Styryl Dye Research Articles

Using FM Dyes to Study Endomembranes and Their Dynamics in Plants and Cell Suspensions.

FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.

2‐(1,1‐dioxidobenzo[b]thiophen‐3(2H)‐ylidene)malononitrile (BTD) Based Styryl Chromophores‐ Solvatochomic and Computational Investigation of Linear and NLO properties

AbstractLinear and nonlinear optical properties of the twelve styryl dyes are investigated using density functional theory (DFT). Range‐separated hybrid CAM−B3LYP and global hybrid BHandHLYP estimate high second order hyperpolarizability. Mean polarizability (α0), polarizability anisotropy (Δα), static first‐order hyperpolarizability (β0) and second hyperpolarizability (γ) were found to be overestimated in CAM−B3LYP than in BHandHLYP. As bond length alternation (BLA) increases first order hyperpolarizability increases. Multilinear regression analysis (MLR) shows the solvent basicity, polarizability and dipolarizabilty are responsible for absorption solvatochromism. The ratio of the dipole moments increases as donor strength and conjugation increases which support charge transfer. The figure of merit (FOM) and intrinsic hyperpolarizability found more in chromophores with dialkylamine donors. As the stability indicated by electrophilicity indices decreases the hyperpolarizability increases. Interrelationships between α0, β0 and γ were evaluated and it shows good linear fit. Absorption is red shifted and hyperpolarizability increases with the amplitude of the sine‐shaped potential along the conjugation chain. A high molecular electrostatic potential was observed in dyes containing 2‐piperidinothiophene donor.

Cubic plasma membrane domains stabilize and restrict zones for pH band formation in Chara internodal cells

ABSTRACTCharacean internodal cells display acid and alkaline regions along their surface when exposed to light. The acidification facilitates uptake of carbon required for photosynthesis. In the genus Chara, cubic plasma membrane domains, known as charasomes, were first described five decades ago. Their size and abundance varies along the cell surface and it has been conjectured that the charasome coverage correlates with the pH banding pattern. In this study, we used fluorescein isothiocyanate-dextran (FITC-dextran) and confocal laser scanning microscopy to map the pH profile at high spatial and temporal resolution. In parallel, we stained the charasomes with styryl dyes and investigated their distribution in relation to the fluorescence pattern of FITC-dextran. In both internodal cells of the branchlets and of the main axis, we found that charasomes were small and sparse at the alkaline bands but large and abundant at the acidic regions. Our findings confirm the existence of cell surface regions predisposed to generate either high or low pH zones. We provide evidence that areas with high charasome abundance stabilize and restrict the position of the acidic bands whereas regions with low charasome abundance allow the formation of either stable or unstable alkaline bands.

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.

Synthesis and photophysical studies on 2‑styryl phenanthro[9,10‑d]oxazole derivatives.

A new series of 2‑styryl phenanthro[9,10‑d]oxazoles was readily accessible from the condensation reaction of 9,10‑phenanthroquinone with cinnamaldehydes in the presence of lactic acid. All these styryl dyes were isolated in good yields and characterized by various analytical and spectroscopic techniques. One of the dyes containing NO2 group (3d) was structurally characterized by single crystal X-ray analysis. These dyes displayed emission in blue to green region with larger Stokes shift values characteristic to the nature of substituents. In addition, positive solvatochromic trend was observed by increasing the solvent polarity suggestive of a more stabilized polar excited state. Moreover, the addition of trifluoroacetic acid leads to a prominent blue-shift in visible and emission color changes owing to the protonation of the nitrogen atom of oxazole moiety. Among the all, the oxazole derivative having NMe2 group (3b) exhibits good response to acidic pH in the range of 3.0 to 5.6 with a good linearity upon decreasing the pH from 8.0 to 2.16. The absorption studies were further supported by density functional theory calculations.

Nonlinear Optical Chromophores with Aggregation Induced Emission Enhancement Based on 2-N,N-Dibutylamino-4-Phenyl Thiazole with FMR Characteristics.

