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PubMed1991

Determination of the membrane potential of cultured mammalian Schwann cells and its sensitivity to potassium using a thiocarbocyanine fluorescent dye.

The membrane potential of cultured rat sciatic nerve Schwann cells was determined with conventional microelectrode and voltage-sensitive fluorescent dye, Di-S-C3(5), optical techniques. The value for membrane potential obtained with microelectrodes was -42.1 +/- 4.7 mV (n = 8). Using optically determined fluorescent intensity changes caused by changes in external potassium ion concentration, in the presence or absence of valinomycin (null point method), the membrane potential was estimated at -45.7 +/- 6.2 mV (n = 7); with a gramicidin and valinomycin double ionophore method it was -52.2 +/- 9.1 (n = 4). The membrane potential of Schwann cells was found to be potassium sensitive at and above the physiological range of [K+] at 27.5 mV/10x delta[K+], which is approximately half the Nernstian value. This result suggests that other ion permeabilities strongly influence the resting membrane potential of cultured Schwann cells. Since Na+ had little effect on the membrane potential, it is concluded that Cl- is a likely candidate for the other permeant ionic species. The optical method has been shown to be a useful tool for the systematic study of the membrane potential of Schwann cells in culture and for the characterization of its ionic basis and regulation.

Animals↗

PubMedDate not supplied

Spectral properties of fluorescent dyes in lecithin vesicles. Probes for the structure of lipid bilayer membranes and for membrane potentials.

Three fluorescent dyes (a merocyanin, a cyanin and a modified umbeliferon) have been incorporated into vesicles from three different lecithins. Their absorption spectrum, emission spectrum and fluorescence polarization in the lipid vesicles has been studied and compared with the spectral properties in homogeneous organic solutions. The reactivity of lipid bound dyes with reagents in the aqueous phase was investigated. The location of various dyes in the lipid membrane and their use as indicators of phase transitions or membrane potentials are discussed.

Fluorescent Dyes↗

PubMed2006

Novel 2,1,3-benzothiadiazole-based red-fluorescent dyes with enhanced two-photon absorption cross-sections.

This paper reports the two-photon absorbing and orange-red fluorescence emitting properties of a series of new 2,1,3-benzothiadiazole (BTD)-based D-pi-A-pi-D-type and star-burst-type fluorescent dyes. In the D-pi-A-pi-D-type dyes 1-6, a central BTD core was connected with two terminal N,N-disubstituted amino groups via various pi-conjugated spacers. The star-burst-type dyes 8 and 10 have a three-branched structure composed of a central core (benzene core in 8 and triphenylamine core in 10) and three triphenylamine-containing BTD branches. All the BTD-based dyes displayed intense orange-red color fluorescence in a region of 550-689 nm, which was obtained by single-photon excitation with good fluorescent quantum yield up to 0.98 as well as by two-photon excitation. Large two-photon absorption (TPA) cross-sections (110-800 GM) of these BTD dyes were evaluated by open aperture Z-scan technique with a femtosecond Ti/sapphire laser. The TPA cross-sections of D-pi-A-pi-D-type dyes 2-6 with a benzene, thiophene, ethene, ethyne, and styrene moiety, respectively, as an additional pi-conjugated spacer are about 1.5-2.5 times larger than that of 1c with only a benzene spacer. The TPA cross-sections significantly increased in three-branched star-burst-type BTDs 8 (780 GM) with a benzene core and 10 (800 GM) with a triphenylamine core, which are about 3-5 times larger than those of the corresponding one-dimensional sub-units 9 (170 GM) and 11 (230 GM), respectively. The ratios of sigma/e(pi) between three-branched and one-dimensional dyes were 6.5:3.8 (for 8 and 9) and 6.0:4.0 (for 10 and 11), which are larger than those predicted simply on the basis of the chromophore number density (1:1), according to a cooperative enhancement of the two-photon absorbing nature in the three-branched system.

Color↗

PubMed1978

Identification of intrafusal muscle fibres activated by single fusimotor axons and injected with fluorescent dye in cat tenuissimus spindles.

