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At least 19 recordsLinked to original sources
PubMed1996

Marking microelectrode penetrations with fluorescent dyes.

Fluorescent dyes were used to mark and identify the tracks left by extracellular microelectrodes in neurophysiological experiments. Forty-two penetrations were made into the postcentral gyrus of 3 Macaque monkeys with electrodes coated with 1 of 5 fluorescent dyes (DiI, DiO, DiI-C5, PyPO, and Fast Blue). The electrodes were driven at rates ranging from 10 to 1000 microns/min, to a depth of about 4000 microns, where a small electrolytic lesion was made. Histological sections were viewed under fluorescent optics and the electrode tracks were reconstructed from the dye traces. Fluorescent traces (width 50-400 microns) were observed in 41 of 42 penetrations with 24 traces extending to the lesion site. Of the electrodes driven in less than 3 h, those coated with DiI (8/8) and DiI-C5 (8/8) left a trace to the lesion site, while 57% (4/7) of the DiO, 40% (2/5) of the Fast Blue and only 11% (1/9) of the PyPO tracks were fully marked. This method of marking penetrations can be used with any extracellular recording configuration, does not require tissue sections to be processed or stained, does not require electrical lesions, and causes no detectable tissue damage. Because the dyes fluoresce at different wavelengths, closely spaced tracks can be uniquely identified.

Animals↗
PubMed1985

[Hydrophobic acridine dyes for fluorescent staining of mitochondria in living cells. 3. Specific accumulation of the fluorescent dye NAO on the mitochondrial membranes in HeLa cells by hydrophobic interaction. Depression of respiratory activity, changes in the ultrastructure of mitochondria due to NAO. Increase of fluorescence in vital stained mitochondria in situ by irradiation].

The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1 X 10(-8) M is sufficient for vital staining and at 5 X 10(-7) M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days. The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction. With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5 X 10(-7) M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size. After incubation of the HeLa-cells with relatively high NAO concentrations, 5 X 10(-6) M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappears within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1 X 10(-7) M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoacridines↗
PubMed1989

Alcian yellow as a fluorescent dye.

Fluorescence characteristics of the cationic dye Alcian yellow are described. Under ultraviolet excitation, the chromatin and basophilic cytoplasm from cell smears show a blue-white emission, which depends on the presence of nucleic acids. Glycosaminoglycans-containing structures (mast cell granules, cartilage matrix) appear brightly fluorescent. The excitation at 320 less than or equal to lambda less than or equal to 340 nm is the most suitable, and the emission wavelength shows dependence on the dye concentration.

Animals↗
PubMed1981

Lucifer dyes--highly fluorescent dyes for biological tracing.

Lucifer dyes are intensity fluorescent 4-aminonaphthalimides which are readily visible in living cells at concentrations and levels of illumination at which they are nontoxic. Because of their low molecular weight they frequently pass from one cell to another; this widespread phenomenon, termed dye-coupling, is thought to reveal functional relationships between cells. Lucifer dyes can also be used for ultrastructural tracing by comparison of electron micrographs with light micrographs of the same thin section. In addition, they show promise for backfilling neurones through cut nerves, for visualizing the results of retrograde axonal transport and for the covalent labeling of macromolecules.

Animals↗
PubMed1986

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids.

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.

Estriol↗
PubMed2003

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

Animals↗
PubMed1984

Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes.

Certain fluorescent dyes, previously reported to localize mitochondria, when used at higher concentrations also localize a continuous net-like structure in both living and glutaraldehyde-fixed cells. A similar reticular structure can be detected by phase-contrast microscopy and whole-mount electron microscopy in potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patch-like areas, and is likely to be the endoplasmic reticulum (ER). The organization of the reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monensin, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone.

Animals↗
PubMed1990

A model study for quantification of approximal caries with a fluorescent dye.

The fluorescence intensity of the dye Fluorol 7GA in in vitro caries lesions was measured through a strip of plastic either 2 or 1.5 mm in thickness. The light scattering in the plastic strips was comparable with light scattering in sound enamel. This method was chosen as a model for fluorescent dye measurement at approximal sites, where the fibreoptic probe cannot be placed directly on the lesion, so that measurements have to be made through a thin layer of sound enamel. It appeared that the fluorescent dye can be measured through a layer of scattering material and that a linear correlation exists between fluorescence intensity and calcium loss as measured by longitudinal microradiography. The correlation coefficient for the 2-mm plastic strip was r = 0.76. With the 1.5-mm plastic strip the fluorescence intensity recordings were about twice as high, and the correlation coefficient was r = 0.81. From these measurements a proposal for an application procedure emerged for the use in a clinical situation. Also the transmission of light at 550 and 650 nm, at which wavelengths the fluorescent dye does not absorb, through plastic strips of varying thickness was measured. It is shown that by measurement of transmission of a non-adsorbing wavelength, the fluorescence intensity measured through a layer of scattering material can be corrected for this layer.

