Electrochromic Dye Research Articles

Fluorescent Probes for Lipid Membranes: From the Cell Surface to Organelles

Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. The development of chemical tools─fluorescent probes─for the sensing and imaging of biomembranes is a rapidly growing research direction, stimulated by a high demand from cell biologists and biophysicists. This Account focuses on advances in these smart molecules, providing a voyage from the cell frontier─plasma membranes (PM)─toward intracellular membrane compartments─organelles. General classification of the membrane probes can be based on targeting principles, sensing profile, and optical response. Probes for PM and organelle membranes are designed based on multiple targeting principles: conjugation with natural lipids or synthetic targeting ligands and in situ cell labeling by bio-orthogonal chemistry, conjugation to protein tags, and receptor-ligand interactions. Thus, to obtain membrane probes targeting PM with selectivity to one leaflet, we designed membrane anchor ligands based on a charged group and an alkyl chain. According to the sensing profile, we define basic membrane markers with constant emission and probes for biophysical and chemical sensing. The markers are built from classical fluorophores, exemplified by a series of bright cyanines and BODIPY dyes bearing the PM anchors (MemBright). Membrane probes for biophysical sensing are based on environment-sensitive fluorophores: (1) polarity-sensitive solvatochromic dyes; (2) viscosity-sensitive fluorescent molecular rotors; (3) mechanosensitive fluorescent flippers; and (4) voltage-sensitive electrochromic dyes. Our solvatochromic probes based on Nile Red (NR12S, NR12A, NR4A), Laurdan (Pro12A), and 3-hydroxyflavone (F2N12S) through polarity-sensing can visualize liquid ordered and disordered phases of lipid membranes, sense lipid order and its heterogeneity in cell PM, detect apoptosis, etc. Chemically sensitive probes, combining a dye, membrane-targeting ligand, and molecular recognition unit, enable the detection of pH, ions, redox species, lipids, and proteins at the biomembrane surface. In terms of the optical response profile, we can identify (1) fluorogenic (turn-on) probes, allowing background-free imaging; (2) ratiometric probes, e.g., solvatochromic probes, which enable ratiometric imaging by changing their emission/excitation color; (3) fluorescence lifetime-responsive probes, e.g., fluorescence molecular rotors and flippers, suitable for fluorescence lifetime imaging (FLIM); and (4) switchable probes, important for single-molecule localization microscopy. We showed that combining solvatochromic probes with on-off switching through a reversible binding specifically to cell PM enables the mapping of their biophysical properties with superior resolution. While the majority of efforts have been focused on PM, the probes for cellular organelles, such as endoplasmic reticulum, mitochondria, Golgi apparatus, etc., emerge rapidly. Thus, nontargeted solvatochromic probes can distinguish organelles by the emission color. Targeted solvatochromic probes based on Nile Red revealed unique signatures of polarity and lipid order of individual organelles and their different sensitivities to oxidative or mechanical stress. Lipid droplets, which are membraneless lipidic structures, constitute another interesting organelle target for probing the cell stress. Currently, we stand at the beginning of a long route with big challenges ahead, in particular (1) to achieve superior organelle specificity; (2) to label specific biomembrane leaflets, notably the inner leaflet of PM; (3) to detect lipid organization in a proximity of specific proteins; and (4) to probe biomembranes in tissues and animals.

Spectroelectrochemical and Theoretical Study of [Si(ttpy)2](PF6)4: A Potential Polychromatic Electrochromic Dye

Complexes consisting of earth-abundant main group metals such as silicon with polypyridine ligands are of interest for a variety of optical and electronic applications including as electrochromic colorants. Previous spectroelectrochemical studies with tris(2,2'-bipyridyl)silicon(IV) hexafluorophosphate, [Si(bpy)3](PF6)4, demonstrated an ability to control the color saturation of the potential electrochromic dye, with the intensity of the dye's green color increasing as the charge state sequentially reduces from 4+ to 1+. In this study, the synthesis of bis(4'-(4-tolyl)-2,2':6',2″-terpyridine)silicon(IV) hexafluorophosphate, [Si(ttpy)2](PF6)4, is reported along with electrochemical and spectroelectrochemical analyses. Computational modeling (density functional theory) is used to further elucidate the electrochromic properties of previously reported Si(bpy)3n+ species and the new Si(ttpy)2n+ species. While the homoleptic tris(bidentate)silicon(IV) complexes are attractive as electrochromic dyes for tunable color saturation, the bis(tridentate)silicon(IV) complexes are attractive as polychromatic electrochromic dyes.