1. Intrafusal muscle fibres of cat tenuissimus spindles have been injected with the fluorescent dye Procion Yellow and identified histologically after recording their changes in membrane potential during 1/sec stimulation of single static or dynamic gamma axons. 2. Thirteen intrafusal muscle fibres innervated by static gamma axons were identified as eight bag2 and five chain fibres. The fact that none proved to be a bag1 fibre is not regarded as significant, for reasons given in the Discussion. 3. In one spindle Procion Yellow was injected into two intrafusal muscle fibres activated by the same static gamma axon; they were identified as a bag2 and a chain fibre. 4. Nine intrafusal muscle fibres innervated by dynamic gamma axons were identified as seven bag1 fibres, one bag2 fibre, and one long chain fibre. 5. In one spindle two bag fibres were injected, one activated by a dynamic gamma axon, the other by a static gamma axon; the former proved to be a bag1 fibre, the latter a bag2 fibre. 6. Stimulation of static gamma axons elicited junctional potentials in seven bag2 fibres and one damaged chain fibre, and action potentials in one bag2 and four chain fibres. In the whole sample of impaled intrafusal muscle fibres (identified and unidentified) activated by static axons, junctional potentials were recorded from twenty-three (62.2%), and action potentials from fourteen (37.8%). Stimulation of dynamic gamma axons always elicited junctional potentials. 7. In a number of instances it was possible to examine the ultrastructure of motor endings belonging to the stimulated gamma axon. The myoneural junctions of trail endings supplied by static gamma axons to bag2 and chain fibres were both smooth and folded; the deepest and most regular folding occurred on chain fibres. The terminals of p2 plates supplied to bag1 fibres by dynamic gamma axons had smooth myoneural junctions.

Action Potentials↗

PubMed1985

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9.

The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.

Complement Membrane Attack Complex↗

PubMed1980

The origins of the centrifugal fibers to the olfactory bulb in the goldfish, Carassius auratus: an experimental study using the fluorescent dye primuline as a retrograde tracer.

The origin of the centrifugal fibers to the olfactory bulb in the goldfish was examined by injecting a fluorescent dye, primuline, into the olfactory bulb by means of hydraulic pressure. Three to five days after the primuline injection, which was confined to the unilateral olfactory bulb, a large number of primuline-labeled perikarya appeared in two telencephalic areas on the ipsilateral side: the area ventralis telencephali pars dorsalis (Vd) and the ventral part of the area dorsalis telencephali pars lateralis (Dlv). No labeled cells were observed in the other olfactory bulb or in the contralateral telencephalic hemisphere or more posterior part of the brain. A combination of the primuline injection and the transection of either the medial or lateral olfactory tract showed that the main course of the centrifugal fibers was through the medial olfactory tract. These results may support Sheldon's opinion that the so-called tractus olfactorius ascendens, which corresponds to the medial part of the medial olfactory tract, is a centrifugal bundle originating from the cells in the nucleus medianus. The areas of the telencephalic hemisphere into which the olfactory bulbar fibers projected were also examined and compared with the originating areas of the centrifugal fibers. There was a considerable overlap between these two areas. This suggests a reciprocal connection between the olfactory bulb and the telencephalon.

Animals↗

PubMed1985

Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes.

We previously showed that liposomes composed of dioleoylphosphatidyl-ethanolamine and palmitoyl-homocysteine (8:2) are highly fusion competent when exposed to an acidic environment of pH less than 6.5. (Connor, J., M. B. Yatvin, and L. Huang, 1984, Proc. Natl. Acad. Sci. USA. 81:1715-1718). Palmitoyl anti-H2Kk was incorporated into these pH-sensitive liposomes by a modified reserve-phase evaporation method. Mouse L929 cells (k haplotype) treated with immunoliposomes composed of dioleoylphosphatidylethanolamine/palmitoyl-homocysteine (8:2) with an entrapped fluorescent dye, calcein, showed diffused fluorescence throughout the cytoplasm. Measurements by use of a microscope-associated photometer gave an approximate value of 50 microM for the cytoplasmic calcein concentration. This concentration represents an efficient delivery of the aqueous content of the immunoliposome. Cells treated with immunoliposomes composed of dioleoylphosphatidylcholine (pH-insensitive liposomes) showed only punctate fluorescence. The cytoplasmic delivery of calcein by the pH-sensitive immunoliposomes could be inhibited by chloroquine or by incubation at 20 degrees C. These results suggest that the efficient cytoplasmic delivery involves the endocytic pathway, particularly the acidic organelles such as the endosomes and/or lysosomes. One possibility is that the immunoliposomes fuse with the endosome membranes from within the endosomes, thus releasing the contents into the cytoplasm. This nontoxic method should be widely applicable to the intracellular delivery of biomolecules into living cells.

