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At least 19 recordsLinked to original sources

PubMed2003

Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit.

The present study examined the efficiency of fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylinodocarbocyanine perchlorate and cholera toxin B subunit in tracing the crossed tectal projection to the nucleus rotundus of the thalamus (tectorotundal pathways) of paraformaldehyde-fixed and living chick embryos. The tracers were injected into the optic tectum under three experimental conditions (carbocyanine postfix, carbocyanine in vivo, and cholera toxin B subunit in vivo) and the anterograde transport of the nucleus rotundus was monitored and compared. In the carbocyanine postfix method, small crystals of carbocyanine dye were inserted into the tectum of paraformaldehyde-fixed embryos. A 6-month post-insertion period was required to label the crossed tectorotundal pathway. Results showed that tectal neurons did not begin to innervate the ipsilateral nucleus rotundus until embryonic day 9 and the contralateral nucleus rotundus until embryonic day 17. This slow progression of labeling through the crossed tectal projection resulted in significant contrast of the labeling between the ipsilateral and contralateral nuclei rotundus. In the carbocyanine in vivo method, a small volume of carbocyanine dye solution was injected into the tectum of living embryos. A 8- to 12-h survival period was sufficient enough to label the tectorotundal pathway. By embryonic day 8, the labeled axons terminated in the ipsilateral nucleus rotundus and the crossed tectorotundal projection was first detected by embryonic day 10. Similarly, in the cholera toxin B subunit in vivo method, a small volume of cholera toxin B subunit solution was injected into the tectum of living embryos. After a 6- to 10-h survival period, heavily labeled axons were found to innervate bilaterally the nucleus rotundus by embryonic day 8. This appeared to be the earliest schedule for detecting the crossed tectorotundal projection, compared with that of both the postfix and in vivo methods of carbocyanine dye. Based on the differences in the detectability of the crossed tectorotundal projection between the postfix and in vivo methods, the present data suggest that the former method is of limited purpose for labeling tectal collaterals during embryogenesis. Moreover, given the rapid transport rate and absence of photobleaching, which is often seen when using carbocyanine dye, the cholera toxin B subunit in vivo method appears to be the tracer of choice for investigating embryonic pathways.

Animals↗

PubMed1986

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.

Animals↗

PubMed1994

Evaluation of four new carbocyanine dyes for photodynamic therapy with lasers.

The search for improved photosensitizers for laser phototherapy of malignancies has led to the examination of a new group of carbocyanine dyes as effective fluorochromes. In this study, four carbocyanine dyes with different absorption maxima of 483 nm [DiOC6(3)], 545.5 nm (DiIC5(3)], 556.6 nm [DiSC5(3)], and 651.0 nm [DiSC3(5)] were tested in vitro. The kinetics of uptake and toxicity of these four dyes were assessed for P3 human squamous cell carcinoma, HT29 colon carcinoma, M26 melanoma, and TE671 fibrosarcoma cell lines at 15, 30, 45, 60, and 180 minutes after exposure with each dye. After sensitization with DiOC6(3), the P3 and M26 cell lines were also tested for phototherapy by treatment with 488-nm light from an argon laser. The results showed that these four carbocyanine dyes had rapid and significant uptake by the carcinoma cell lines with no toxicity at concentrations < 0.1 micrograms/mL. Nontoxic DiOC6(3) levels in sensitized tumor cells after laser phototherapy resulted in approximately 85% inhibition of P3 and approximately 95% inhibition of M26 cell lines by MTT assays. The results suggest that these carbocyanine dyes can be used for tumor photosensitization and wavelength-matched laser photodynamic therapy. Further in vivo studies will be necessary to define the clinical potential of carbocyanine dyes as tumor-targeting agents for phototherapy of cancer.

Adenocarcinoma↗

PubMed1995

Carbocyanine dyes with long alkyl side-chains: broad spectrum inhibitors of mitochondrial electron transport chain activity.

