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PubMed1983

Localization of renal sensory neurons using the fluorescent dye technique.

The location of cell bodies of renal sensory neurons was studied. Small injections of a fluorescent dye (True Blue or Fast Blue) were placed into either the right or left kidney of male or female rats. Whereas no differences were detected in the labeling patterns of males vs females, right kidney injections did label slightly higher dorsal root ganglia than left injections. In all cases the labeling was confined to the T6-L2 ganglia ipsilateral to the injection.

Animals↗

PubMed1985

Fluorescent dyes demonstrate the uniform expansion of the growing rabbit cornea.

Thiocyanate and triazinyl chloride derivatives of fluorescent dyes have been employed for the covalent labeling of components of the connective tissue of the rabbit cornea. Collagen is the major macromolecular component that becomes dyed. Some of the stromal components that are labeled by these reagents in the first two weeks of life persist over many months, but are progressively dispersed over a greater area consistent with a uniform expansion of the tissue with growth without complete turnover. These dyes may find useful application in the morphologic study of other persistent connective tissue components during development and wound healing.

Animals↗

PubMed1998

Near-infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination.

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencing format. The heavy-atom modification consisted of an intramolecular halogen situated on a remote section of the chromophore in order to minimize the perturbation on the lifetimes and fluorescence quantum yields. In addition, the dye series possessed an isothiocyanate functional group to allow facile attachment to sequencing primers. The unconjugated dyes showed similar absorption and emission maxima (lambda abs = 765-768 nm; lambda em = 794-798 nm) as well as fluorescence quantum yields that were invariant, within experimental error, with the heavy atom. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution (I, tau f = 947 ps; F, tau f = 843 ps, measured in methanol), with an average variation within the dye series of 35 ps. The spectroscopic properties of the free dyes and the dyes conjugated to sequencing primers on the 5'-end of the oligonucleotide were determined in a DNA-sequencing matrix (denaturing gels containing formamide). The results indicated slight differences in the fluorescence properties of the free dyes compared to those of the dye/ primer conjugates in this particular matrix. Inspection of the ground-state absorption spectra showed significant aggregation for the free dyes in this solution, but the conjugated dyes exhibited no sign of aggregation due to the highly anionic nature of the oligonucleotide. The fluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, with an average variation of 51 ps, an adequate difference to allow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyes were found to be very similar. When the dye/primer conjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, they comigrated, indicating that, in single-lane sequencing applications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependent mobility shifts.

DNA↗

PubMed1999

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.

Adenoviridae↗

PubMed1991

Cytotoxic action of triphenyltin on mouse thymocytes: a flow-cytometric study using fluorescent dyes for membrane potential and intracellular Ca2+.

Effects of triphenyltin on mouse thymocytes were examined using fluorescent dyes to monitor membrane potential and intracellular Ca2+. Triphenyltin at 3 x 10(-7) M to 1 x 10(-6) M hyperpolarized thymocytes and depolarized them at 3 x 10(-6) M or more, associated with increasing intracellular Ca2+. Hyperpolarization was suppressed by quinine, but not by tetraethylammonium and 4-aminopyridine, suggesting the involvement of Ca(2+)-activated K+ current. Triphentyltin failed to hyperpolarize thymocytes in Ca(2+)-free solution. Results indicate that triphenyltin promotes Ca(2+)-influx to thymocytes. Such an action of triphenyltin may be related to the immunotoxicity of organotins.

4-Aminopyridine↗

PubMed1989

Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Animals↗

PubMed1988

Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis.

We have stripped small (3 x 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.

Journal Article↗

PubMed2003

Fluorescent dyes as probes to study lipid-binding proteins.

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.

Anilino Naphthalenesulfonates↗

PubMed1989

Interaction of the fluorescent dye 1-N-phenylnaphthylamine with Escherichia coli cells during heat stress and recovery from heat stress.

The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.

1-Naphthylamine↗

PubMed2005

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein.

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.

Arabidopsis↗

PubMed1983

Advantages of preelectrophoretic conjugation of polypeptides with fluorescent dyes.

A rapid simple procedure is described for the conjugation of proteins, glycoproteins, and peptides with the fluorescent dye fluorescein isothiocyanate during the time required to polymerize a polyacrylamide gel. Such conjugation does not perturb the electrophoretic mobility of the polypeptides in detergent containing gels. The location of polypeptide . dye conjugate is evident by inspection immediately upon removal of a gel from an electrophoresis cabinet avoiding the time required for postelectrophoretic staining and destaining procedures. The sensitivity of detection of polypeptide . fluorescein conjugates is at least equivalent to that obtained using Coomassie blue.

Animals↗

PubMed1983

Kinetics of sodium-dependent solute transport by rabbit renal and jejunal brush-border vesicles using a fluorescent dye.

The kinetics of Na-coupled solute transport by renal and jejunal brush-border vesicles in the rabbit were examined using the potential-sensitive fluorescent dye diS-C3-(5). All organic solutes known to be transported across these membranes by Na-coupled mechanisms increase the fluorescence of the dye in the presence of Na, but not K. An increase in fluorescence (delta F) corresponds to a depolarization of the electrical potential difference (5-60 mV) across the brush-border membrane in the intact cell. delta F was independent of the valency of the transported solute. The fluorescence response was saturable, and for twelve solutes the Kf, i.e. the concentration of the substrate generating 50% of the maximal response, agreed quite closely with the Kt values reported from tracer studies. For six solutes increasing the Na concentration decreased Kf, and this agrees with the effect of Na on the kinetics of succinate transport in renal vesicles. We conclude that D-glucose, neutral amino acids and imino acids are co-transported with Na across both renal and jejunal brush-border membranes, and that carboxylic acids, beta-amino acids, and dibasic amino acids are co-transported with Na across the renal, but not jejunal, membranes.

