Cyclo-Ketal Xanthene Dyes: A New Class of Near-Infrared Fluorophores for Super-Resolution Imaging of Live Cells

Newly emerging super-resolution imaging techniques provide opportunities for precise observations on cellular microstructures. However, they also impose severe demands on fluorophores. Here, we develop a new series of NIR xanthene dyes, named as KRh s, by replacing the 10-position O of rhodamines with a cyclo-ketal. KRh s display an intense NIR emission peak at 700 nm with fluorescence quantum yields up to 0.64. More importantly, they, without the aid of enhancing buffer, exhibit stochastic fluorescence off–on switches to support time-resolved localization of single fluorophore. KRh s are functionalized into KRh-MitoFix , KRh-Mem and KRh-Halo that demonstrate mitochondria, plasma membrane and fusion protein targeting ability, respectively. Consequently, these KRh probes demonstrate straightforward usage for super-resolution imaging of these targets in live cells. Therefore, KRh s merit future development for fluorescence labeling and super-resolution imaging in the NIR region.     

Surface-Enhanced Raman Scattering of Rhodamine 123 in Silver Hydrosols and in Langmuir-Blodgett Films on Silver Islands

Surface-enhanced Raman scattering(SERS) of the Rhodamine 123 (Rh 123) molecule on ion-induced silver colloids has been studied. A time-dependent study of the SER spectra at a particular pH confirms charge transfer interaction between the probe molecule and the metal. The SER spectra of Rh 123 in Ag sol is compared with that of the molecules organized in a monolayer on silver island films by the Langmuir-Blodgett (LB) technique. The origin of high SERS activity of Rh 123 molecules in a monolayer on a silver island film is shown to be due to physisorption whereas in the ion-induced colloidal SERS both physisorption and chemisorption machanisms are involved. From these results, the contribution of charge transfer interaction to SERS in Ag sol has been estimated. In monolayer SERS, all the in-plane and out-of-plane (of xanthene ring) modes are more or less equally enhanced. This indicates that the xanthene plane of Rh 123 molecule organized in a LB film is oriented neither flat nor perpendicular to the silver island surface but is tilted. Copyright 2001 Academic Press.     

吨染料与反胶束体系中CdS的光敏电子转移

用ＡＯＴ／异辛烷／水反胶束体系制备出了不同尺寸大小的纳米ＣｄＳ,并以曙红（ＥＯ）和孟加拉玫瑰红（ＲＢ）为探针分子,研究了它们与硫化镉微粒间的光致电荷转移相互作用,用光照实验和ＥＳＲ技术研究了这些染料向ＣｄＳ的导带注入电子的过程并讨论了作用机理     

镉(Ⅱ)-碘化物-碱性呫吨染料体系的倍频散射和二级散射及其 分析应用

在稀磷酸介质中,当镉(Ⅱ)与过量Ⅰ^-离子形成[CdI4]^2-配阴离子并进一步与罗丹明B(RhB)、罗丹明6G(Rh6G)、乙基罗丹明B(ERhB)和丁基罗丹明B(BRhB)等碱性呫吨染料(BXD)形成离子缔合配合物[BXD]2[CdI4]时,在产生强烈共振瑞利散射(RRS)的同时,也会产生强烈的倍频散射(FDS)和明显的二级散射(SOS)。在一定条件下散射强度与镉的浓度成正比。方法有高灵敏度和较好的选择性,可用于纯锌中痕量镉的测定。文中对于FDS和SOS产生的原因进行了初步的探讨。     

Fluorescence lifetimes and quantum yields of rhodamine derivatives: new insights from theory and experiment

Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S(0) ← S(1) de-excitation, and to the xanthene/phenyl relative orientation assumed in the S(1) minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors.     

Heteroatom-substituted rhodamine dyes: Structure and spectroscopic properties

Rhodamine is one class of most popular dyes used in fluorescence imaging due to the outstanding photoproperties including high brightness and photostability. In recent years, replacement the xanthene oxygen with other elements, especially silicon, has attracted great attentions in the development of new rhodamine derivatives. This review summarized the structures and photophysical properties of heteroatom-substituted rhodamines. We hope this review can help to understand the structure-property relationships of rhodamine dyes and then elucidate the way to create derivatives with improved photoproperties.     

