Fluorescence Probing of Cell Membranes with Azacrown Substituted Ketocyanine Dyes (original) (raw)
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Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-l-␣-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.
Electrochimica Acta, 2003
Potentiometric optical probes gain importance. Here, we report on the application of those probes to study transmembrane potentials using nonlinear optical (NLO) and fluorescence techniques. A NLO spectroscopy method is presented which allows for precise recording of membrane potential changes. To this end the signal intensity of second harmonic generation (SHG) mediated by novel acetylenic hemicyanine dye molecules embedded in the membrane of Retzius neuron cells was used. Bichromophoric systems have been designed for efficient energy transfer measurements. In many cells these bichromophores change their behaviour from stationary fast-response dyes to slow-response redistribution dyes. Completely novel potentiometric probes acting in a different and most efficient way were constructed using one aminostyrylpyridinium (ASP) bichromophoric dye and one oxonol dye. The redistribution ability of the ASP dye can be optimised by controlled and tailor-made modification of its substituents. In combination with the anionic oxonol dye DiBAC 4 (3) a bichromophoric system results with two chromophores showing opposite redistribution behaviour. Fluorescence ratio changes of 100% for a 100 mV depolarization can be recorded.
ABA-C 15 : A New Dye for Probing Solvent Relaxation in Phospholipid Bilayers
Langmuir, 2002
We synthesized and studied N-palmitoyl-3-aminobenzanthrone (ABA-C-15), which we proved to be an advantageous new fluorescent phospholipid membrane label. While the absorption of ABA-C-15 in protic solvents shows negative solvatochromism, its fluorescence emission is substantially red-shifted when the polarity of the solvent is increased. ABA-C15 is excitable by lasers emitting in the range between :390 and 190 nm; it exhibits reasonable quantum yields in protic solvents and binds with high affinity to small unilamellar phospholipid vesicles. Absorption, steady state fluorescence, and solvent relaxation data indicate that the aminobenzanthrone chromophore is located in the headgroup region of phospholipid bilayers in the liquid crystalline state of small unilamellar vesicles. The solvent relaxation kinetics probed by ABA-C-15 in the liquid crystalline state is characterized by three solvent relaxation times in the order of 0.05. 0.2, and 1.5 ns, respectively. We observed that the relative contribution of the 0.05 ns component and the overall Stokes shift became larger with increasing difference between the experimental temperature and the main phase transition temperature; this suggests that the chromophore becomes more accessible by water molecules. In the gel phase, a component faster than 30 ps significantly contributes to the solvent relaxation kinetics. However, the solvent relaxation on the nanosecond time seat(., appears to be slower than in the liquid crystalline phase. The shape and time evolution of the time-resolved emission spectra suggest that two distinct microenvironments of the dye might be responsible for the atypical solvent relaxation characteristics in the gel phase.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1994
Fluorescent amphiphilic hemicyanine dyes were adsorbed to the plasma membrane of isolated Retzius neurons of the leech. Voltage steps were applied to the neuron by the patch-clamp technique in whole-cell configuration. The change of fluorescence was observed as induced by the voltage jump. The relative changes of the excitation spectrum and of the emission spectrum of fluorescence were recorded. The complete set of spectral data for each dye was fitted by five parameters: shifts of the emission and the excitation spectrum, a change of fluorescence quantum yield and changes of the widths of the excitation and of the emission spectrum. The only common feature for all dyes was a blue-shift of the excitation spectrum and a drop of the yield when the neuron was stained from the outside and a positive voltage was applied to the inside. With respect to the shift of the emission spectrum and the changes of width qualitatively different results were obtained for different dyes. It is not attempted to assign a physical mechanism-probably a superposition of several mechanisms-of voltage-sensitivity.
Biochemistry, 1991
The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized positive charge. It formed under favorable conditions a concentration-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained constant. The membrane potential of energized mitochondria (negative inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to be pH independent within the physiological p H range of 7.1 5-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.
