. The presence of pi electrons in the solvent molecules results in relatively small but still significant changes in the spectra. When BP-tetrol is bound to its MAb, however, neither of these effects is observed; its spectrum is very similar to the one obtained with an aprotic solvent, methylcyclohexane. Therefore, we can conclude that this MAb has an internal binding site in which the interaction with BP-tetrol is of a hydrophobic character. For BP-6-N7Gua, however, there is a strong effect of the presence of pi electrons in the solvent molecules. The FLN spectrum of this antigen bound to its MAb is very similar to its spectrum in acetone, indicating that pi-pi interactions play an important role in the binding.

Spectral hole burning and fluorescence in femtosecond laser induced Sm 2+-doped glasses

The femtosecond laser was used to irradiate sol–gel derived Sm 3+-doped Al 2O 3–SiO 2 glasses, in which the Sm 3+ was reduced into Sm 2+ ions. The fluorescence line narrowing was applied to investigate the coordination sphere of the Sm 2+ ion. The spectral hole burning was performed on 7F 0→ 5D 0 transition of the Sm 2+. The depth and width of the burnt holes were ∼27% and ∼4 cm −1 FWHM at 7 K, respectively. Hole spectra were stable up to room temperature. The hole-burning efficiency was superior to that of Sm 2+ in H 2 treated glasses and comparable to that in X-ray in terms of hole-burning dynamics.

Red Spectral Forms of Chlorophylls in Green Plant PSI− A Site-Selective and High-Pressure Spectroscopy Study

One of the special spectroscopic characteristics of photosystem I (PSI) complexes is that they possess absorption and emission bands at lower energy than those of the reaction center. In this paper, the red pigment pools of PSI-200, PSI-core, and LHCI complex from Arabidopsis thalianahave been characterized at low temperatures by means of spectrally selective (hole-burning and fluorescence line-narrowing) and high-pressure spectroscopic techniques. It was shown that the green plant PSI-200 complex has at least three red pigment pools, from which two are located in the PSI-core and one, in the peripheral light-harvesting complex I (LHCI). All of the red pigment pools are characterized by strong electron-phonon coupling. A Huang-Rhys factor of 2.9 found for the red pigments of LHCI is the largest found for any photosynthetic antenna system. This contrasts with the bulk pigments in the main Qy absorption band of chlorophyll a pigments for which the HuangRhys factors of less than unity are observed. This electron-phonon coupling difference of the red and bulk pigments is well reflected by the spectral dependence of the hole-burning efficiency, which is significantly reduced in the red absorption region. As a result of extremely low hole-burning efficiency in the red absorption band of LHCI, the hole-burning spectra of the PSI-200 complex mainly originate from the red pigments of the PSI-core complex. At the same time, the source of the red emission in PSI-200 is the red pigments of LHCI, in agreement with previous studies. The hole-burning spectra of PSI-core complexes from green plant and cyanobacteria are similar, both in red and bulk absorption regions. High-pressure spectroscopy data reveal dramatically larger pressure-induced linear shift rates for the redmost absorption and emission bands relative to those of bulk absorption bands. This is interpreted as due mostly to increased conformational mixing between the locally excited and charge transfer configurations of the red pigment aggregates. On the basis of analysis of available experimental data, we suggest that pigment dimers are probably responsible for the redmost states. Consequently, the excited red states can be interpreted as excimer states.

Site selective study of Eu3+-doped transparent oxyfluoride glass ceramics

Optical properties of Eu3+ ions in oxyfluoride glasses and glass ceramics doped with two different concentrations, 0.1 and 1 mol %, have been analyzed and compared with previous results for higher concentrated samples, 2.5 mol %. The Eu3+ ions in the 0.1 mol % doped glass ceramics are diluted into like crystalline environments with higher symmetry and lower coupled phonon energy than in the precursor glasses; meanwhile in the 1 mol % doped glass ceramics the presence of EuF3 clusters has been observed in addition to diluted ions. Fluorescence line narrowing measurements indicate the presence of two main fluoride site distributions for the diluted Eu3+ ions in both glass ceramics.

Inhomogeneous and homogeneous linewidths in Er3+-doped chalcogenide glasses

Abstract The erbium 4 I 13/2 – 4 I 15/2 transition around 1.5 μm is of prim interest for telecommunications and depends on the erbium ions surrounding. In glasses, the broadening of a transition comes from two contributions: inhomogeneous (due to the disorder) and homogeneous (due to the electron phonon interaction) broadening. Resonant Fluorescence Line Narrowing (RFLN) is a useful tool to separate this two parameters. We will show in this paper that the 4 I 13/2 – 4 I 15/2 transition in chalcogenide glass (GeGaSSb) presents a strong homogeneous character and a smaller inhomogeneous contribution compared to aluminosilicate and fluoride glasses. Consequences on gain saturation will also be discussed.