Certain indocarbocyanine, thiacarbocyanine, and oxacarbocyanine dyes possessing short alkyl side-chains (one to five carbons) are potent inhibitors of mammalian mitochondrial NADH-ubiquinone reductase (EC 1.6.99.3) activity (Anderson et al., Biochem Pharmacol 41: 677-684, 1991; Anderson et al., Biochem Pharmacol 45: 691-696, 1993; Anderson et al., Biochem Pharmacol 45: 2115-2122, 1993), and act similarly to rotenone. This study examines the inhibitory capacities of twelve other carbocyanine dyes (six indocarbocyanines, four oxacarbocyanines, and two thiacarbocyanines) possessing long alkyl side-chains (seven to eighteen carbons with both saturated and unsaturated side-chains) on mitochondrial NADH, succinate and cytochrome c oxidase activities. Three of the indocarbocyanines inhibited electron transport chain activity, while three were non-inhibitory. Two of the oxacarbocyanines also inhibited electron transport chain activity, while the other two were without effect. Both the thiacarbocyanines were non-inhibitory. In contrast to previous studies, the long alkyl side-chain carbocyanines exhibited a broad spectrum of inhibition of respiratory chain activity, affecting either oxidation of all three substrates or of NADH and cytochrome c, rather than specific inhibition of mitochondrial NADH-ubiquinone reductase activity, indicating that there could be multiple binding sites for these compounds. The five inhibitory long side-chain carbocyanines also inhibited reduction of ferricyanide and coenzyme Q1 by NADH, using submitochondrial particles, but not when tested with purified complex I, indicating that the mitochondrial inner membrane was an integral component in their inhibitory capacity. No general correlation of side-chain length or degree of unsaturation and inhibitory capacity was discernible.

Animals↗

PubMed2005

Carbocyanine dyes as efficient reversible single-molecule optical switch.

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

Biotin↗

PubMed2002

Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.

Animals↗

PubMed2004

Does aliphatic chain length influence carbocyanines' orientation in supported lipid multilayers?

UV-Vis linear dichroism was used to study the orientation of 3,3'-dihexyloxacarbocyanine (DiOC6(3)) and 3,3'-dihexadecyloxacarbocyanine (DiOC16(3)) in supported multilayers of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC). Orientational probability density functions were similar for the two carbocyanines in both lipids. Multimodal distributions were found in all cases. The main peak is at 9 degrees-11 degrees relative to the bilayer normal axis, except for DiOC16(3) in DLPC multilayers (main peaks at 13 degrees and 90 degrees). Quenching studies revealed that the two carbocyanines are localized at the interface region of the membrane regardless of the lipid matrix they are inserted in. Combining these data with linear dichroism results lead to the conclusion that both the aliphatic chain length of carbocyanines and the lipid phase have little influence in the structural organization of these probes in lipidic bilayers. The partition constants of DiOC6 (3), K(p), were determined from fluorescence anisotropy measurements; the values obtained were K(p) (DPPC) = (2.39 +/- 0.05) x 10(3) and K(p) (DLPC) = (5.01 +/- 1.15) x 10(3).

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Carbocyanine postmortem neuronal tracing. Influence of different parameters on tracing distance and combination with immunocytochemistry.

Carbocyanines (DiI, DiA, DiO) are able to travel along membranes by diffusion and therefore have been used as postmortem neuronal tracers in aldehyde-fixed tissues. Surprisingly, detailed data on the influence of different parameters on tracing distances are still missing. This study was carried out to optimize tracing procedures and to reveal the validity of the combination of postmortem tracing with immunocytochemistry. Carbocyanine crystals were applied to the cervical spinal cord, sciatic nerves, and brachial plexuses of humans and guinea pigs. Incubation in the dark at 37C for 12-15 weeks proved optimal to achieve longest tracing distances (28.9 +/- 2.2 mm) in human and animal tissues. Longer incubation times and incubation temperatures higher than 37C did not result in longer tracing distances. No differences were evident between adult and newborn animals and between central and peripheral nervous system. The diffusion coefficient for DiI was calculated to be 2.5 x 10(-7) cm2 sec-1. After application of DiI to nerves of guinea pig extraocular muscles, DiI-positive afferent perikarya were observed in the anteromedial part of the trigeminal ganglion. These perikarya were identified by calcitonin gene-related peptide immunoreactivity (CGRP-IR). The percentage of CGRP-IR neurons after tracing was concordant with the percentage of CGRP-IR in trigeminal ganglia exclusively processed for CGRP-IR without previous postmortem tracing. These results demonstrate carbocyanines to be specific tracers for exact neuronal mapping studies. (J Histochem Cytochem 46:901-910, 1998)

