Fluorescence Excitation Spectra Research Articles

Lack of Excitation Energy Transfer from the Bacteriochlorophyll Soret Band to Carotenoids in Photosynthetic Complexes of Purple Bacteria.

The excitation energy transfer (EET) from the bacteriochlorophyll (BChl) Soret band to the second excited state(s) (S2) of carotenoids in pigment-protein complexes of purple bacteria was investigated. The efficiency of EET was determined, based on fluorescence excitation and absorption spectra of chromatophores, peripheral light-harvesting complexes (LH2), core complexes (LH1-RC), and pigments in solution. Carotenoid-containing and carotenoid-less samples were compared: LH1-RC and LH2 from Allochromatium minutissimum, Ectothiorhodospira haloalkaliphila, and chromatophores from Rhodobacter sphaeroides and Rhodospirillum rubrum wild type and carotenoid-free strains R-26 and G9. BChl-to-carotenoid EET was absent, or its efficiency was less than the accuracy of the measurements of ∼5%. Quantum chemical calculations support the experimental results: The transition dipole moments of spatially close carotenoid/BChl pairs were found to be nearly orthogonal. The structural arrangements suggest that Soret EET may be lacking for the studied systems, however, EET from carotenoids to Qx appears to be possible.

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system.

Different impact of high light on the response and recovery of wild type and lut2 mutant of Arabidopsis thaliana at low temperature

Carotenoid composition is important for plant protection against unfavorable environment. In the present study the effect of several days treatment with high light illumination at low temperature (500 µmol photons m−2 s−1 and (12/10 °C) on Arabidopsis thaliana plants—wt and mutant lut2, was investigated. In addition, a comparative study of the ability of wt and lut2 plants to recover after one week return to control conditions was performed. The response of wt and lut2 to both stress factors was evaluated by changes in net photosynthesis, photochemistry and energy distribution and interaction between both photosystems. The alterations in oxygen evolving capacity were evaluated by registration of flash-induced oxygen yields and kinetics of initial oxygen burst. For the first time the effect of exposure at low temperature/high light on the fluidity of thylakoid membranes isolated from wt and lut2 mutant was followed. The analysis of low temperature (77 K) fluorescence emission and excitation spectra revealed that during exposition to both stress factors the antenna size of photosystem I and photosystem II were affected. Lut2 mutant showed higher sensitivity to the inhibitory action of high light at low temperature, with the grana-situated photosystem II centers being more affected. However, lut2 mutant exhibited a higher ability to restore the photosynthetic activity after recovery period under control conditions. Data presented showed that at low temperature, complete replacement of lutein in lut2 mutant by xanthophyll cycle-associated carotenoids did not result in higher protection against high light, but facilitated better recovery during the recovery period.

Quantifying the uncertainties in multi-wavelength PAM fluorometry due to innate and irradiance-induced variability of fluorescence spectra

Multi-wavelength Chl a fluorometers are increasingly applied to assess phytoplankton photosynthetic capacity and composition, but their usefulness is limited by uncertainties in fluorescence excitation spectra (FES). We investigated this issue using the Phyto-PAM fluorometer to evaluate the effects of innate and irradiance-dependent variations in background (F) and variable (Fv) FES on analysis of three pigment groups (cyanobacteria, chlorophytes and chromophytes). The effects on group-specific estimates of minimum fluorescence (F0), a proxy for biomass, and Fv/Fm, the quantum yield of photochemistry, presented some challenges to the interpretation of group-specific results. F0 estimates usually had a 5–15% margin of error, even when measuring highly uneven mixtures, and applying imperfectly matched calibration FES or stressing samples with photosynthetically active and ultraviolet radiation; errors in Fv/Fm were commonly < 15%. Despite such relatively good accuracy, estimates for F0 and, especially, Fv/Fm are unreliable for groups at low relative abundance, and results can sometimes be reported for groups not actually present. We report margins of error for different levels of relative abundance to inform interpretation of measurements from natural communities and show that F and Fv spectra for some taxa can differ in ways that produce severe errors in F0 and Fv/Fm estimates if used uncritically.