Temperature dependence of phonon sidebands in line-narrowed fluorescence spectra of chromophores in glasses

Site-selective line-narrowed fluorescence spectra have been measured from 1.5 to 60–90 K for the S1→S0 electronic transitions of phenanthrene and of perylene doped into four amorphous or nearly amorphous matrices: polyethylene, polystyrene, poly(methyl methacrylate), and ethanol. The band shapes (zero-phonon line plus phonon wing) are examined for the best-isolated strong vibronic band in the emission spectrum of each chromophore and compared with simulations using two different models that assume harmonic phonons linearly coupled to the electronic transition. One model extracts the spectral density of coupled phonons from the lowest-temperature experimental data, while the other employs an empirically determined set of harmonic phonon frequencies and coupling strengths. Good fits between simulation and experiment are obtained over the full temperature range for all chromophore–matrix combinations examined, which vary considerably in electron–phonon coupling strength. The assumption of linearly coupled harmonic phonons therefore appears to be reasonably good for these systems. Some broadening of the zero-phonon lines with increasing temperature in polystyrene and in ethanol suggests a contribution from quadratically coupled phonons as well in these matrices.

Self-trapped excitons in circular cacteriochlorophyll antenna complexes

Fluorescence line narrowing and hole-burning spectroscopic studies of excitons in the LH2 pigment-protein complex, which is a part of the light harvesting system of purple bacteria, are combined with straightforward numerical simulations of the emission spectra based on exciton polaron model. The analysis provides evidence for self-trapping of all the excitons, except the lowest one.

Non-photochemical spectral hole-burning mediated by water molecules in interligand pockets of [Cr(terpy)2]3+

Low temperature fluorescence line-narrowing (FLN) and spectral hole-burning experiments (SHB) were performed in the 2E←4A2 spin–flip transition of [Cr(2,2′:6′,2″-terpyridine)2]3+ in frozen ethylene glycol/water (2 ∶ 1) and DMSO/water (2 ∶ 1) glasses. In the FLN experiments an average 2E splitting of 23 cm−1 is observed. It is concluded that the interaction with water molecules in pockets provided by the ligands is most likely to be responsible for the relatively efficient non-photochemical hole-burning. Fast spectral diffusion and spontaneous hole-filling prevent the observation of holes above 20 K. The FLN and SHB experiments were performed by using a diode laser.

Consequences of Inhomogeneous Broadening on Fluorescence Line Narrowing Spectra

Fluorescence line narrowing is a sensitive tool to monitor subtle changes in protein conformation. We restudied the consequences of inhomogeneous broadening on the observed spectra. Vibronic absorption bands of systems with weak electron-phonon coupling can be described by a lorentzian function with width of the order of a few centimeters. For an ensemble of molecules with closely spaced electronic transitions, a single laser frequency will excite many molecules through the broad base (“tail”) of the lorentzian function. This, along with excitation of low-frequency modes, contributes to the unresolved background observed in line-narrowed spectra. Examples are shown for Zn cytochrome c, a fluorescent derivative of Fe cytochrome c. Spectra are compared for the protein in two solvents: glycerol/water or trehalose. Two types of cytochrome, from horse and yeast, are also compared.

Optical Properties of Rare Earth Doped Transparent Oxyfluoride Glass Ceramics

Optical properties of Eu3☎ ions in oxyfluoride glasses and glass ceramics doped with low concentration (0.1 mol%) have been analysed and compared with previous results for high concentrated samples (2.5 mol%). The Eu3☎ ions in the low dopant concentration glass ceramics are diluted into like crystalline environments with higher symmetry and lower coupled phonons energy than in the precursor glasses. Fluorescence line narrowing measurements indicate the presence of two main fluoride site distributions for the Eu3☎ ions in these low concentrated glass ceramics.

Resonant fluorescence line narrowing measurements in erbium-doped glasses for optical amplifiers

Rapid development of optical fiber amplifiers, and more especially of the erbium-doped fiber amplifiers (EDFA) during the last decade, has benefited from extensive work on the configuration of the system itself: determination of pump wavelength, pumping configuration, improvement of noise performance, etc. However, less effort have been dedicated to study the interaction that influences the spectral profile of the gain bandwidth and particularly the relation between the glass composition and the spectroscopy of erbium. Here, we report a systematic determination of key parameters responsible for the global profile of the gain bandwidth: stark splitting, homogeneous broadening, and inhomogeneous broadening. The correlation between these parameters and the glass structure from one part and the behavior of the EDFA in amplification regime from another part is discussed. The quasiregular crystal-field splitting of tellurite and fluoride glasses is found to correlate well with the flatness of the gain profile. Moreover, the low-temperature homogeneous bandwidths extrapolated to room temperature can be correlated to the saturation spectral hole widths measured in the amplification regime.