Animals↗
PubMed1999

Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.

Animals↗
PubMed1998

Advances in the automated detection of metaphase chromosomes labeled with fluorescence dyes.

Applications of fluorescence in situ hybridization (FISH) for translocation studies and biological dosimetry would benefit substantially from reliable and efficient automatic detection of metaphase chromosomes labeled with fluorescent dyes. We replicated and evaluated a fluorescence metaphase finder previously developed at the Medical Research Council (MRC), Human Genetics Unit (Scotland) and at Lawrence Berkeley Laboratory (LBL; California). The MRC/LBL system seemed to detect nearly all of the metaphases on the test slides, but it presented an unacceptable number of false positives (about five false positives per one true positive). Furthermore, we determined that the system actually overcalled true detections by counting certain metaphase spreads twice (duplicates). Through modifications of the MRC/LBL system, we developed the Lawrence Livermore National Laboratory (LLNL) system, which minimizes the detection of duplicates, incorporates new detection features, uses a binary decision tree (BDT) for classification, and provides functionalities to improve scanning accuracy and improve the post-detection review. To test the new system, DAPI-stained preparations of metaphase chromosomes from blood lymphocytes of four unrelated donors were placed on slides in drops ranging from 7 mm to 20 mm in diameter. Drops contained between 5 and 200 scorable metaphases each. The LLNL system achieved approximately 90% detection of non-duplicated metaphases as verified by an expert cytogeneticist, with typically less than one false positive per every one true positive detected.

Automation↗
PubMed1986

Two rapid methods of counterstaining fluorescent dye tracer containing sections without reducing the fluorescence.

A method is described for counterstaining neural tissue containing cells that are retrogradely labeled by fluorescent dyes or horseradish peroxidase (HRP). Specifically, protocols are detailed for the combined use of the tracers with Methylene blue for a Nissl stain or with silver methods for the detection of acetylcholine esterase. The usefulness of these techniques is evaluated in relation to cortico-cortico and thalamocortico projections. The findings indicate that the methods do not mask the labeling of the most sensitive fluorescent dyes or by HRP. Only the yellow dyes are significantly affected by the Methylene blue counterstain. Further, Fast blue labeling in neurons is not significantly diminished by the Bodian fiber stain. The effect of coverslipping sections containing fluorescent dye labeled cells also was evaluated and found to significantly extend the life of the labeling while not reducing the sensitivity. Thus the two counterstaining techniques provide excellent structural information, do not seriously affect tracer labeling and have few of the disadvantages common to other counterstaining methods.

Acetylcholinesterase↗
PubMed1991

Detection of Plasmodium falciparum infection with the fluorescent dye, benzothiocarboxypurine.

The fluorescent dye benzothiocarboxypurine (BCP) intensely stains nucleic acids. The dye does not penetrate viable white blood cells but does stain these cells following fixation. It has also been found that the dye stains the nucleic acid of viable Plasmodium falciparum. We have subsequently evaluated the staining of P. falciparum by benzothiocarboxypurine within red blood cells and have found that the red blood cell membrane is freely permeable to this dye and consequently P. falciparum is stained within the red blood cell. This finding prompted an in-depth analysis of the dye in the laboratory and in a field study as an alternative to Giemsa-stained blood smears and as a means of enhancing the microscopic diagnosis of malarial infection. In a field study the BCP dye allowed detection of malaria in fresh blood at a level equivalent to the Giemsa method (parasitemia ranged from 0.01% to 30%). The BCP staining procedure could also be used with fixed specimens although the differential staining characteristics were lost following specimen preparation. Of 111 blinded samples obtained in the field 22 were negative by Giemsa-stained thin smear, 16 were negative on thick smear and the same 16 were negative by BCP analysis. We have found that the BCP dye offers many advantages compared with the microscopic diagnosis of P. falciparum infection with standard Giemsa stains. These advantages are especially evident in conditions of low parasitemia, in the speed of staining and evaluation, and the relatively low level of training required to provide consistent results.

Animals↗
PubMed1993

The concentration jump method. Kinetics of vital staining of mitochondria in HeLa cells with lipophilic cationic fluorescent dyes.

Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; Rm values and the partition coefficients Po/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye CD can be increased from CD = 0 to CD > 0 within a few seconds (concentration jump). Thus, the time t = 0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, CD = const., are unambiguously defined. The concentration of the dye, Cb, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence Ib. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, I(f) << Ib. The intensity of the fluorescence I = Ib was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t-->infinity. In the linear range of I(t) the flow J(o) = (dI/dt)o of the dye into the cell depends strongly on the dye concentration and increases linearly with CD. The concentration range CD = 10(-9)-10(-5) M at 37 degrees C was investigated. From the linear correlation between J(o) and CD it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t = 0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJ(o)/dCD of the plot J(o) vs CD describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.