Verification of Microwave Effects on Molecular Clusters by Using Supersonic Molecular Jets.

The origin of the specific effect of microwaves on chemical reactions (the microwave effect) was investigated by examining the effect of microwaves on small groups of molecules such as clusters. The origin of the effect was verified by introducing 2.45 GHz microwaves into a system equipped with a supersonic molecular jet and a special microwave feedthrough to record the fluorescence excitation spectrum of molecules. The carrier gas was bubbled through water and introduced into a phenol-filled sample holder to generate phenol-water clusters. Subsequently, it was confirmed that exposure of the phenol-water clusters contained in the molecular jet ejected from the pulse valve to microwave radiation increased the fluorescence derived from the phenol monomer. This is considered to occur because the phenol-water clusters in the molecular jet absorb microwaves and collapse, thereby increasing the abundance of phenol monomers. This result suggests that microwaves affect not only bulk systems but also small groups of molecules, and that local selective heating, which is one of the causes of the microwave effect, may occur.

Laser-induced fluorescence spectroscopy of uranium monofluoride

The laser-induced fluorescence excitation spectra of uranium monofluoride have been recorded in the range of 17000-19000 cm−1 using two-dimensional spectroscopy. High resolution dispersed fluorescence spectra and time-resolved fluorescence spectroscopy were also recorded. Three rotationally resolved bands were intensively analyzed, and all bands were found to be derived from the ground state X(1)4.5 with a rotational constant of 0.23421 cm−1. The low-lying electronic states were observed near 435 and 651 cm−1 in the dispersed fluorescence spectra, which were assigned as Ω′ = 3.5 and 2.5, respectively. The vibrational constants for the X(1)4.5 and X(1)3.5 states were calculated. The branching ratios of the dispersed fluorescence spectra for the [18.62]3.5, [17.72]4.5, and [17.65]4.5 states were reported. Radiative lifetime of 332(9) ns, 825(49) ns, and 433(15) ns for the [18.62]3.5, [17.72]4.5, and [17.65]4.5 states were obtained by fitting the time-resolved fluorescence spectroscopy, respectively. Transition dipole moments were performed using the branching ratios and the radiative lifetimes.

Switching resonance character within merocyanine stacks and its impact on excited-state dynamics

Summary In this study, the optical properties and excited-state dynamics of the unique self-assembled donor-acceptor (DA) merocyanine dye stacks from dimer up to octamer, prepared via dipole-dipole interactions, are reported in terms of coherent exciton dynamics and formation of an excimer-like state. Our findings are based on the steady-state absorption/emission, time-resolved fluorescence, and transient absorption (anisotropy) measurements, including wavepacket analysis and quantum mechanical calculations. Coherent exciton of torsional motions-restricted dye stacks rapidly localizes into the weakly emissive excimer-like state, by shortening the inter-moiety distance and changing the bond-length alternation pattern. The inner merocyanine moiety, having two neighboring units, has a reversed resonance character (non-polar (N) Z) in the ground state. This difference has led to two conclusions: (1) tetramers and octamers exhibit different features of excimer-like state than the dimer, and (2) octamers exhibit slower localization dynamics due to the enhanced homogeneity (six inner-moieties) compared with tetramers (two inner moieties).

Fluorescence excitation properties of bilirubin in solution and in serum

The bichromophore nature of bilirubin explains the presence of at least two partially overlaying bands in both absorption and fluorescence emission spectra, and accounts for interchromophore exciton transfer events responsible for the emission sensitivity to the molecular environment and excitation wavelength. These concepts were likely responsible for the previously reported good yield of the unexpected remarkable bilirubin fluorescence emission under excitation at 366 nm, at which bilirubin absorption is very low. In this connection, aim of this work is to further investigate bilirubin spectral excitation properties and their diagnostic potential, until now poorly considered. Fluorescence excitation spectra of pure bilirubin in solution with solubilizing agents observed at 520 and 570 nm showed a wide region in the 430–510 nm range, similar to the absorption profile. In addition, an excitation band centered at about 400 nm was detected. Comparable excitation features were detected in rat serum. The 430–510 nm excitation region was well separated from a main band at shorter wavelength, ascribable to other endogenous fluorophores, with a shoulder at about 400 nm which was also easily discriminated by spectral fitting analysis. The bands ascribable to bilirubin showed changes of their relative contribution to the overall spectral region after liver ischemia/reperfusion, comparable to bilirubin biochemical data. Excitation spectra proved to discriminate the fluorescence of serum bilirubin at levels much lower than emission spectra, opening promising perspectives to improve the real time fluorescence analysis of crude serum in the absence of any exogenous labelling agent, and advance the diagnostic application of optical-biopsy in experimental hepatology and biomedicine.