Amino Acids↗

PubMed1999

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.

Electron Spin Resonance Spectroscopy↗

PubMed2001

Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes.

The excitation spectra of Nile red and SYPRO red, two currently used dyes for the fluorescent staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels, show an excitation peak in the UV region and another in the visible region (maximum at about 550 nm). Ethidium bromide and other intercalating dyes, e.g. propidium iodide, ethidium dimers, and benzoxazolium-4-quinolinium dimer-3 (YOYO), used for the fluorescent staining of DNA bands in agarose gels also show an excitation peak in the same region of the visible spectrum. We have designed and constructed a green-light transilluminator with an emission maximum at 542 nm. This visible transilluminator allows the detection of protein bands stained with Nile red and SYPRO red with the same sensitivity obtained with a 300 nm UV transilluminator. The green-light transilluminator also allows the detection of about 2 ng of DNA per band in gels stained with ethidium bromide and the other intercalating dyes indicated above. In contrast to the UV transilluminators, the green-light transilluminator does not produce photodamage of DNA even after long exposures (10 min). This makes this transilluminator very useful for preparative work. Furthermore, the green-light transilluminator does not require UV safety equipment and, consequently, it can be very convenient for teaching laboratories.

Animals↗

PubMed1995

"Satellite cells" and nerve terminals in the crayfish opener muscle visualized with fluorescent dyes.

Nerve terminals and associated cells on the muscle's surface were visualized in the crayfish opener muscle with several fluorescent dyes in conjunction with confocal microscopy and conventional fluorescence microscopy. The nerve terminals of the excitatory and inhibitory axons were best seen with 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). This dye is selectively accumulated in mitochondria, which are numerous both in the axons and in synapse-bearing terminal varicosities. Muscle nuclei were also clearly visualized, because they excluded 4-Di-2-Asp but were stained by acridine orange (AO). A positive attraction between muscle nuclei and nerve terminals was evident by visual inspection and was confirmed by spatial statistics. Additional flat cells on the muscle's surface appeared as bright rings with elongated processes that were often close to or overlapped nearby nerve terminals. The structure of these cells was established by electron microscopy after labeling them with fluorescent polystyrene beads, which could be found over structures on the muscle surface in sections of embedded specimens. The flat surface cells were distinct from peripheral glial cells closely associated with axons and nerve terminals. Nevertheless, spatial statistics showed that the surface cells were grouped near nerve terminals. They occupied a small fraction of the muscle cell's surface. Their functional role has not been determined in crustacean muscles.

Animals↗

PubMed2006

Resonance energy transfer from a fluorescent dye to a metal nanoparticle.

A quantum mechanical theory of the rate of excitation energy transfer from a fluorescent dye molecule to the surface plasmonic modes of a spherical metal nanoparticle is presented. The theory predicts the distance dependence of the transfer rate to vary as 1/d(sigma), with sigma=3-4 at intermediate distances, in partial agreement with the recent experimental results. Förster's 1/d(6) dependence is recovered at large separations. The predicted rate exhibits nontrivial nanoparticle size dependence, ultimately going over to an asymptotic, a(3) size dependence. Unlike in conventional fluorescence resonance energy transfer, the orientational factor is found to vary between 1 and 4.

Energy Transfer↗

PubMed2005

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes.

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.

Animals↗

PubMed2003

Confocal laser scanning microscopy of urinary bladder after intravesical instillation of a fluorescent dye.

OBJECTIVES: To assess the potential of confocal laser scanning microscopy for imaging of the urinary bladder after intravesical instillation of a fluorescent dye. METHODS: The study was performed on the bladder of male Copenhagen rats. For confocal fluorescence microscopy (CFM), a standard confocal laser scanning microscope (Zeiss LSM 410) was used. Before measuring, the fluorescent marker SYTO 17 was instilled intravesically. After 2 hours of incubation, the rat was killed, the bladder excised and opened, and CFM was performed starting from the surface going through the urothelium and superficial layers of the lamina propria. Except for the opening incision, the bladder was left intact and no biopsies were taken. After imaging, the bladder was sent for conventional histologic studies. RESULTS: CFM allows imaging of cellular details of the entire urothelium (superficial umbrella cells, intermediate, and basal urothelial cells) and superficial layers of the lamina propria. CFM images are close to those obtained by standard microscopy after conventional hematoxylin-eosin staining. Cell structure (eg, shape, size, chromatin texture, nucleoli, mitotic figures, nuclear/cytoplasmic ratio), as well as the structure of the connective tissue (eg, collagen fibers, blood vessels, erythrocytes), can be studied, allowing a standard histologic evaluation. Furthermore, in contrast to conventional histologic evaluation, CFM provides three-dimensional information and allows the study of intact tissue representing the true in vivo situation. CONCLUSIONS: CFM enables the study of the microscopic anatomy of bladder mucosa in its in vivo state. In combination with optical fiber bundles, endoscopic microscopy of the bladder may be possible in the future.

Administration, Intravesical↗