Site-selective fluorescence spectroscopy of Eu 3+ and Sm 2+ ions in glass

Fluorescence line–narrowing or site-selective fluorescence experiments on Eu 3+ and Sm 2+ ions in glass are reviewed. It is shown that this technique gives detailed information that is difficult to obtain by other means, such as site-dependent homogeneous widths, inhomogeneous broadening mechanisms, distribution of crystal-field components in glass, and so on. A simple theoretical treatment to consider only the second-order terms of the crystal-field potential is discussed and is shown to be very effective. It will be emphasized that J-mixing, i.e., wave function mixing between 4f N states through the static crystal-field potential, plays a significant role in the optical properties of rare-earth ions in glass.

Line-narrowed fluorescence spectra of phenanthrene and perylene in polymers: Chromophore, polymer, and vibrational state dependence of coupling to matrix phonons

Site-selective line-narrowed fluorescence spectra have been obtained for the S1↔S0 transitions of phenanthrene and perylene in polyethylene, polystyrene, and poly(methyl methacrylate) films at 1.5 K. The spectral distributions and intensities of the phonon sidebands on each vibronic transition are analyzed to examine the chromophore, matrix, and vibrational level dependence of the coupling of the chromophore’s electronic transition to matrix and/or intermolecular vibrations. The frequency distribution of the coupled phonons (spectral density) and the strength of the coupling vary considerably among polymers. Different vibronic transitions of the same chromophore in the same matrix exhibit similar spectral densities but slightly different coupling strengths. The coupling strengths appear to correlate with the extent to which the normal mode changes the molecule’s overall dimensions. The more redshifted absorbers within a given chromophore and matrix also exhibit slightly stronger coupling to matrix phonons. Computational simulations of the emission spectra have been performed in order to examine transferability of the spectral densities between different vibronic bands of the same chromophore and between chromophores in the same matrix. Somewhat different spectral densities are required to model vibronic bands of different chromophores in the same polymer.

Resonant Energy Transfer in the Mixed Crystal Series [Rh(bpy)3][NaAlxCr1-x(ox)3]ClO4 (bpy = 2,2‘-bipyridine, ox = Oxalate, x = 0.05−1)

Efficient resonant energy transfer occurs within the R1 line of the 4A2 → 2E transition of the [Cr(ox)3]3- chromophore in mixed crystal [Rh(bpy)3][NaAl1-xCrx(ox)3]ClO4 (x = 0.05−0.9, ox = oxalate, bpy = 2,2‘-bipyridine). This manifests itself in the form of multiline patterns in resonant fluorescence line narrowing (FLN) experiments at 1.5 K. The conditions for such a resonant process to occur are that the inhomogeneous line width of the R1 line is larger than the zero-field splitting of the ground state, which, in turn, is larger than the homogeneous line width of the transition. The number of lines and their relative intensities depend critically upon the [Cr(ox)3]3- concentration and the excitation wavelength within the inhomogeneous distribution. The basic model for resonant energy transfer as presented by von Arx et al. (Phys. Rev B 1996, 54, 15800) is extended to include the effects of diluting the chromophores in an inert host lattice and of nonresonant R2 excitation. In addition, Monte Carlo simulations serve to explain the temporal evolution of the multiline pattern following pulsed excitation.

Resonant and Phonon-Assisted Excitation Energy Transfer within the R1 Lines of [Cr(Ox)3]3- in a System with Two Crystallographically Non-equivalent Lattice Sites

The [Ru(bpy)3][LiCr(ox)3] system (bpy = 2,2'-bipyridine, ox = oxalate) has two crystallographically non-equivalent [Cr(ox)3]3- sites. In steady-state resonant and nonresonant fluorescence line narrowing (FLN) experiments on the R1 lines of the two non-equivalent [Cr(ox)3]3- chromophores, multiline spectra are observed at 1.6 K. Such multiline spectra are clear evidence for resonant energy transfer processes within the inhomogeneously broadened R1 lines. In addition, time-resolved experiments show that also site-to-site energy transfer occurs, which turns out to be resonant, too, however with a non-negligible phonon-assisted contribution even at 1.5 K.