Electrochromic Device Demonstrator from Household Materials.

Electrochromism encompasses reversible changes of material’s optical properties (color, opacity) under the influence of an external electric current or applied voltage. The effect has been known for decades, but its importance continues to grow due to the rapid development of smart systems and the accompanying demand to build devices that consume less power. Most commercial electrochromic devices (ECDs) require sophisticated chemicals and advanced material preparation techniques. Also, the demonstration of electrochromism in chemistry classes mainly uses expensive WO3 films, intrinsically conductive polymers, and/or optically transparent electrodes (OTEs). The aim of this article is to present a simple and fast educational method to build ECDs from household materials without the need for OTEs: unsharpened kitchen knives are used as electrodes, curcumin from turmeric is used as the electrochromic dye, and baking soda is used as the electrolyte. The laboratory experiments presented will help students gain a deeper understanding of the fundamentals of electrochemistry (electrolysis, pH change) and electrochromism (in our case, color changes due to pH-induced keto-enol tautomerism of curcumin).

Intercalation-free, fast switching of mesoporous antimony doped tin oxide with cathodically coloring electrochromic dyes.

Mesoporous nanoparticle layers of transparent conductive oxides (TCOs) with anchored organic dyes are of great interest for electrochromic applications. Herein, we prepared mesoporous layers of antimony doped tin oxide (ATO) consisting of only 5 nm large particles with a low Sb concentration (2% antimony). The particles were prepared via a modified synthesis procedure based on hexahydroxostannate and pure Sb(v) hexahydroxoantimonate(v). We show that the ATO layers benefit from using a non-intercalating electrolyte such as tetrabutylammonium perchlorate (TBAP) compared to lithium perchlorate. Especially in the negative potential range, negative side effects, such as degradation due to lithium intercalation, are reduced. Furthermore, comparing the behavior of particles with varying antimony doping concentrations showed that the particles doped with 2% Sb are most suitable with respect to their conductivity and transparency. When modified with an electrochromic dye (viologen), the hybrid electrodes allow fully reversible (de)coloration with the non-intercalating electrolyte. Similar viologen/TiO2 electrodes on the other hand show severely restricted performance with the non-intercalating electrolyte as the oxidation of the dye is partially inhibited. Finally, we built a full electrochromic device composed of two ATO electrodes, each bearing a different electrochromic dye with TBAP as the electrolyte. Despite the dense morphology of the layers due to the small particle size as well as the large size of the electrolyte cation, the device displays remarkable switching times below 0.5 s.

Redox-induced reversible [2 + 2] cycloaddition of an etheno-fused diporphyrin.

3,5-Ethenoporphyrin is a π-extended porphyrin containing a fused ethene unit between the meso- and β-positions, exhibiting unique contribution of macrocyclic antiaromaticity. We have recently reported that its analogue, etheno-fused diporphyrin, underwent thermal [2 + 2] cycloaddition to furnish X-shaped cyclobutane-linked tetraporphyrins. Here we demonstrate that the cyclobutane-ring formation is dynamically redox-active. Namely, the tetraporphyrin underwent two-step four-electron oxidation to afford two etheno-fused diporphyrin dications. The reduction of the resulting dication regenerated the cyclobutane-linked tetraporphyrin. The dication was sufficiently stable to allow its isolation under ambient conditions. The structure of the dication has been confirmed by 1H NMR spectroscopy and X-ray diffraction analysis. Importantly, the simultaneous double C–C bond cleavage in the cyclopropane ring in the tetraporphyrin is exceptional among dynamic redox (dyrex) systems to achieve large structural changes, thus offering new insights for the design of novel redox-active functional organic materials for electrochromic dyes, organic batteries, and organic memories.

