Potential-modulated fluorescence spectroscopy of the membrane potential-sensitive dye di-4-ANEPPS at the 1,2-dichloroethane/water interface

The spectrofluorometric behavior of a membrane potential-sensitive dye, 1-(3-sulfonatopropyl)-4-[β-{2-(di-N-butylamino)-6-naphthyl}vinyl]pyridinium betaine (di-4-ANEPPS), at the polarized 1,2-dichloroethane/water interface was studied by means of potential-modulated fluorescence (PMF) spectroscopy. The results, combined with those from cyclic and alternating current voltammetry, clearly suggested that the dye adsorbed at the interface underwent a reorientation with increasing the interfacial potential, giving a well-developed PMF response as well as a voltammetric response. In addition to the PMF response, another PMF response was observed by addition of dilauroyl phosphatidylcholine (DLPC). This additional response was well explained in terms of a reorientation of di-4-ANEPPS at the interface, which would be induced by the potential-dependent desorption of DLPC from the interface. Thus, the present study supported the reorientation/solvatochromic mechanism for the membrane potential-sensitive dye rather than the electrochromic mechanism.     

Combined use of two membrane-potential-sensitive dyes for determination of the Galvani potential difference across a biomimetic oil/water interface

The fluorescence behavior of anionic membrane-potential-sensitive dyes, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) and bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC₂(3)), at a biomimetic 1,2-dichloroethane (DCE)/water (W) interface was studied by the mean of potential-modulated fluorescence (PMF) spectroscopy. The respective dyes gave a well-defined PMF signal due to the adsorption/desorption at the DCE/W interface. It was also found that the potentials where the two dyes gave the PMF signals were different by about 100 mV. We then attempted a combined use of the two dyes for determination of the Galvani potential difference across the DCE/W interface. When 40 μM DiBAC₄(3) and 15 μM DiSBAC₂(3) were initially added to the W phase, distinctly different spectra were obtained for different interfacial potentials. The ratio of the PMF signal intensities at 530 and 575 nm (the fluorescence maximum wavelengths for the respective dyes) showed a clear dependence on the interfacial potential. These results suggested the potential utility of the combined use of two dyes for the determination of membrane potentials in vivo.

Interfacial behavior of sulforhodamine 101 at the polarized water/1,2-dichloroethane interface studied by spectroelectrochemical techniques

The transfer mechanism of an amphoteric rhodamine, sulforhodamine 101 (SR101), across the polarized water/1,2-dichloroethane (DCE) interface was investigated using cyclic voltammetry, differential voltfluorometry and potential-modulated fluorescence (PMF) spectroscopy. The voltammetric response for the ion transfer of SR101 monoanion from water to DCE was observed as the diffusion-controlled transfer process. An unusual voltammetric response was found at 0.15 V more negative than the formal transfer potential of SR101⁻ in the cyclic voltammogram and voltfluorogram. The frequency dependence of the PMF responses confirmed the presence of the adsorption processes at negative potentials. In addition, a further transient adsorption step was uncovered at The interfacial mechanism of SR101 is discussed by comparing the results obtained from each technique.     

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.     

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.     

Time-resolved total internal reflection fluorescence spectroscopy. Part I. Photophysics of Coumarin 343 at liquid/liquid interface

Pico-second time-resolved time-correlated single photon counting (TCSPC) technique under the total internal reflection (TIR) condition has been used to study the photophysical properties of Coumarin 343 (C343) dye molecules adsorbed at the water/1,2-dichloroethane (DCE) interface. The fluorescence decay profile of C343 under TIR condition at the water/DCE interface was non-exponential and fitted to the double exponential decay function with the fluorescence lifetimes 0.3 and 3.6 ns, which proved the existence of two different forms of C343 species having largely different lifetimes at the interface. The longer fluorescence lifetime component of C343 at the interface is attributed to the emission from the monomeric form of the dye molecules and the shorter lifetime component is due to the aggregation of dye molecules. The penetration depth dependence of decay curves indicated no change in the fluorescence lifetime components, however, the amplitude corresponding to the lifetime of aggregate increased and the amplitude corresponding to the lifetime of monomer decreased with the decrease in penetration depth of the aqueous phase from the interface. Aggregation is significant in the interfacial layer. The decrease in monomer lifetime at the interface compared to that in the bulk solution is interpreted in terms of excitation energy migration between the dye molecules.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