Nanostructured Polymeric Micelles Carrying Xanthene Dyes for Photodynamic Evaluation

It was evaluated the properties of the xanthene dyes Erythrosin B, Eosin Y and theirs Methyl, Butyl and Decyl ester derivatives as possible photosensitizers (PS) for photodynamic treatments. The more hydrophobic dyes self‐aggregate in water/ethanol solutions above 70% water (vol/vol) in the mixture. In buffered water, these PS were encapsulated in Pluronic polymeric surfactants of P‐123 and F‐127 by two methodologies: direct addition and the thin‐film solid dispersion methods. The thin‐film solid method provided formulations with higher stabilities besides effective encapsulation of the PS as monomers. Size measurements demonstrated that Pluronic forms self‐assembled micelles with uniform size, which present slightly negative surface potential and a spherical form detected by TEM microscopy. The ester length modulates xanthene localization in the micelle, which is deeper with the increase in the alkyl chain. Moreover, some PS are distributed into two populations: one on the corona micelle interface shell (PEO layer) and the other into the core (PPO region). Although all PS formulations show high singlet oxygen quantum yield, promising results were obtained for Erythrosin B esters with the hydrophobic P‐123, which ensures their potential as drug for clinical photodynamic applications.     

Heteroatom-substituted rhodamine dyes: Structure and spectroscopic properties

This review summarized the structures and photophysical properties of heteroatom-substituted rhodamines.     

PHOTODYNAMIC INACTIVATION OF SARCOPLASMIC RETICULUM VESICLE MEMBRANES BY XANTHENE DYES

Abstract— Photosensitized oxidation of sarcoplasmic reticulum (SR) vesicle membranes by a series of xanthene dyes was investigated. With increasing dye concentration and illumination time, the calcium ion uptake, ATPase activity and UV fluorescence intensity of SR decreased, and the absorbance at 241 nm increased. In xanthene dyes, the order of inactivation of the calcium uptake and ATPase activity of SR was in accord with decreasing order, of fluorescence intensity and increasing order of absorbance at 241 nm. Some regular relationships exist between the molecular structures of xanthene dyes and the photodynamic inactivation of SR membranes.     

Quantitative assessment of rhodamine spectra

With the development of single-molecule detection and super-resolution fluorescence imaging, rhodamine dyes gain new life. Through the modification of the N-substituents and the replacement of the oxygen atom in xanthene, the wavelength and brightness can be effectively changed. However, the spectra of rhodamine, especially due to the balance between ring-closed non-fluorescent lactone and ring-opened fluorescent zwitterion/cation, are sensitive to interference from various environmental factors. In this way, the spectral data of various rhodamines reported by different research groups under different test conditions lacked comparability, sometimes even lacked accuracy. In order to meet the requirements for the accuracy and uniformity of spectral data in the research of single molecule imaging and dye structure-fluorescence relationship study, we have tested the spectra of fifteen rhodamine dyes that cover the visible and near-infrared regions under exactly the same conditions. By studying the dependence of the spectra on dye concentrations, it was confirmed that 1 μmol/L was ideal for detection less from the interference of dye molecule aggregation. We provide comprehensive and reliable spectral data of these fifteen dyes, which are expected to be used as references for future research. And the direct comparison of different rhodamine spectra would help to understand the structure-fluorescence relationship of rhodamines.     

Rhodamine-inspired far-red to near-infrared dyes and their application as fluorescence probes

Probes to dye for: Rhodamine-inspired Si-pyronine, Si-rhodamine, Te-rhodamine, and Changsha NIR dyes have been developed recently. These dyes show fluorescence in the far-red to near-infrared region, while retaining the advantages of the original rhodamines, such as high fluorescence quantum yield, tolerance to photobleaching, good water solubility, and exhibit great potential for biological application.     

Polyunsaturated fatty acids in lipid bilayers: intrinsic and environmental contributions to their unique physical properties.