Aged↗

PubMed1993

Carbocyanine dye labeling reveals a new motor nucleus in octopus brain.

This work aims at a better understanding of the organization of the brain of Octopus vulgaris, emphasizing the touch and visual learning centers. We injected the carbocyanine dye, DiI, into the cerebrobrachial connectives and, separately, into the brachial nerves of living octopuses. In both experiments, retrogradely transported granules of DiI appeared in motor neurons in the superior buccal, posterior buccal and subvertical lobes and in a hitherto unsuspected motor nucleus of several hundred neurons in the posterior dorsal basal and median basal lobes. In addition we labeled afferent fibers by injecting DiI into the caudal (sensory) division of the cerebrobrachial connective on one side; the label spread throughout the superior buccal, posterior buccal and the lateral and median inferior frontal lobes mainly on the injected side. It extended through the cerebral tract into the subvertical lobe, into the superior frontal lobe through the interfrontal tract, through the posterior buccal commissure into the opposite posterior buccal lobe and into the median inferior frontal lobe. The work suggests a new function for the posterior dorsal and median basal lobes, which are shown for the first time to project through the inferior frontal lobe system into the brachial nerves. In addition it represents the first full report of the successful use of the carbocyanine dyes DiI and DiO for labeling nerve tissue in a live invertebrate animal.

Animals↗

PubMed1987

Characterization of a carbocyanine derivative as a fluorescent penetration probe.

The fluorescent carbocyanine dye 3,3-diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence-activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.

Animals↗

PubMed1978

Differential effects of erythrocyte lysates on spectra of potential-sensing carbocyanine dyes.

A recent report by Hladky, S.B. and Rink, T.J. ((1976) J. Physiol. 263, 287--319) demonstrates the binding of a thiocarbocyanine dye (di-S-C3 (5)) to an intracellular constituent from human erythrocytes. Evidence presented below shows that the binding of carbocyanine dyes depends on the particular type of dye used and on the species of erythrocyte from which cellular constituents are prepared. It is recommended that dyes which show minimal binding to intracellular components be used for investigations where carbocyanine dyes are employed as probes of membrane potential.

Animals↗

PubMed1983

Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential. Evidence for interactions between carbocyanine dyes and lipophilic anions.

The accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3'-diethylthiadicarbocyanine iodide and by 3,3'-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.

Animals↗

PubMed1996

The septointerpeduncular projection in the rat: tracing with the carbocyanine dye Dil.

The fluorescent carbocyanine dye Dil has been used to retrogradely label the neuronal connections between the forebrain septal area and the interpeduncular nucleus. Previous works based on retrograde horseradish peroxidase transport have identified that only the diagonal band nucleus is a source of the septointerpeduncular projections, but anterograde tracing with labeled amino acids and selective lesions with colchicine have shown that also the posterior septal nuclei project to the interpeduncular nucleus. In the present study, the retrograde labeling in septal nuclei after placing the carbocyanine Dil in the interpeduncular nucleus resulted in the fluorescent labeling of numerous neurons of the diagonal band nucleus. Our results, in addition, showed the labeling of some scattered neurons in the ventral portion of the triangular nucleus of the septal area and in the septofimbrial nucleus, confirming the presence of a previously controversial septointerpeduncular projection.

Animals↗

PubMed2004

Carbocyanine labeled LDL for optical imaging of tumors.