Cell Biochemistry and Biophysics, 2013
Two novel precursors of fluorescent dyes (PFD813 and PFD814) have been studied for their ability to photo-activation, transfer across the biomembrane and cells staining. The fluorescent dyes Rho813 and Rho814 formed by photo-activation of their precursors PFD813 and PFD814 inside cells were used for the optical detection of particular features in vitro (HaCat cells, human epithelial carcinoma A431, epidermoid carcinoma of the cervix HeLa and chinese hamster ovary CHO cells). One of the possibilities to visualize and track the pathways of macromolecules or organelles in a "living" cell is to monitor them after staining with these PFDs during the real time measurements. A bright fluorescent signal from the photoactivated dye molecules inside the small spot in the cell can be monitored during their movement into the cell dark region (where the dye was not activated and did not fluoresce). The obtained data are important for further application of these precursors of the fluorescent dyes ("caged" dyes) for microscopic probing of biological objects.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
The emission and polarization spectra of 1-phenyl-3-(2-naphthyl)-2-pyrazoline (PNP) in various environments were studied. Compared to the widely used orientational membrane probe 1,6-diphenylhexatriene (DPH), PNP is five times less photolabile and since its fluorescence emission maximum is at longer wavelengths (Xma x ~ 445 nm), it is more suitable for use with intact erythrocytes. The limiting fluorescence anisotropy of PNP is 0.385. In erythrocyte ghosts, the steady-state emission anisotropy of PNP is a decreasing function of wavelength and its temperature dependence parallels that of DPH, dropping from 0.298 at 2°C to 0.185 at 38°C when averaged between 420 and 470 nm.
Journal of Luminescence, 2018
From electrooptical absorption measurements (EOAM) follows that the dipole moments of azacrown ketocyanine dyes 2,5-di{(E)-1-[4-(4,7,10,13-tetraoxa-1azacyclopentadecyl)phenyl]methylidene}-1-cyclopentanone (Compound 1) and (E)-1-(2hydroxy-4methoxyphenyl)-3-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecyl)phenyl]-2-propen-1one (Compound 2) in the equilibrium ground state μ g and the change of dipole moments upon transition to the excited Franck-Condon state Δ a μ are large. This alteration causes a significant long-wavelength shift of the absorption and emission spectra, as well as large fluorescence Stokes shift with increasing polarity of the solvent. From the DFT calculations follows that the terminal groups of the dyes are located outside the plane of the central part of the molecules by the torsion angles that allow good conjugation of the nitrogen atoms lone pairs with π-system of the molecule. The transition dipole moment of Compound 1 m a is perpendicular to the dipole moment g in the equilibrium ground state and for Compound 2 m a is parallel to g. This is due to the differences in the geometric structure of the dyes. The distribution of fluorescence lifetime of Compound 1 significantly depends on the polarity of the solvent. Thus, ketocyanine dyes with azacrown cycles can serve not only as ion indicators but also as fluorescent probes for studying the polarity of the environment.
2014
Indian Chemical Society, 92, Acharya Prafulla Chandra Road, Kolkata-700 009, India <em>E-mail</em> : drrahulchem@rediffmail.com Department of Chemistry, West Bengal State University (Barasat), Kolkata-700 126, India <em>E-mail </em>: ranjan.das68@gmail.com <em>Manuscript received online 07 December 2012, revised 09 January 2013, accepted 11 March 2013</em> <strong>The photophysics of a dye, <em>N</em>-[[4'-<em>N,N</em>-diethylamino-3-hydroxy-6-flavonyl]methyl]-<em>N</em>-methyl-<em>N</em>-(3-sulfopropyl)- 1-dodecanaminium, inner salt (F2N12S), in the large unilamellar vesicles of neutral egg yolk phosphatidylcholine (EYPC) and negatively charged egg yolk phosphatidylglycerol (EYPG) lipids were investigated by steady state and time-resolved fluorescence spectroscopy. The fluorescence spectra display a dramatic variation of the relative intensity of dual emission bands attributed to the norma...