Evidence for ATP Interaction with Phosphatidylcholine Bilayers

ATP is a fundamental intracellular molecule and is thought to diffuse freely through the cytosol. However, evidence obtained from nucleoside-sensing sarcolemmal ion channels and red blood cells suggest that ATP is compartmentalised or buffered, especially beneath the sarcolemma. Macromolecular crowding of the cytosolic compartment, tortuosity of cytoskeletal structures and non-hydrolysing binding sites on cytoskeletal proteins are some of the physical barriers proposed that could potentially reduce free diffusion of adenine nucleotides through the cytosol. In this study we compared ATP and adenosine using a spectroscopic study of RH421, a membrane bound electrochromic dye, impedance spectroscopy measurements with tethered phospholipid bilayers and differential scanning calorimetry to determine the interaction with zwitterionic phospholipid bilayers. We report a concentration dependent shift of the excitation spectrum of RH421 indicating an interaction between ATP and DOPC unilammelar vesicles, with little effect observed from adenosine. This was supported by a concentration dependent change in the calculated membrane conductance of tethered phospholipid bilayers with ATP and not adenosine. Concentration dependent alterations to the measured enthalpy of the DMPC main phase transition with ATP was also observed, which weren’t replicated with adenosine. These observations provide strong evidence for the interaction of ATP with zwitterionic phospholipid bilayers at physiologically relevant concentrations. An interaction between ATP and the phospholipid bilayer could create a population of ATP molecules with a divergent diffusion rate with respect to “free” ATP and thus establish spatially distinct areas of compartmentalised ATP beneath the sarcolemma.

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens.

P1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn2+ and toxic Cd2+, but not at deficient Zn2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn2+ and AhHMA4 in Zn2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn2+ when compared to replete Zn2+ and toxic Cd2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn2+ supply, but were up-regulated by 10 μM Cd2+. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri.

Structure-Dependent Response Characteristics of Electrochromic Dye-Modified Transparent Conductive Oxide Porous Electrode

Fast-response electrochromic (EC) cell composed of EC dye-modified TiO2-nanoparticle-based transparent porous electrode was reported by R. Cinnsealach et al. (Sol. Energy Mater. Sol. Cells, 55, 215, 1998). From then, TiO2 porous electrodes modified with EC dyes have been regarded as a promising candidate for fast-response EC display (ECD) applications. Our research purpose is the realization of flexible fast-response ECD applications. However, TiO2 porous electrodes require high sintering temperature (>400 °C) to use as EC electrode because TiO2 has high resistivity. This high process temperature required by TiO2 porous electrode was an important obstacle for their application as transparent electrodes in plastic substrate-based flexible ECDs. To solve this problem, we substituted transparent conductive oxides (TCO) for TiO2 as the porous electrode material. TCO materials have high electrical conductivity than TiO2, so they have the potential to work as the EC electrode even if they are sintered at low temperature. Previously, we reported that the porous electrodes consisting of indium tin oxide (ITO) nanoparticles exhibit good optical transparency and high electric conductivity. Moreover, EC response characteristics of EC-dye modified ITO porous electrode sintered at 150 °C was improved than that of conventional TiO2 porous electrode due to its high conductivity (AIP Adv., 6, 065121, 2016). Therefore, TCO porous electrodes have potential for application in porous electrode for flexible ultrafast-response ECDs. To achieve such applications, it is necessary to analyze the factors controlling the EC response speed in TCO porous electrodes. In this study, we investigated the response characteristics of EC dye-modified ITO, antimony-doped tin oxide (ATO), and TiO2 porous electrodes. ITO and ATO porous films have low electric resistivity due to their high electric conductivity. As mentioned above, ITO porous electrode exhibited fast response. On the other hand, low-resistivity ATO porous electrode exhibited slower response than that of a conventional high-resistivity TiO2 porous electrode. This clearly indicates that the EC response speed of the ATO porous electrode is dependent on a factor other than the electric resistance of the porous film. From the structural comparisons, it is revealed that the pore size of the film consisting of small nanoparticles is one of the key factors controlling the EC response speed of porous electrode. The details about the effect of porous film structure on EC response characteristics will be discussed.