Polyunsaturated lipids are an essential component of biological membranes, influencing order and dynamics of lipids, protein-lipid interaction, and membrane transport properties. To gain an atomic level picture of the impact of polyunsaturation on membrane properties, quantum mechanical (QM) and empirical force field based calculations have been undertaken. The QM calculations of the torsional energy surface for rotation about vinyl-methylene bonds reveal low barriers to rotation, indicating an intrinsic propensity toward flexibility. Based on QM and experimental data, empirical force field parameters were developed for polyunsaturated lipids and applied in a 16 ns molecular dynamics (MD) simulation of a 1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC) lipid bilayer. The simulation results are in good agreement with experimental data, suggesting an unusually high degree of conformational flexibility of polyunsaturated hydrocarbon chains in membranes. The detailed analysis of chain conformation and dynamics by simulations is aiding the interpretation of experimental data and is useful for understanding the unique role of polyunsaturated lipids in biological membranes. The complete force field is included as Supporting Information and is available from http://www.pharmacy.umaryland.edu/faculty/amackere/research.html.     

New Fluorinated Rhodamines for Optical Microscopy and Nanoscopy

New photostable rhodamine dyes represented by the compounds 1 a – r and 3 – 5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N′,N‐bis(2,2,2‐trifluoroethyl) derivatives 1 e , 1 i , 1 j , 3 ‐H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4′ and 5′. Two fluorine atoms were introduced into the positions 2′ and 7′ of the 3′,6′‐diaminoxanthene fragment in compounds 1 a – d , 1 i – l and 1 m – r . The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512–554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited‐state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO₃ in H₂SO₄ is compatible with the secondary amide bond (rhodamine‐CON(Me)CH₂CH₂COOH) formed with MeNHCH₂CH₂COOCH₃ to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very‐high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live‐cell‐tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments.     

Xanthene dyes induce membrane permeabilization of bacteria and erythrocytes by photoinactivation

We analyzed the photoinactivation of the membrane functions of bacteria and erythrocytes induced by xanthene dyes. The dyes tested were rose bengal, phloxine B, erythrosine B and eosin B. These dyes induced the leakage of K(+) from Staphylococcus aureus cells within minutes of photoirradiation, in the order of rose bengal > phloxine B > erythrosine B > eosin B. The ability of dyes to inhibit respiration was weak, except for rose bengal, and the dyes dissipated the membrane potential in similar time traces with changes in K(+) permeability. The xanthene dyes also induced the leakage of K(+) from bovine erythrocytes upon photoirradiation in the same order as that observed with bacteria. Furthermore, we found that the ability to cause the leakage of K(+) from erythrocytes was associated with dye-induced morphological changes, forming a crenated form from the normal discoid. These results are discussed in connection with the ability of xanthene dyes to generate singlet oxygen and bind to bacterial cells, and further compared with the actions of cationic porphyrins, which induced photoinactivation of bacteria through respiratory inhibition.     

Interfacing Click Chemistry with Automated Oligonucleotide Synthesis for the Preparation of Fluorescent DNA Probes Containing Internal Xanthene and Cyanine Dyes

Double‐labeled oligonucleotide probes containing fluorophores interacting by energy‐transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2′‐O‐propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid‐phase copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy‐transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40–110 nm), quenched fluorescence of single‐stranded probes accompanied by up to 7.7‐fold light‐up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single‐nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM ).     

Spectral properties and orientation of voltage-sensitive dyes in lipid membranes

Voltage-sensitive dyes are frequently used for probing variations in the electric potential across cell membranes. The dyes respond by changing their spectral properties: measured as shifts of wavelength of absorption or emission maxima or as changes of absorption or fluorescence intensity. Although such probes have been studied and used for decades, the mechanism behind their voltage sensitivity is still obscure. We ask whether the voltage response is due to electrochromism as a result of direct field interaction on the chromophore or to solvatochromism, which is the focus of this study, as result of changed environment or molecular alignment in the membrane. The spectral properties of three styryl dyes, di-4-ANEPPS, di-8-ANEPPS, and RH421, were investigated in solvents of varying polarity and in model membranes using spectroscopy. Using quantum mechanical calculations, the spectral dependence of monomer and dimer ANEPPS on solvent properties was modeled. Also, the kinetics of binding to lipid membranes and the binding geometry of the probe molecules were found relevant to address. The spectral properties of all three probes were found to be highly sensitive to the local environment, and the probes are oriented nearly parallel with the membrane normal. Slow binding kinetics and scattering in absorption spectra indicate, especially for di-8-ANEPPS, involvement of aggregation. On the basis of the experimental spectra and time-dependent density functional theory calculations, we find that aggregate formation may contribute to the blue-shifts seen for the dyes in decanol and when bound to membrane models. In conclusion, solvatochromic and other intermolecular interactions effects also need to be included when considering electrochromic response voltage-sensitive dyes.     