RATIONALE AND OBJECTIVES: The purpose of this study was to define and characterize carbocyanine labeled low-density lipoprotein (LDL) to be used in the optical imaging of LDL receptor (LDLr)-overexpressing tumor models. MATERIALS AND METHODS: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label LDL (DiI-LDL). Scatchard plots were generated to determine the maximum binding capacity B(max) and dissociation constants K(D) of DiI-LDL in B16 melanoma (B16) and hepatoblastoma G(2) (HepG(2)) cell lines. Selective uptake of DiI-LDL into both tumor cells and corresponding subcutaneous tumors in mice were demonstrated by confocal microscopy and three-dimensional Cryo-imaging, respectively. RESULTS: The labeling efficiency of DiI-LDL was 61 ng DiI/microg LDL protein (34 mol DiI/mol LDL protein). B(max) and K(D) for B16 cells were 6.311 ng LDL/mg cell protein and 60.38 microg protein/mL (117 nM), respectively. B(max) and K(D) were 7.573 ng LDL/mg cell protein and 26.79 microg protein/mL (52 nM) for HepG(2) cells, respectively. Confocal microscopic images showed specific uptake of DiI-LDL throughout the cytoplasm in the B16/HepG(2) cells. Cryo-imaging demonstrated preferential accumulations of DiI-LDL in the viable tumor regions of both B16 and HepG(2) tumors compared with their adjacent normal tissues and corresponding necrotic tumor regions. In addition, uptake of DiI-LDL by the HepG(2) tumor was much higher than that of the B16 tumor, consistent with the fact that the probe binding affinity for LDLrs of HepG(2) cells is 2.3 times that of B16 cells. CONCLUSION: This study suggested that carbocyanine labeled LDL could be used for optical imaging of tumors overexpressing LDLr.

Animals↗

PubMed1999

Interaction of cyanine dyes with nucleic acids. XII.beta-substituted carbocyanines as possible fluorescent probes for nucleic acids detection.

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.

Carbocyanines↗

PubMedDate not supplied

Multivalent carbocyanine molecular probes: synthesis and applications.

Synergistic multivalent interactions can amplify desired chemical or biological molecular recognitions. We report a new class of multicarboxylate-containing carbocyanine dye constructs for use as optical scaffolds that not only serve as fluorescent antennas but also participate in structural assembly of the multivalent molecular construct. Three generations of carboxylate-terminating multivalent near-infrared carbocyanine probes from a dicarboxylic acid precursor dye (cypate) were prepared via its imino diacetic acid derivatives. Conjugation of the probes with D-(+)-glucosamine afforded dendritic arrays of the carbohydrates on an inner NIR chromophore core. All the multicarboxylate probes and their glucosamine conjugates have similar NIR spectral properties because conjugation occurred at distal positions to the inner chromophore core, thereby providing consistent and predictable spectral properties for their biological applications. Although light-induced photodamage equally affected the precursor dye, multicarboxylate probes, and their glucosamine derivatives, we observed that octacarboxylcypate (multivalent probe) was remarkably stable in different mediums at physiologically relevant temperatures relative to cypate, especially in basic mediums. Biodistribution studies in tumor-bearing nude mice show that all the glucosamine conjugates localized in the tumor but cypate was almost exclusively retained in the liver at 24 h postinjection. The tumor uptake does not correlate with the number of glucosamine tether on the multicarboxylate probe. Overall, the triglucosamine derivative appears to offer the best balance between high tumor uptake and low retention in nontarget tissues. These results suggest that multivalent molecular beacons are useful for assessing the beneficial effects of multivalency and for optimizing the biological and chemical properties of tissue-specific molecular probes.

Animals↗

PubMed1991

J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential.

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 pH range of 7.15-8.0 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.

Animals↗

PubMed1984

Staining glycol methacrylate embedded cartilage with triethyl-carbocyanin DBTC ("ethyl-stains all") with special reference to the interlacunar network.

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.

Animals↗