Buffers↗
PubMed1984

The cortical projection of the basolateral amygdaloid nucleus in the rat: a retrograde fluorescent dye study.

The fluorescent dye, retrograde labeling technique was used to determine the extent of the projection from the basolateral nucleus of the amygdala to the neocortex in the rat. Each rat received a single cortical injection of fast blue, and in one-half of the animals, a subsequent injection of nuclear yellow was placed in a different cortical region. An analysis of the results demonstrates that the projection to the midline cortex arises in the medial neurons within the caudal two-thirds of the basolateral nucleus. This projection is directed to the anterior cingulate cortex, but not to the posterior cingulate cortex. The primary motor cortex receives a basolateral amygdala projection which originates from neurons in two areas, (1) the medial part of the anterior one-third of the nucleus and (2) the center (in the lateral to medial axis) portion of the posterior two-thirds of the nucleus. The latter neurons are situated lateral to the neurons projecting to the cingulate cortex. Somatosensory cortex injections label many fewer basolateral nucleus neurons than do motor cortex injections, but these neurons are located in a position similar to that of those labeled by motor cortex injections. Finally the gustatory cortex, which lies just dorsal to the rhinal sulcus, receives a basolateral projection from neurons in the lateroventral one-half of the basolateral nucleus. These results demonstrate that the basolateral nucleus gives rise to a rather widespread and topographically organized projection to the anterior half of the neocortex of the rat.

Amidines↗
PubMed1987

The organization of trigeminotectal and trigeminothalamic neurons in rodents: a double-labeling study with fluorescent dyes.

Retrogradely transported fluorescent dyes (fast blue and diamidino-dihydrochloride yellow) were used to compare the distributions of trigeminofugal neurons that project to the superior colliculus and/or the thalamus in three rodent species. The objective was to determine what the projection and collateralization patterns of these trigeminofugal pathways are and whether they are similar among different species. In each anesthetized animal, one dye was injected into the superior colliculus and the other into the topographically congruent area of the thalamus. Counts of the numbers of yellow, blue, and double-labeled neurons were made throughout the trigeminal complex: principalis, pars oralis, pars interpolaris, and pars caudalis. Trigeminothalamic projections were similar in each of the rodent species studied. The densest concentration of retrogradely labeled neurons was in principalis, with substantially fewer neurons in pars interpolaris, and fewer still in pars oralis and pars caudalis. These neurons were generally small and tended to have round or fusiform somata. A common pattern was also noted among the three species for trigeminotectal neurons. Most trigeminotectal projections originated from neurons in pars interpolaris, somewhat fewer from pars oralis, and the fewest from principalis and pars caudalis. These neurons tended to be the largest in each subdivision and were often multipolar. Following paired injections of the tracers, double-labeled neurons were scattered throughout the sensory trigeminal complex and had morphologies characteristic of single-labeled trigeminotectal neurons. Although comparatively few double-labeled neurons were observed in any species, most of those seen were restricted to the ventrolateral portion of pars interpolaris, a position that corresponds to the representation of the vibrissae. These data indicate that, regardless of the rodent species, the vast majority of labeled trigeminal neurons project either to the superior colliculus or the thalamus, but not to both targets. This might be expected on the basis of the very different behavioral roles these structures play. On the other hand, a subpopulation of trigeminal neurons exists (mainly in pars interpolaris) that does project to both the superior colliculus and the thalamus, perhaps because both structures require some of the same somatosensory information to perform their behavioral functions.

Animals↗
PubMed1993

Synthesis and characterization of 5'-fluorescent-dye-labeled oligonucleotides.

Fluorescent-dye-labeled oligonucleotides are used in many procedures, including DNA sequencing, PCR, restriction mapping, the study of genetic disease, and forensics identification. In this paper, we describe detailed methods for the synthesis, purification, and quantification of 5'-fluorescent-dye-labeled oligonucleotides. The relationship of specific and nonspecific dye attachment to synthetic oligonucleotides is discussed, and the importance of primer design is considered as a way to prevent chemical problems from interfering with biological reactions.

Chromatography, High Pressure Liquid↗
PubMed1998

Synthesis of novel fluorinated coumarins: excellent UV-light excitable fluorescent dyes.

Two new fluorinated fluorescent dyes, 6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue), exhibit excellent photophysical properties among a series of novel fluorinated 7-hydroxycoumarins. Most of these fluorinated coumarins have quantum yields (0.63 to 0.89) equal to or higher than that of the parent compound (0.63), which, in combination with their lower pKaS and higher photostability, make them superior fluorescent dyes for use as reporter molecules in biological systems.

Coumarins↗