Animals↗

PubMed1993

Variation with age in the labelling of amoeboid microglial cells in rats following intraperitoneal or intravenous injection of a fluorescent dye.

Amoeboid microglial cells (AMC) in the corpus callosum were selectively labelled following a single intraperitoneal (i.p.) injection of the fluorescent dye, rhodamine isothiocyanate (RhIc) into postnatal rats. The frequency of RhIc-labelled cells varied with age, with the largest number occurring in 7-d-old animals. Thereafter, the labelled cells declined drastically in number and fluorescence and were barely detectable in 12-d-old injected rats. Labelled cells were absent in 13-d or older rats given an RhIc injection. When the injected RhIc was followed over a time course sequence, it was first detected in the cerebral blood vessels and their lining endothelia within 5 min after the injection. A variable number of AMC emitting a weaker fluorescence were closely adherent to the outer walls of the blood vessels. With time, the fluorescence in the AMC was progressively enhanced, but that in the blood vessels showed a concomitant reduction. In the rats that received an intravenous (i.v.) injection of RhIc, the labelling pattern of AMC, both in terms of its variation with age and in temporal sequence, paralleled that in rats given i.p. injections. In 12-d-old rats subjected to a stab wound coupled with an i.p. injection of RhIc, a considerable number of AMC not normally labelled at this age were activated. The cells exhibited an intense fluorescence and expressed MHC surface antigen immunoreactivity. It is concluded from this study that when injected i.p. or i.v., RhIc is readily circulated to the cerebral vessels, where it enters brain tissue by transendothelial transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1983

Non-fluorescent dye staining of primate blue cones.

The intravitreal injection in macaque retina of the fluorescent dye Procion yellow can selectively label a specific cone population whose eccentricity distribution and angular separation are consistent with those of the blue-sensitive cones of human and non-human primate retinas. Because at the concentrations used the dye is poorly visible in conventional light microscopy, fluorescence microscopy is required for the observation of the stained cones. In this paper we describe several alternative methods for the staining of blue cones in primate retina, staining that can be visualized in conventional light microscopy and, with some methods, electron microscopy.

Animals↗

PubMed2005

Spectroscopic studies of fluorescent dye N, N'- bis(2-hydroxy-5-methyl-benzylidene)-1,2-ethanediamine and its DNA complex in solution.

Absorption and fluorescence emission properties of an N-salicylideneamine fluorescent dye molecule N, N'-bis(2-hydroxy-5-methylbenzylidene)-1,2-ethanediamine (1) have been studied in three typical solvents--2-methylbutane, ethanol, dimethyl sulphoxide (DMSO), and its DNA complex in methanol/H2O mixed solvent. The normal absorption band of 1 is observed in both aprotic and protic solvents and has been assigned to the l --> a(pi) transition in the enol form of 1. The long-wavelength absorption band of 1, which is caused by the formation of a cis-keto species in the ground state, is absent in aprotic solvents, but is observable in protic ones. Normal fluorescence emission from the excited enol state of 1 is obtained only when the normal absorption band is excited, while the excited-state intramolecular proton transfer (ESIPT) emissions from both cis- and trans-keto species are recorded in all cases, being acceptable for the variation of the relative emission intensities. A preliminary spectroscopic study of the 1-DNA complex indicates an intercalation-binding mode, the convincing supporting evidence being the enhanced ESIPT fluorescence intensity of 1 when complexed with DNA. Finally, a universal energy-state diagram is given to interpret the experimental results.