The synthesis, photophysical, linear and nonlinear optical (NLO) properties of novel fluorescent styryls based on 2-N,N-dibutylamino-4-phenyl thiazole as donor attached to different acceptor groups via conjugated π-bridge were studied with the help of solvatochromic model. The NLO properties of extended styryls based on benzylidene malononitrile and isophorone were more enhanced than the simple styryls based on malononitile and ethylcyano acetate which was attributed to the longer conjugation path. The benzylidene based extended styryl dye showed maximum value of two-photon absorption cross section σ2PA 98.85GM in dichloromethane. The 2-amino thiazole derivatives were responsive towards the viscosity of the medium hence investigated for the fluorescent molecular rotor (FMR) properties wherein almost 2.07 to 3.65 fold increase in emission intensity was observed. Also, the aggregates of these dyes in N,N-dimethyl formamide-water mixture showed enhancement in emission on aggregates formation like 3.73 fold increase in emission intensity was observed for benzylidene based extended styryl dye. Thus NLOphoric AIEgens with FMR properties emitting in the red region of the electromagnetic radiation were developed.

Adsorption and photodynamic efficiency of meso-tetrakis(p-sulfonatophenyl)porphyrin on the surface of bilayer lipid membranes

The adsorption and photodynamic efficiency of 5,10,15,20-tetrakis(p-sulfonatophenyl)porphyrin (H2TPPS4) on bilayer lipid membranes (BLM) have been studied. The adsorption of H2TPPS4 on BLM leads to rising of the potential drop on the membrane/water interface which has been detected either by the intramembrane field compensation (IFC) method, or as ζ-potential of liposomes measured by the dynamic light scattering method. The dependence of this potential on the concentration of H2TPPS4 and KCl in the solution can be described in the frame of Gouy-Chapman model of diffuse double layer assuming that the molecules of H2TPPS4 adsorb on the surface of BLM as an anions with four charged groups. The potential disappeared when the pH of solution decreased from 6 to 3 allowing the conclusion that the protonated forms of H2TPPS4 can not adsorb on the BLM probably due to change of conformation or aggregation of the molecules. The photodynamic efficiency of H2TPPS4 was evaluated by measuring the rate of damage of the targets - molecules of styryl dye (di-4-ANEPPS) by singlet oxygen generated under illumination on the surface of BLM. This rate was proportional to the surface density of H2TPPS4 molecules on the membrane which was determined from the change of surface charge of the membrane due to adsorption of the H2TPPS4. These results indicate that the di-4-ANEPPS molecules are damaged by singlet oxygen generated by monomers of H2TPPS4 molecules adsorbed on the membrane. The rate of oxidation of di-4-ANEPPS molecules adsorbed on the same (cis) side of the membrane together with the H2TPPS4 molecules was either the same or higher than that when di-4-ANEPPS molecules were adsorbed on opposite (trans) side. It indicates that the quenching of singlet oxygen by the di-4-ANEPPS molecules at cis side of the membrane was negligible, in contrast to our earlier study when singlet oxygen was generated by aluminum(III) phthalocyanines with one or two peripheral sulfo groups. The difference between these phthalocyanines and H2TPPS4 was explained by their different adsorption depth in the membrane.

Annelated tricyclic thiophenes and their photophysical properties

Photochemical oxidative cyclization of 3-[(E)-2-(3,4-di-methoxyphenyl)vinyl]thiophene and its 15-crown-5-analogue (15-[(E)-2-(3-thienyl)vinyl]-2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-benzopentaoxacyclopentadecine) affords the isomeric thiophene-containing fused aromatic compounds demonstrating photophysical properties different from those of initial styryl derivatives. E-Configuration of the initial styryl dye, 3-[(E)-2-(3,4-dimethoxyphenyl)vinyl]thiophene, has been proved by X-ray analysis.