Interfacial complexation reactions of Sr2+ with octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide for understanding its extraction in reprocessing spent nuclear fuels

The complexation reactions between strontium (Sr(2+)) and octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) were studied at the aqueous|1,2-dichloroethane (w|DCE) and aqueous|room-temperature ionic liquid (w|RTIL) microinterfaces, in order to understand its extraction in reprocessing spent nuclear fuels, remediation of environmental contamination, and potential radiological isotope feed stock for (90)Y from its isotope (90)Sr in fission byproducts. The stoichiometry (or metal to ligand ratios) and overall complexation constant (β) for these reactions at these two interfaces are described herein. Two stoichiometries at the w|DCE interface were discovered, that is, [Sr(CMPO)(2)](2+) and [Sr(CMPO)(3)](2+) with β values of 4.5×10(19) and 5.5×10(25), respectively. Only one stoichiometry was observed at the w|RTIL interface: [Sr(CMPO)(3)](2+) with β equal to 1.5×10(34). The larger complexation constant for [Sr(CMPO)(3)](2+) at the w|RTIL interface than those found at the w|DCE interface supported the previous observation of a greater distribution ratio in the aqueous-RTIL metal extraction than that in the aqueous-alkane processing. The kinetics of the reactions at the w|RTIL interface was slow. The stoichiometries at the w|DCE interface were confirmed using biphasic electrospray ionization mass spectrometry (BESI-MS) as well as direct injection of Sr(2+) and CMPO mixture by means of a "shaking flask" experiment to conventional ESI-MS.     

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.     

Single Fusion Events at Polarized Liquid–Liquid Interfaces

A new electrochemical framework for tracking individual soft particles in solution and monitoring their fusion with polarized liquid–liquid interfaces is reported. The physicochemical principle lies in the interfacial transfer of an ionic probe confined in the particles dispersed in solution and that is released upon their collision and fusion with the fluid interface. As a proof‐of‐concept, spike‐like transients of a stochastic nature are reported in the current–time response of 1,2‐dichloroethane(DCE)|water(W) submilli‐interfaces after injection of DCE‐in‐W emulsions. The sign and potential dependence of the spikes reflect the charge and lipophilicity of the ionic load of the droplets. A comparison with dynamic light scattering measurements indicates that each spike is associated with the collision of a single sub‐picoliter droplet. This opens a new framework for the study of single fusion events at the micro‐ and nanoscale and of ion transport across biomimetic soft interfaces.     

Encapsulation of anilinonaphthalenesulfonates in carboxylate-terminated PAMAM dendrimer at the polarized water|1,2-dichloroethane interface

Molecular encapsulation of water-soluble anionic fluorescent dye molecules, 8-anilino-1-naphthalenesulfonic acid (ANS), and its bimolecular derivative (bis-ANS), in the generation 3.5 polyamidoamine (G3.5 PAMAM) dendrimer was investigated in the bulk aqueous phase and at the polarized water|1,2-dichloroethane interface. ANS(-) was electrostatically incorporated in the dendrimer, and the fluorescence enhancement with a blue shift of the emission maximum was observed at pH values <6, where the interior of the dendrimer was positively charged. The fluorescence enhancement of ANS was maximized around pH 3 and then decreased under more acidic conditions. The potential dependences of the molecular encapsulation and the interfacial mechanism were studied in detail by means of potential modulated fluorescence (PMF) spectroscopy. Under acidic conditions, the dendrimer incorporated ANS(-) at the positively polarized interface as well as in the aqueous phase. ANS(-) was released from the dendrimer at the intrinsic transfer potential and independently transferred across the interface. Bis-ANS exhibited relatively strong interaction with the dendrimer over a wide pH range (1 < pH < 8), and a negative shift of the transfer potential was observed under the corresponding pH condition. The PMF analysis clearly demonstrated that the interfacial mechanism of the dendrimer involves transfer and adsorption processes depending on the pH condition and the Galvani potential difference.     