Animals↗

PubMed1992

Visualization of enteric and gallbladder ganglia with a vital fluorescent dye.

A method is described here for the vital staining of the nerve plexuses of the gastrointestinal tract with the fluorescent dye 4-(4-diethylaminostyryl)-methylpyridinium iodide (4-Di-2-ASP). This dye stained nerve fibers in the myenteric and submucosal plexuses and in the intramural ganglia of the gallbladder. In addition to staining the ganglia, the dye clearly revealed the secondary and the fine tertiary fibers of the myenteric plexus. In the submucosal plexus thin fibers innervating the blood vessels were also stained. This method proved to be effective in tissues that are relatively thick and in which the visualization of the nerves is quite difficult without staining--the myenteric plexus of rat small intestine, guinea-pig gallbladder and human large intestine. The staining did not fade within at least 6-8 h, or after long exposure to bright light. Intracellular recordings from myenteric neurons in the guinea pig ileum showed that cellular properties were not significantly altered after incubation in the dye solution. 4-Di-2-ASP thus appears to be suitable for rapid vital staining of nerves in whole mount from many parts of the gastrointestinal tract.

Animals↗

PubMed2002

Evidence of collateralization of vagal efferents innervating subdiaphragmatic segments of the gastrointestinal tract in the rat using the double labelling fluorescent dyes technique.

Collateralization of the abdominal vagal trunks was investigated in the rat using double labelling fluorescence dye technique. A total of 20 adult male and female rats were used for the study. The anterior and posterior walls of the corpus and fundus of the stomach were injected with 0.1 microliter (microliter) of 5% aqueous solution of diamidino yellow (Dy) in eight rats. The same quantity and percentage of fast blue (Fb) was injected into the walls of the duodenum and proximal jejunum in the same eight rats that were injected with Dy. The anterior and posterior walls of the stomach of four rats were injected with 0.1 microliter of 5% Dy only while four other rats had injections of 0.1 microliter of Fb only into the duodenum and proximal jejunum. Two control rats had stomach injections with normal saline, and two rats had saline injections into the intestine. Each rat was perfused with a preservative 14 days after injection and serial sections taken for examination with a fluorescence microscope. The results of the experiment revealed that in the eight rats injected simultaneously with Dy and Fb, some neurons of the dorsal motor nucleus of the vagus nerve (DMX) were labelled with Dy only, some with Fb only and some were doubly labelled with Dy and Fb. No double-labelled neurons were seen in the rat injected with one dye only and no labellings at all were seen in the controls. The pattern of labelling obtained in the study is suggestive of collateralization of axons of the abdominal vagal trunks.

Afferent Pathways↗

PubMed1998

Interactions of DNA with fluorescent dyes: by scanning tunneling microscopy.

Genomic DNA was obtained from peripheral blood samples of healthy volunteers and interacted with two fluorescent dyes (i.e. Hoechst 33,258 and ethidium bromide) in aqueous media. These media containing DNA-dye complexes deposited on the gold coated mica surfaces. Then, STM images were obtained in which the STM was operated in air at atmospheric pressure with a tip-to-substrate bias voltage of 250-1000 mV (sample positive) and the tunneling currents in the range of 10-20 pA by using etched tips of Pt/Ir, in constant current mode. Both dyes from molecular clusters on DNA. While, the Hoechst molecules were observed on the DNA chains at regular distances, the ethidium bromide molecular clusters did not.

Bisbenzimidazole↗

PubMed1990

The use and effects of vital fluorescent dyes: observation of motor nerve terminals and satellite cells in living frog muscles.