Electronic structure and spectral properties of indole based fluorescent styryl dyes: Comprehensive study on linear and non-linear optical properties by DFT/TDDFT method

The photophysics, linear and non-linear optical properties of indole based fluorescent styryl dyes are investigated with density functional theory and time-dependent density functional theory. The performance of seven global hybrid functionals - B3LYP, PBE0, BHHLYP, M06, M06-L, M06-2X, M06-HF and six range separated hybrid functionals - CAM-B3LYP, HISSbPBE, HSEH1PBE, ωB97, ωB97X, ωB97XD with poples [6-311++G(d,p)] and correlation consistence (cc-pVDZ, cc-pVTZ) basis sets is analyzed. The linear (vertical excitations, oscillator strengths) and non-linear optical parameters (first and second order hyperpolarizabilities) are calculated with each functional in combination with different basis sets for comparison. The vertical excitation of all the dyes are shown to have good correlation with the experimental values by the M06-2X/6-311++G(d,p) and HISSbPBE/cc-pVDZ combination with the least deviations (mean standard error) of 8.67 nm and 4.34 nm with respect to global and range separated hybrid functionals respectively. On the other hand, polarizability and hyperpolarizability values evaluated for all the dyes suggested that the dye which contain N-ethyl group as a donor and nitro group as an acceptor is found to have the largest NLO response as compared to other dyes. Mean standard error was calculated for each system with respect to global and range separated hybrid functionals to evaluate the accuracy of the DFT methods. Mean standard error of polarizability and hyperpolarizability obtained using global hybrids and range separated hybrid functionals are found to be basis sets dependent i.e. each functional and basis set combination perform differently.

Effect of methoxy group on NLOphoric properties of fluorescent 7-arylstyryl-2-methoxyphenylimidazo[1,2-a]pyridine - Solvatochromic and computational method

Extensively conjugated donor-π-acceptor-donor (D-π-A-π-D) type fluorescent styryl dyes bearing imidazo[1,2-a]pyridine units have been investigated for their nonlinear optical properties (NLO). Enhanced Stokes shift (2486–2843 cm−1) brought about by replacing methoxy group by diethyl, dimethyl, cyclohexylamino substituents at C-2 and C-7 positions of imidazo[1,2-a]pyridine core in styryl dyes have been understood in elucidating the electronic structures. The position of methoxy group acting as auxiliary donor largely influences the nonlinear optical (NLO) properties of imidazo[1,2-a]pyridine derivatives. Polarizabilities and hyperpolarizabilies were estimated by solvatochromic and DFT method using BHandHLYP and CAM-B3LYP functionals with the 6-311 + G(d,p) basis set. Imidazo[1,2-a]pyridine derivatives showed minimum ten-fold higher NLO parameters compared to reminiscent of charge transfer contribution of donor groups. The dyes have two-photon cross-section values 66–189 GM and comparable with commercial two photon absorbing styryl dyes.

Enhancement of NIR emission by a tight confinement of a hemicyanine dye within zeolitic MgAPO-5 nanochannels.

The encapsulation of a hemicyanine dye, LDS 730, into the 1D nanochannels of MgAPO-5 aluminophosphate by "one-pot" synthesis, based on "in situ" occlusion via a crystallization inclusion method, has led to a hybrid material with emission in the NIR region. The tight fitting between the molecular size of the guest dye and the pore dimensions of the host has enabled a rigid conformation of the LDS 730 dye within the nanochannels. Consequently, fluorescence in the NIR range of the spectra is enhanced with respect to the dye in solution. The synthesis of the hybrid material was optimized through a systematic variation of the gel composition via MW in order to obtain a pure phase.

The Time-Resolved Fluorescence Stokes Shift of Cucurbit[6]Uril Complexes with a Pyridinium Styryl Dye.