Silicon functionalization expands the repertoire of Si-rhodamine fluorescent probes

Fluorescent dyes such as rhodamines are widely used to assay the activity and image the location of otherwise invisible molecules. Si-rhodamines, in which the bridging oxygen of rhodamines is replaced with a dimethyl silyl group, are increasingly the dye scaffold of choice for biological applications, as fluorescence is shifted into the near-infrared while maintaining high brightness. Despite intense interest in Si-rhodamines, there has been no exploration of the scope of silicon functionalization in these dyes, a potential site of modification that does not exist in conventional rhodamines. Here we report a broad range of silyl modifications that enable brighter dyes, further red-shifting, new ways to modulate fluorescence, and the introduction of handles for dye attachment, including fluorogenic labeling agents for nuclear DNA, SNAP-tag and HaloTag labeling. Modifications to the bridging silicon are therefore of broad utility to improve and expand the applications of all Si-dyes.

Functionalization of the bridging silicon atom of Si-rhodamine dyes allows tuning of dye performance, the attachment of sensors, and the addition of biomolecular targeting ligands useful for the construction of live cell imaging probes.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Modeling the electrostatic potential of asymmetric lipopolysaccharide membranes: The MEMPOT algorithm implemented in DelPhi

Four chemotypes of the rough lipopolysaccharides (LPS) membrane from Pseudomonas aeruginosa were investigated by a combined approach of explicit water molecular dynamics (MD) simulations and Poisson–Boltzmann continuum electrostatics with the goal to deliver the distribution of the electrostatic potential across the membrane. For the purpose of this investigation, a new tool for modeling the electrostatic potential profile along the axis normal to the membrane, MEMbrane POTential (MEMPOT), was developed and implemented in DelPhi. Applying MEMPOT on the snapshots obtained by MD simulations, two observations were made: (a) the average electrostatic potential has a complex profile but is mostly positive inside the membrane due to the presence of Ca²⁺ ions, which overcompensate for the negative potential created by lipid phosphate groups; and (b) correct modeling of the electrostatic potential profile across the membrane requires taking into account the water phase, while neglecting it (vacuum calculations) results in dramatic changes including a reversal of the sign of the potential inside the membrane. Furthermore, using DelPhi to assign different dielectric constants for different regions of the LPS membranes, it was investigated whether a single frame structure before MD simulations with appropriate dielectric constants for the lipid tails, inner, and the external leaflet regions, can deliver the same average electrostatic potential distribution as obtained from the MD‐generated ensemble of structures. Indeed, this can be attained by using smaller dielectric constant for the tail and inner leaflet regions (mostly hydrophobic) than for the external leaflet region (hydrophilic) and the optimal dielectric constant values are chemotype‐specific. © 2014 Wiley Periodicals, Inc.     

Triangular Rhodamine Triads and Their Intrinsic Photophysics Revealed from Gas-Phase Ion Fluorescence Experiments

When ionic dyes are close together, the internal Coulomb interaction may affect their photophysics and the energy-transfer efficiency. To explore this, we have prepared triangular architectures of three rhodamines connected to a central triethynylbenzene unit (1,3,5-tris(buta-1,3-diyn-1-yl)benzene) based on acetylenic coupling reactions and measured fluorescence spectra of the isolated, triply charged ions in vacuo. We find from comparisons with previously reported monomer and dimer spectra that while polarization of the π-system causes redshifted emission, the separation between the rhodamines is too large for a Stark shift. This picture is supported by electrostatic calculations on model systems composed of three linear and polarizable ionic dyes in D_3h configuration: The electric field that each dye experiences from the other two is too small to induce a dipole moment, both in the ground and the excited state. In the case of heterotrimers that contain either two rhodamine 575 (R575) and one R640 or one R575 and two R640, emission is almost purely from R640 although the polarization of the π-system expectedly diminishes the dipole-dipole interaction.