Several different fluorescent mitochondrial dyes were tested as vital stains for motor nerve terminals and other cells in frog skeletal muscles. It was found that 3,3' diethyloxadicarbocyanine iodide and 4-(4-diethylaminostyryl)-N-methylpyridinium iodide were most useful. Both dyes labelled motor nerve terminals with high reliability. Electrophysiological and morphological control experiments showed that these dyes could be used to repeatedly observe neuromuscular junctions in living animals without affecting synaptic growth or remodelling. The importance of appropriate controls was emphasized by the finding that illumination, if excessively intense or prolonged, can cause physiological and structural damage to nerve terminals. Additional observations indicated that these dyes may be useful for determining the mitochondrial content, and therefore oxidative capacity, of living muscle fibres. It was also found that the fluorescent dyes labelled cells identified as muscle satellite cells, and that these myoblast precursors could be visualized in fixed whole mounts with a nitroblue tetrazolium stain. Both methods were used to study reactive cells that were closely associated with muscle fibres in lesioned muscles. Mitochondrial dyes also labelled the microvasculature, associated axons and other cells.

Animals↗

PubMedDate not supplied

Spectrofluorimetric techniques in the study of the interaction between fluorescent dyes and proteins or nuclear acids.

After a review of the applications of spectrofluorimetric techniques to the study of ligand--biological substrate interactions, mention is made of the main fluorescent dyes used for the demonstration of structural characteristics and conformational alterations in proteins, nucleic acids, etc. The possibilities of using new compounds recommended in the literature (fluorescamine, fluoropa, a.o.) for the identification and study of proteins are analysed and original investigations with newly synthesized fluorescent compounds (anthraquinone dyes, anhydrides of peridicarboxylic acids, Tb3+ and Sm3+ complexes of the pyridine -2,6-dicarboxylic acid) are presented.

Chemical Phenomena↗

PubMed1981

Effect of liposome encapsulation of a fluorescent dye on its uptake by the lymphatics of the rat.

The fluorescent dye carboxyfluorescein (CF), entrapped in liposomes and administered by intraduodenal injection to thoracic duct-cannulated rats, was not detected in thoracic duct lymph at any time after dosing. However, the dye was present in blood, and in higher concentration in portal than in systemic blood. Similarly, free CF given intraperitoneally (i.p.) appeared to be absorbed into portal blood and was quickly cleared from the circulation, with less than 1% of the injected dye being recovered in thoracic duct lymph within 24 h after dosing. In contrast, 27% of the injected CF was recovered in thoracic duct lymph of rats receiving CF/liposomes i.p. These findings indicate that liposomal entrapment effectively limits passage of CF into the splanchnic blood vessels while enhancing the lymphatic uptake of the dye from the peritoneal cavity.

Animals↗

PubMed2005

The effect of DNA labeling with the fluorescent dyes R110 and R6G on genotype analysis using capillary electrophoresis.

We investigated the mobility of DNA fragments labeled with the fluorescent dyes R110 and R6G, specifically for use in genotyping using capillary electrophoresis. Genomic DNA was isolated from blood, and a highly polymorphic region of the HLA-C gene was amplified by PCR with the use of either [F]-dNTP-[R110] or [F]-dNTP-[R6G]. Pre-diluted (30-fold) PCR products were mixed with formamide, denatured (at 95 degrees C for 2 min.), and rapidly cooled on ice before being subjected to electrophoresis. The results showed that the number and mobility of allele-specific DNA fragments were independent of the two dyes used. Both dyes were equally efficient at differentiating homozygous or heterozygous allelic presentation. An additional dye-specific peak of 132-base-pair mobility was observed with the use of [F]-dNTP-[R110]; it significantly impaired the resolution of one allele-specific peak. The electropherograms obtained with [F]-dNTP-[R6G] were free from any interfering peaks within the target region, thus the [R6G]-based procedure is more preferable for genotype analysis. As this procedure does not involve any post-PCR cleanup, it is simple, rapid and cost-effective.

Alleles↗

PubMed1991

Characterization of multidrug resistance by fluorescent dyes.

Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype. P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes. But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative. In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil. Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR. None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport. But accumulation of both neutral and cationic dyes was impaired in CEM/VLB100, a subline of CCRF-CEM expressing mdr. These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype. With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB100. The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

Animals↗