The time-resolved Stokes shift of fluorescence of styryl dye, trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (DASPI), and its host-guest complexes with cucurbit[6]uril (CB[6]) was studied by up-conversion technique on a 5ps time scale. In all cases, the solvation time correlation function - that describes relaxation of the fluorescent state - contains two components. The time constant of the first component, which is related to the vibrational relaxation of the dye cation, increases from 118fs without CB[6] to 224fs in the presence CB[6]. It suggests the production of CB[6] complexes to disturb the hydrogen-bond net in the vicinity of DASPI cation. The time constant of the second component, which is associated with solvation, changes from 0.81ps without CB[6] to 1.15ps in the presence of the cavitand. The observed increase can be related to the displacement of the dye cation deeper into CB[6] that allows considering DASPI-CB[6] complexes as a light-controlled molecular machine with a response time on the picosecond scale.

Carbazole based NLOphoric styryl dyes- synthesis and study of photophysical properties by solvatochromism and viscosity sensitivity

We have synthesized push-pull chromophore based on carbazole moiety. The intramolecular charge transfer (ICT) of these dyes were examined by photophysical properties such as absorption, emission and fluorescence quantum yield in varying solvents of different polarities. They show absorption and emission in the range of 390–460 nm and 510–600 nm respectively. Oscillator strengths, transition dipole moments, different solvent polarity functions and frontier molecular orbital (FMO) analysis have been studied for understanding charge transfer from donor to the acceptor moiety. Density Functional Theory (DFT) revealed dyes with phenyl group in conjugation path with an acceptor (Dyes 9c and 10c) exhibited a relatively lower σ2PA cross-section in comparison to their counterparts devoid of conjugation (Dyes 9a, 9b, 10a, and 10b). Viscosity induced emission intensity enhancement has been observed for all dyes at 0–100% PEG-400 in ethanol 9c and 10c are good FMR as compared to the other due to the presence of extra-π-conjugation. Bond length, bond order alternation analysis and non-linear polarizability studies of these dyes were calculated by DFT. The spectral variations were analyzed by means of linear solvation energy relationships concept proposed by Kamlet-Taft and Catalan parameters which indicate that solvent polarizability/dipolarity play an important role as compared to solvent acidity or basicity. Thus, linear and non-linear optical properties of styryls have been determined.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation.

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Effect of Heavy Water on Ultrafast Dynamics of the Fluorescence Stokes Shift for a Styryl Dye and its Complexes with Cucurbiturils

Effect of Heavy Water on Ultrafast Dynamics of the Fluorescence Stokes Shift for a Styryl Dye and its Complexes with Cucurbiturils

Viscosity induced emission of red-emitting NLOphoric coumarin morpholine-thiazole hybrid styryl dyes as FMRs: Consolidated experimental and theoretical approach

Four new coumarin hybrid styryl dyes are synthesized by condensing 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-2-morpholinothiazole-5-carbaldehyde with dicyanovinylene containing active methylene intermediates and their linear and non-linear optical properties are studied. The dye having dicyanovinylene-isophorone acceptor displayed a large Stokes shift of 3702–4795 cm−1 in non-polar to polar solvent respectively. The dyes exhibit a good charge transfer characteristics and positive emission solvatochromism (∼50 nm–72 nm) in non-polar to a polar solvent which is well supported by multi-linear regression analysis. Viscosity induced enhancement study in ethanol/polyethylene glycol-400 system shows 2.71–6.78 fold increase in emission intensity. The intra and twisted-intramolecular charge transfer (ICT-TICT) characteristics were established using emission solvatochromism, polarity plots, generalised Mullikan-Hush (GMH) analysis and optimized geometry. A dye having the highest charge transfer dipole moment relatively possess the maximum two-photon absorption cross-section area (KK-1 = 165–207 GM) which was established using theoretical two-level model. The NLO properties have been investigated employing solvatochromic and computational methods and were found to be directly proportional to the polarity of the solvent. Z-scan results reveal that the dyes KK-1 and KK-2 possesses reverse saturable kind of behaviour whereas KK-3 and KK-4 show saturable kind of behaviour. From the experimental and theoretical data, these coumarin thiazole hybrid dyes can be considered as promising candidates for FMR and NLOphores.