Asymmetry in the Q y Fluorescence and Absorption Spectra of Chlorophyll a Pertaining to Exciton Dynamics.

Significant asymmetry found between the high-resolution Qy emission and absorption spectra of chlorophyll-a is herein explained, providing basic information needed to understand photosynthetic exciton transport and photochemical reactions. The Qy spectral asymmetry in chlorophyll has previously been masked by interference in absorption from the nearby Qx transition, but this effect has recently been removed using extensive quantum spectral simulations or else by analytical inversion of absorption and magnetic circular dichroism data, allowing high-resolution absorption information to be accurately determined from fluorescence-excitation spectra. To compliment this, here, we measure and thoroughly analyze the high-resolution differential fluorescence line narrowing spectra of chlorophyll-a in trimethylamine and in 1-propanol. The results show that vibrational frequencies often change little between absorption and emission, yet large changes in line intensities are found, this effect also being strongly solvent dependent. Among other effects, the analysis in terms of four basic patterns of Duschinsky-rotation matrix elements, obtained using CAM-B3LYP calculations, predicts that a chlorophyll-a molecule excited into a specific vibrational level, may, without phase loss or energy relaxation, reemit the light over a spectral bandwidth exceeding 1,000 cm−1 (0.13 eV) to influence exciton-transport dynamics.

Invisible dye tracing – an efficient technique for study of groundwater channels

We studied excitation spectra of uranine fluorescence in eluates from charcoal traps saturated in cave waters and in reference uranine solutions and found that the most appropriate excitation wavelength is 295±5 nm, because it excites fluorescence of uranine many times stronger than fluorescence of natural fluorescent compounds in groundwater. This allows us to lower the detection limit of uranine fluorescence with one order of magnitude, even below the level of natural fluorescence of karst waters at integral UV excitation.

Spectroelectrochemical Study of Organic-Inorganic Hybrid Nano-Molecular Clusters Exhibiting Voltage-Gated Fluorescence Switching and Electrochromism

Polyhedral boranes especially the twelve vertex closo-dodecaborate and the ten vertex closo-decaborate anions exhibit remarkably high thermal stability and resistance to oxidative degradation. Such high stability is attributed to the extensive delocalization of their framework bonding electrons, giving rise to three dimensional aromaticity. The substitution chemistry of polyhedral boron chemistry using aromatic ligands was previously limited in scope. However, we recently reported new and extensive polysubstitution chemistry for these ions. The structural characterization of the products revealed that up to ten of B-H vertices may be replaced with phenyl, substituted phenyl, or polycyclic aromatic hydrocarbons as ligands through the formation of stable boron-carbon bonds. The resulting organic-inorganic hybrid nano-molecular ions are pseudospherical whose diameters range between 1.3 – 3.5 nanometers depending on the ligands involved. These polyarylborane clusters are found to exhibit interesting electronic and spectroscopic properties, including high solution-phase fluorescence quantum yields, high molar absorptivities, and extraordinarily large stoke shifts. Furthermore, the fluorescence emission, excitation and absorption spectra for most of these products change reversibly upon applying product-specific electrochemical potentials, thus resulting in redox-controlled fluorescence switching and electrochromism. The properties of each polyarylborane depend upon the composition and the structure of the bound ligands, and these may be tailored through chemical modification of the surface. Our study suggest that these new nanomaterials may have potential for applications in biomedical imaging and sensor development. This presentation will focus on the details of syntheses, structural characterization, electrochemical and spectroelectrochemical properties of polyarylboranes.