Impact of an ionic surfactant on the ion transfer behaviors at meso-liquid/liquid interface arrays

The influence of ionic surfactants,cetyltrimethylammonium bromide(CTAB),self-assembled within silica-nanochannels of a hybrid mesoporous silica membrane(HMSM) on simple ion transfer(IT)behaviors at the meso-water/1,2-dichloroethane(W/DCE) interface arrays supported by such a HMSM was investigated by voltammetry for the first time.Significantly,it is found that the CTAB in HMSM can dramatically enhance the peak-current responses corresponding to ITs of some anions and even lower their Gibbs transfer energies from W to DCE,which could be ascribed to an anion-exchange process between anions and the bromide of CTAB associated with partial ion-dehydration induced by the CTAB.This work will provide a new strategy to study anion transfer processes and improve the electroanalytical performance for anion detection at the liquid/liquid interface.     

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.     

Unfolding dynamics of cytochrome c revealed by single-molecule and ensemble-averaged spectroscopy

Denaturant-induced conformational change of yeast iso-1-cytochrome c (Cytc) has been comprehensively investigated in the single-molecule and bulk phases. By fluorescence-quenching experiments with dye-labelled heme-protein (Alexa 488-labelled Cytc, Cytc-A488), we clearly show that the fluorescence quenching observed from folded Cytc-A488 is due mainly to photoinduced electron transfer (PET) between electron-donating amino acids such as tryptophan and the dye attached to the protein. In addition, the unfolding process of Cytc-A488 observed in the single-molecule and bulk phases can be explained well in terms of a three-state model: Cytc unfolds through an intermediate with a native-like compactness. By quantitative analysis of fluorescence correlation spectroscopy (FCS) data, we were able to observe a relaxation time of ～1.5 μs corresponding to segmental motion and fast folding dynamics of 55 μs in the unfolded state of Cytc. The results presented here also suggest that a combination of single-molecule and ensemble-averaged spectroscopy is necessary to provide convincing and comprehensive assignments of protein kinetics.     

立方体溴化银颗粒光谱增感的电子转移超快动力学研究

本文利用皮秒时间分辨条纹相机技术检测了3种染料在立方颗粒溴化银上吸附后形成聚集体的荧光光谱,分析了3种染料在不同染料浓度下对染料聚集体到溴化银导带的超快电子转移过程的影响,进而分析其对增感效率的影响关系,并探讨了增感过程的微观机理.实验结果表明,荧光衰减的动力学曲线与一个双指数函数拟合得相当好,存在一快一慢两个衰减成分,快衰减成分占拟合较大比例,表明其源于与荧光衰减相竞争的从激发态染料聚集体到AgBr导带的电子转移.光致电子转移的速率及增感效率随着染料相对浓度的增加表现出一定的变化趋势,染料浓度增加,增感效率减小.     

Luminescence Spectroscopy of Rhodamine Homodimer Dications in Vacuo Reveals Strong Dye-Dye Interactions

Being alone or together makes a difference for the photophysics of dyes but for ionic dyes it is difficult to quantify the interactions due to solvent screening and nearby counter ions. Gas-phase luminescence experiments are desirable and now possible based on recent developments in mass spectrometry. Here we present results on tailor-made rhodamine homodimers where two dye cations are separated by methylene linkers, (CH₂)_n. In solution the fluorescence is almost identical to that from the monomer whereas the emission from bare cation dimers redshifts with decreasing n. In the absence of screening, the electric field from the charge on one dye is strong enough to polarize the other dye, both in the ground state and in the excited state. An electrostatic model based on symmetric dye responses (equal induced-dipole moments in ground state) captures the underlying physics and demonstrates interaction even at large distances. Our results have possible implications for gas-phase Förster Resonance Energy Transfer.     