Can fused thiophene–pyrrole-containing rings act as possible new electrochromic dyes? A computational prediction

The computed electronic spectra of neutral, radical cation and dication species for a series of fused thiophene–pyrrole-containing ring systems clearly suggest that these molecules can be used as new electrochromic materials. The calculations have been performed in the framework of the time-dependent density functional linear response theory employing different exchange–correlation functionals. Based on our results, we can indicate the long-chain molecules with twelve and fourteen rings, not yet synthesized, as the best candidates for electrochromic devices operating in the near-infrared region. The calculated properties are in excellent agreement with the available experimental data.

Multicolored, Low-Power, Flexible Electrochromic Devices Based on Ion Gels.

Ion gels composed of a copolymer and a room temperature ionic liquid are versatile solid-state electrolytes with excellent features including high ionic conductivity, nonvolatility, easily tunable mechanical properties, good flexibility and solution processability. Ion gels can be functionalized by incorporating redox-active species such as electrochemiluminescent (ECL) luminophores or electrochromic (EC) dyes. Here, we enhance the functionality of EC gels for realizing multicolored EC devices (ECDs), either by controlling the chemical equilibrium between a monomer and dimer of a colored EC species, or by modifying the molecular structures of the EC species. All devices in this work are conveniently fabricated by a "cut-and-stick" strategy, and require very low power for maintaining the colored state [i.e., 90 μW/cm(2) (113 μA/cm(2) at -0.8 V) for blue, 4 μW/cm(2) (10 μA/cm(2) at -0.4 V) for green, and 32 μW/cm(2) (79 μA/cm(2) at -0.4 V) for red ECD]. We also successfully demonstrate a patterned, multicolored, flexible ECD on plastic. Overall, these results suggest that gel-based ECDs have significant potential as low power displays in printed electronics powered by thin-film batteries.

Role of protons in the pump cycle of KdpFABC investigated by time-resolved kinetic experiments.

The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 μM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P → P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).

Electrochromic and unique chiroptical properties of helically deformed tetraarylquinodimethanes generated from less-hindered dicationic precursors upon reduction

AbstractElectron-donating 1,1,4,4-tetraarylbutadiene is a representative electrochromic dye, and the same chromophore can be found in 9,10-bis(diarylmethylene)-9,10-dihydrophenanthrene (dibenzo-oQD) albeit in a fixed s-cis geometry. Unlike thermodynamically unstable 7,7,8,8-tetraaryl-o-quinodimethane, spontaneous electrocyclization is prohibited by dibenzo-annulation. Several derivatives of dibenzo-oQD were successfully generated despite highly strained geometry caused by steric hindrance between the bulky diarylmethylene units. Their precursors are phenanthrene-9,10-diyl bis(diarylmethylium) dyes (PDM2+) stabilized by four electron-donating alkoxy groups. The redox pairs of dibenzo-oQD/PDM2+ exhibit vivid change in color upon redox interconversion (electrochromism). Both dibenzo-oQD and PDM2+ adopt a helical conformation, whose configuration is unstable. When a chiral alkoxy substituent is attached on each of the aryl groups, the point chirality is successfully transmitted to helicity of PDM2+. Resulting diastereomeric biasing is the key for dibenzo-oQD/PDM2+ to exhibit the two-way-output response (e.g., UV–vis and CD). In addition, much more strained quinodimethane derivatives, 1,2-bis(diarylmethylene)acenaphthene and 1,16-diaryldibenzo[b,n]perylene, were also generated from the dicationic precursors, demonstrating that the reductive transformation can serve as a useful protocol to generate severely deformed π-conjugated systems.

Indocyanine Green is a Voltage-Sensitive Fluorescent Dye

Indocyanine green (ICG) is an infrared fluorescent dye with widespread use in clinical applications, including hepatic function tests, cardiac output monitoring, and ophthalmic angiography. Using Xenopus laevis oocytes under voltage clamp, we have found that ICG is voltage-sensitive and capable of responding to changes in cellular membrane potential with a roughly linear voltage dependency. ICG's voltage response does not display the sub-millisecond kinetics characteristic of electrochromic dyes; however, it is faster than many so-called slow-response voltage probes. Tests of oocytes performed under current clamp-like conditions show that ICG can clearly follow action potentials produced at approximately 70 Hz. ICG has low expected cytotoxicity; an infrared wavelength amenable to deep tissue imaging; and the ability to label excitable cells, including cardiac myocytes and neurons. Consequently, the voltage-sensitivity of ICG presented here may have significant implications for novel applications in research and the clinic. Support: NIH GM030376.

Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (lo) and liquid disordered (ld) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized.N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase.N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label ld domains in yeast membranes.4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to lo domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol–lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires

Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltage-sensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity.

Ionically conducting DNA-based membranes for eletrochromic devices

Ionically conductive membranes based on plasticized DNA with glycerol and containing conducting polymers such as: either PEDOT:PSS (poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)), POEA (poly (o-ethoxyaniline)) or electrochromic dye Prussian Blue (PB) were prepared, characterized and applied in electrochromic devices. The results of ionic conductivity measurements for the studied membranes varied, depending on the added polymer or dye, from 3 × 10 −5 S/cm for the pure DNA-based samples to 5.1 × 10 −4 S/cm for the samples containing PEDOT:PSS or PB, at room temperature. The samples exhibit a good thermal stability up to 170 °C, with variable transparency range, depending on the added molecule. Moreover, the samples exhibit also a semicrystalline structure and in the case of pure DNA- and DNA–PB-based compounds two glass transition temperatures of −75 °C and 60 °C were observed. The membranes were applied in small electrochromic devices and have shown an up to 15% transmission change from colored to bleached states. The inserted/extracted charges were 0.7–3.0 mC/cm 2 after 15 s of applied potential of −2.5 V for membranes with thickness of 72–80 μm. All obtained results indicate that DNA-based gel-polymer electrolytes (GPEs) are good candidates to be used in electrochemical devices.

Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ∼30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)β(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.

Action-Spectra of Electrochromic Voltage-Sensitive Dyes in an Intact Excitable Tissue

Voltage-sensitive dyes VSDs provide a spatially resolved optical read-out of electrical signals in excitable tissues. Several com- mon fluorescent VSDs display electrochromic shifts of their emission spectra, making them suitable candidates for ratiometric measure- ments of transmembrane voltages. These advantages of VSDs are tem- pered by tissue-specific shifts to their fluorescence emission. In addi- tion, the optimal electrochromic dye response occurs in wavelength bands distinct from the dye's maximal resting emission. This spectrum can undergo tissue-specific shifts as well. We have devel- oped a technique for in situ measurements of the action spectra of VSDs in intact excitable tissues. Fluorescence emission spectra of VSDs during action-potential depolarization were obtained within a single sweep of a spectrophotometer equipped with a change-coupled device CCD array detector. To resolve the subtle electrochromic shifts in voltage-induced dye emission, fluorescence emission spectra measured right before and during field-induced action-potential de- polarization were averaged over about 100 trials. Removing white- noise contributions from the spectrometer's CCD detector/amplifier via low-pass filtering in Fourier space, the action spectra of all dyes could be readily determined. © 2008 Society of Photo-Optical Instrumentation En-

Multi-Color Electrochromic Device Based on Organic Electrochromic Materials

Multi-color electrochromic (EC) device was fabricated on a patterned ITO glass. Organic electrochromic dyes were encapsulated within nano-sized porous TiO2 nanoparticles, and their relative electrochromic (EC) properties were determined using spectroelectrochemical method. Blue, green, and red colored organic dye doped TiO2 nanoparticles were coated on a patterned ITO substrate to afford multi-colored EC electrode, which was coated with a photo-curable polymer electrolyte and then covered with a bare ITO glass as a counter electrode. Upon photocuring of the electrolyte layer under UV light, an all-solid state multi-colored electrochromic device (ECD) was obtained. The resultant ECD showed reversible multi-color change within 8 sec and high optical contrast.

Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation

SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-binding sites in the P-E2 conformation of the Ca-ATPase. The open membrane fragments showed about 80% of the enzyme activity in the untreated membranes. Pump function was investigated by using electrochromic styryl dyes. The kinetic properties of cytoplasmic ion binding showed no significant differences between the Ca-ATPases in SR vesicles and in membrane fragments. From pH-dependent Ca2+ binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules. Major differences between Ca-ATPase from SR vesicles and membrane fragments were the respective fluorescence amplitudes. This effect is, however, produced by dye-lipid interaction and not by pump function. It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.