Structural and spectrophotometric characterization of 2-[4-(dimethylamino)styryl]-1-ethylquinolinium iodide as a reagent for sequential injection determination of tungsten

Structure, spectrophotometric and protolytic properties of the styryl dye 2-[4-(dimethylamino)styryl]-1-ethylquinolinium iodide (R) as well as its complex with tungsten were studied. The selective protonation of dimethylamino group was confirmed by density functional theory investigation through the computation of Fukui function, NPA partial atomic charges, and NICS(0) aromaticity indexes. The TD-DFT study explains the experimental change of color by excluding the dimethylamino group from HOMO orbital upon protonation. The acid dissociation constant, the optimum wavelength and the molar absorptivity of R were found to be: 3.02, 501nm and 4.0×104Lmol−1cm−1, respectively. The protolytic properties of the reagent were found to change significantly in the presence of tungsten(VI). Analysis of bond critical points between the anions and Quinaldine Red cation gives the selectivity raw HWO4−>MoO4−>H2VO4−>ReO4−>ClO4−, that perfectly match with the experimental data. Based on this observation, a non-extractive sequential-injection spectrophotometric method for the determination of tungsten was developed. The absorbance of the colored extracts obeys Beer's law up to 55.2mgL−1 of W at 520nm wavelength. The limit of detection calculated from a blank test (n=10) based on 3s was 0.96mgL−1. The developed method was applied for the determination of tungsten in model samples.

Novel pH-Sensitive Lipid Based Exo-Endocytosis Tracers Reveal Fast Intermixing of Synaptic Vesicle Pools.

Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation. Measuring the incorporation efficacy of different dye-labeled lipids into budding SVs, we did not observe an enrichment of lipids with affinity for liquid ordered membrane domains. But most importantly, we found no evidence for a static segregation of SVs into recycling and resting pools. A small but significant fraction of SVs that is reluctant to release during a first round of evoked activity can be exocytosed during a second bout of stimulation, showing fast intermixing of SV pools within seconds. Furthermore, we found that SVs recycling spontaneously have a higher chance to re-occupy release sites than SVs recycling during high-frequency evoked activity. In summary, our data provide strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs.

NLO properties of ester containing fluorescent carbazole based styryl dyes – Consolidated spectroscopic and DFT approach

The linear and nonlinear optical (NLO) properties of new fluorescent styryl dyes based on anchoring ester containing carbazole as donor appended to different acceptor groups to have a conjugated π-system with push-pull geometry are studied. The NLO properties have been determined using solvatochromic and computational methods. Three different TD-DFT functional are used namely, B3LYP, BHandHLYP, and CAM-B3LYP, with aim of elucidating better functional for NLOphores. Further, the two photon properties (σ2PA) have been described theoretically by two level model considering the dipole moment difference between the ground and the final electronic states and bypassing the intermediated resonance state. The compounds with a high charge transfer from the acceptor group to the carbazole ring have relatively high two-photon absorption cross-sections (60–317 GM). The linear polarizability (αCT), first order hyperpolarizability (β) and second order hyperpolarizability (ɣ) for 4c dye was the highest among the studied dyes which is attributed to the lesser energy gap evident by both the methods. But in contrary, the σ2PA cross-section value was low for dye 4c which is due to the presence of freely rotatable twisted phenyl ring in the conjugation path, pulling the electron density towards itself and thus lead to decrease in σ2PA cross-section. Structure-property relationship is better understood by the correlation of bond length alternation/bond order alternation (BLA/BOA) with NLO properties of dyes. Thus by simple solvatochromic method and computational method, we have screened the carbazole styryls as NLO candidates with good first order hyperpolarizability and good two photon cross-section.

Supramolecular tuning of energy transfer efficiency and direction in a bis(styryl) dye–crown ether conjugate

The control of the energy transfer (FRET) in a novel bischromophoric styryl(pyridinium) dye bearing two different crown ether residues is presented. Complexation of the dye with metal cations allows to tune the FRET efficiency without changing the transfer direction or to switch the FRET off, whereas protonation launches the FRET process in the opposite direction relative to the molecular structure.