Covalent anchoring of atomically precise glutathione-protected gold nanoclusters on graphene oxide nanosheets

This paper describes the development of a novel method of producing nanocomposites consisting of gold nanoclusters anchored on graphene oxide nanosheets in a cost-effective and reproducible manner. The novelty of the technique hinges on the covalent functionalization of atomically precise subnanometer gold clusters protected by glutathione (Au15SG13 and Au25SG18) on to graphene oxide (GO) nanosheets according to the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride crosslinking method, using the existing carboxylic groups present both at the surfaces of the nanoclusters and the GO nanosheets. The atomic precision of glutathione-protected gold nanoclusters was evidenced by electrospray ionization mass spectrometry. The formed hybrid nanocomposites were characterized by TEM measurements and exhibit nonlinear optical properties characteristic of GO, in particular a strong second harmonic scattering response as well as a multi-photon excited fluorescence spectrum characterized by a broad band in the visible range between 350 and 700 nm. Atomically precise nanoclusters covalently linked to GO nanosheets are therefore promising for new applications in the areas of optoelectronics and photovoltaics.

FRET-based fluorometry assay for curcumin detecting using PVP-templated Cu NCs

A novel fluorometric strategy is proposed for detecting curcumin by polyvinyl pyrrolidone-templated Cu NCs (PVP–Cu NCs) as a fluorescent probe which exhibits excitation/emission peaks at 380/510 nm. The fluorescent excitation and emission spectra of PVP-Cu NCs have a striking overlap with the UV–vis spectrum of curcumin, and the fluorescence lifetime of PVP-Cu NCs decreases after the addition of curcumin. Curcumin leads to fluorescence quenching based on fluorescence resonance energy transfer. This method allows for the determination of curcumin in the range of 0.1–10 μg mL−1 and the detection limit is 21 ng mL−1. Furthermore, this method displays good selectivity and is successfully applied for real sample analysis.

J-like aggregation of a cationic polythiophene with hydrogen-bonding capabilities due to 1,4-dioxane: Solution excitation spectra and fluorescence, morphology and surface free energy of films

This work presents solution- and solid-state evidence of the enhancement of J-like aggregation of a cationic polythiophene (CPT) with isothiouronium functionalities (PT1), caused by a decrease in the polarity and hydrogen-bonding (H-bonding) capacity of the solvent, generated by using a 50:50 v/v 1,4-dioxane-water mixture (W-DI) instead of water.In solution, the presence of 1,4-dioxane (DI) seems to generate selective solvation, tuning the energy transfer within PT1 from inter-chain into intra-chain, enhancing J-like aggregation. On the other hand, during the casting process, the presence of DI directs the interaction with solid-substrates, generating an increase in the solid-state fluorescence, modifying the morphology from one similar to ballistic-aggregation (BA) into one similar to attachment limited aggregation (ALA), DI also modifies the SFE by increasing slightly its polar contribution (γSp) and decreasing the dispersive one (γSd). These results can be explained to be caused by a “coating” effect in presence of DI (as proposed before experimentally and computationally).Our results show a clear correlation between the solution- and solid-state properties of PT1 in each solvent, further validating the use of the fluorescence excitation spectra to trace J-like aggregation of water-soluble conjugated polymeric fluorophores in solution. This information could be useful for predicting and designing specific mesoscopic architectures of CPTs (and conjugated polyelectrolytes in general), which are molecules lacking of clear structure-function guidelines for designing high-performance polythiophene-based interlayer materials, especially for CPTs (and conjugated polyelectrolytes (CPEs) in general), particularly those with H-bonding capabilities. To the best of our knowledge the use of solution-state fluorescence excitation spectra to identify J-like aggregation of water-soluble conjugated polymers (CPs) has been scarcely used/discussed in literature and no correlation with solid-state properties was reported previously.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