有机染料敏化太阳电池中激发态弛豫和电子注入的超快光谱研究

为了实现窄能隙有机光敏剂的理性设计,有必要全面理解发生在二氧化钛/染料/电解质复杂界面的激发态演化动力学。本文通过构建分别以苯并噻二唑-苯甲酸(BTBA)和吡啶并噻二唑-苯甲酸(PTBA)为电子受体的有机给受体染料,借助超快瞬态吸收光谱测量与理论模拟,我们发现在实际的二氧化钛/染料/电解质界面存在激发态多步弛豫与多态电子注入的过程。密度泛函理论及含时密度泛函理论计算表明,二氧化钛表面的光激发产生的"热"激发态染料分子会通过分子片段间的扭转运动发生显著的多步结构弛豫,最终形成共轭骨架具有醌式结构、更加平面化的平衡构型。通过对飞秒瞬态吸收光谱进行目标分析,我们发现相对于以苯并噻二唑-苯甲酸为电子受体的染料,以吡啶并噻二唑-苯甲酸为电子受体的染料呈现出较慢的电子注入速率与较短的激发态寿命,导致总的电子注入产率较低,给出了基于该染料所制备的太阳电池的外量子产率峰值低的原因。     

Eosin y triplet state as a probe of spatial heterogeneity in microcrystalline cellulose

The photophysical behavior of eosin Y adsorbed onto microcrystalline cellulose was evaluated by reflectance spectroscopy, steady-state fluorescence spectroscopy and laser induced time-resolved luminescence. On increasing the concentration of the dye, small changes in absorption spectra, fluorescence redshifts and fluorescence quenching are observed. Changes in absorption spectra point to the occurrence of weak exciton interactions among close-lying dye molecules, whereas fluorescence is affected by reabsorption and excitation energy trapping. Phosphorescence decays are concentration independent as a result of the negligible exciton interaction of dye pairs in the triplet state. Lifetime distribution and bilinear regression analyses of time-resolved phosphorescence and delayed fluorescence spectra reveal the existence of two different environments: long-lived, more energetic triplet states arise from dyes tightly entrapped within the cellulose chains, while short-lived, less-energetic states result from dyes in more flexible environments. Stronger hydrogen bond interactions between the dye and cellulose hydroxyl groups lead in the latter case to a lower triplet energy and faster radiationless decay. These effects, observed also at low temperatures, are similar to those encountered in several amorphous systems, but rather than being originated in changes in the environment during the triplet lifetime, they are ascribed in this case to spatial heterogeneity.     

Photosensitization mechanisms in photopolymer coating film containing photoinitiators sensitized by aminochalcone‐type dye for computer‐to‐photopolymer plate

Photosensitization mechanisms in photopolymer coating film containing an aminochalcone‐type dye sensitizer and a radical generating reagent, sensitizer dyes, (E)‐3‐(9‐julolidinyl)‐1‐phenyl‐2‐propen‐1‐one (A), (E)‐2‐(9‐julolidinyl)‐methylene‐1‐indanone (B), 9‐benzoyl‐2,3,6,7‐tetrahydro‐1H,5H‐benzo[i,j]‐furano‐[3,2‐g]quinolizine (C), 4‐(dimethylamino) chalcone (D) and a radical‐generating reagent, 2,4,6‐tris (trichloromethyl)‐1,3,5‐triazine (TCT), were investigated by laser flash photolysis using a total reflection cell. Weak fluorescence and strong broad triplet absorption were detected. The fluorescence was statically quenched by TCT at quenching distances (R_f) of 15, 14, 20 and 14 Å for A, B, C and D as well as the triplet initial absorption, at quenching distances (R_t) of 16, 16, 16 and 14 for A, B, C and D, similar to the fluorescence quenching distances. The triplet decay time of the dyes was inefficiently quenched by TCT with the rate constants (k _q) of 1.9, 3.1, 0.7 and 1.0×10⁵ mol⁻¹/dm³/s for A, B, C and D. The sensitivity of photopolymers containing a sensitizer dye and a TCT was obtained at an excitation of 488 nm corresponding to the emission peaks of argon ion laser of 1.1, 0.2, 0.54 and 9.1 mJ cm² for A, B, C and D. The results indicated that the static sensitization process from the fluorescent singlet excited state of the dyes to the ground state of TCT was predominant, and the high sensitivity for A and B was caused by the high absorbance at 488 nm and that for C by the high fluorescent quenching distance. Copyright © 1999 John Wiley & Sons, Ltd.     

Carbocyanine dyes as efficient reversible single-molecule optical switch

We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher beta-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of >90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.