The electronic structure of WS molecule below 21,500 cm−1

The low-lying electronic states of the tungsten monosulfide (WS) molecule have been investigated using laser-induced fluorescence excitation spectra and high-level ab initio calculations. 29 vibronic bands in total are experimentally observed in the range of 13,100 – 21,500 cm−1 and grouped into 8 electronic transition systems. The spectroscopic constants including the vibrational frequency, rotational constant and radiative lifetime in the low-lying excited electronic state are obtained by analysis of the rovibrational-resolved spectra. Seven lowest Ω sub-states that belong to the lowest three Λ-S states considering the spin-orbit effect, i.e. the ground state 3Σ−, first and second excited states 5Π and 3Φ, are identified through single-vibrational-level emission spectra. In addition, seven Ω = 1 excited sub-states in the visible energy range are identified, which are consecutively consistent with the ab initio calculation results of the sixth to twelfth Ω = 1 sub-states. The experimental and theoretical results on WS molecule may provide a benchmark to study the electronic structure of those molecules with strong spin-orbit coupling and electron-electron correlation.

MRCI+Q study on the electronic structure and spectroscopy of the low-lying electronic states of HgBr including spin-orbit coupling

Due to attractive candidate for the laser application and searching for the permanent electric dipole moment of the electron (eEDM), mercury bromide (HgBr) is of much interest to researchers. However, detailed information of the electronic structure of HgBr is still lacking, especially for spin-orbit interactions in excited states. In this work, high-level configuration interaction calculations of low-lying states correlating to the lowest two dissociation limits Hg(1S) + Br(2P) and Hg(3P) + Br(2P) of HgBr are carried out. In order to ensure good accuracy, the Davidson correction and spin-orbit coupling (SOC) effect are all taken into consideration in our computations. The potential energy curves (PECs) of 14 Λ-S states and 30 Ω states are determined. Based on the PECs, the spectroscopic constants of the bound states are obtained, most of which have not been reported in previous studies. The calculated SOC integrals of 22Σ+-22Π indicate a strong spin-orbit interaction, which can explain the apparent perturbations between B2Σ+1/2 and C2Π1/2 found in the HgBr fluorescence excitation spectrum. Finally, to reveal more detail on transition properties of excited states, transition dipole moments of C2Π1/2-X2Σ+1/2, D2Π3/2-X2Σ+1/2, and B2Σ+1/2-X2Σ+1/2 transitions and radiative lifetimes of C2Π1/2, D2Π3/2, and B2Σ+1/2 are determined.

Genetic Algorithm for quick finding of diatomic molecule potential parameters

ABSTRACT Application of Genetic Algorithm (GA) for determination of parameters of an analytical representation of diatomic molecule potential is presented. GA can be used for finding potential characteristics of an electronic energy state which can be described by an analytical function. GA was tested on two artificially generated datasets which base on potentials with known characteristics and two laser induced fluorescence excitation spectra recorded using transitions in CdKr and CdAr molecules. Tests on generated datasets showed that GA can properly reproduce parameters of the potentials. Tests on experimental spectra indicated that changing the potential model from a Morse, which is frequently used as a starting potential in the inverse perturbation approach method, to an expanded Morse oscillator leads to noticeable improvement of agreement between simulated and experimental data.

Selecting optimal spectral bands for improved detection of autofluorescent biomarkers in multiphoton microscopy.

.Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing.Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers.Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio.Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images.Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

Investigation on Photophysical, Solvatochromism and Biological Significance of Substituted 2H‐Indazole Derivatives

AbstractThe electronic properties of substituted 2H‐indazoles were investigated for their photophysical behavior under isolated conditions and in solvent environment, both computationally and experimentally. DFT calculations with CAM−B3LYP functional and 6–311++G** basis set were performed in the ground and excited states. The effect of solvents on the low and high energy electronic states was studied by following the steady state absorption and fluorescence excitation and emission spectra. Solvatochromic interactions including general and specific solvent effects are reported along with the results of Lippert and ET(30) plots. The biological significance of these 2H‐indazole derivatives was also investigated through docking studies. The interactions of 2H‐indazole derivatives with the active site of subdomain II of human casein kinase (C2 K) alpha are reported.