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PubMedDate not supplied

Spectroscopic study and evaluation of red-absorbing fluorescent dyes.

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

Biotinylation↗
PubMedDate not supplied

[Use of low-molecular fluorescent dyes for localizing tumor cells].

A novel fluorescence microscopic technique, based on the application of low-molecular fluorescent dyes, was used to study the expression of the enzyme guanidinobenzoatase in malignant tumors of the head and neck area. Frozen sections have shown that moderately and highly differentiated squamous cell carcinomas express this enzyme, particularly in the area of tumor invasion. The protease is capable of breaking down fibronectin, an important component of the extracellular matrix. In less differentiated squamous cell carcinomas as well as malignant melanomas almost all tumor cells exhibited guanidinobenzoatase activity. The fluorescence microscopic technique presented here provides a fast and easy method for the localization of tumor cells. At the same time it provides a better understanding of the biological control of a tumor-associated protease, thus furnishing important new information on the invasion behavior of malignant tumors.

Carcinoma, Squamous Cell↗
PubMed1983

[Mechanisms of the damaging effect of fluorescent dyes on the retina].

Mechanisms of the photo-damage of a fluorescent dye (methylene blue) and of the protective action of antioxidants and quenchers of singlet oxygen on the outer retinal rod segments (ORRS) and retinal function in situ and in vivo were studied. The methylene blue-induced formation of singlet oxygen in the ORRS resulted in accumulation of lipid peroxidation products, oligomerization of rhodopsin, and in a decrease in rhodopsin thermal resistance. Modification of the lipid and protein components of the visual cells by singlet oxygen inhibited the electrical activity of both isolated frog retina in situ and rabbit retina in vivo (waves a and b on the electroretinogram). The antioxidants (alpha-naphthol, alpha-tocopherol, 4-methyl-2,6-di-tert-butylphenol) and singlet oxygen quenchers, [1,4-diazabicyclo (2,2,2)octane] and alpha-tocopherol prevented the damaging effects of the fluorescent dye induced by singlet oxygen formation.

Animals↗
PubMed1982

The use of fluorescent dyes to measure membrane potentials: a response.

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related tot he application of the dyes have been reviewed. These problems include: 1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, 2) the effects of altered energy metabolism on the fluorescent response of the dyes and on Em, and 3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.

Adenosine Triphosphate↗
PubMed2006

Methoxyperylene bisimides and perylene lactame imides: novel, red fluorescent dyes.

The synthesis of methoxyperylene bisimides and perylene lactame imides with aliphatic N-substituents is described. Both classes of dyes exhibit fluorescence in the bathochromic region of visible light so that red light is obtained. The lightfastness of the dyes is very high, thus, there is special interest for diverse applications.

Journal Article↗
PubMed2003

Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes.

Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system.

Animals↗
PubMed2003

Extraction of periodic multivariate signals: mapping of voltage-dependent dye fluorescence in the mouse heart.

In many experimental circumstances, heart dynamics are, to a good approximation, periodic. For this reason, it makes sense to use high-resolution methods in the frequency domain to visualize the spectrum of imaging data of the heart and to estimate the deterministic signal content and extract the periodic signal from background noise in experimental data. In this paper, we describe the first application of a new method that we call cardiac rhythm analysis which uses a combination of principal component analysis and multitaper harmonic analysis to extract periodic, deterministic signals from high-resolution imaging data of cardiac electrical activity, We show that this method significantly increases the signal-to-noise ratio of our recordings, allowing for better visualization of signal dynamics and more accurate quantification of the properties of electrical conduction. We visualize the spectra of three cardiac data sets of mouse hearts exhibiting sinus rhythm, paced rhythm and monomorphic tachycardia. Then, for pedagogical purposes, we investigate the tachycardia more closely, demonstrating the presence of two distinct periodicities in the re-entrant tachycardia. Analysis of the tachycardia shows that cardiac rhythm analysis not only allows for better visualization of electrical activity, but also provides new opportunities to study multiple periodicities in signal dynamics.

Action Potentials↗
PubMed1979

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.

Cells, Cultured↗
PubMed1991

Detection of lymphocyte subsets using three-color/single-laser flow cytometry and the fluorescent dye peridinin chlorophyll-alpha protein.

The fluorescent dye, Peridinin chlorophyll A protein (PerCP) derived from dinoflagellate organisms (Glenodinium sp.) can be excited by a 488 nm laser and emits light with a large Stokes shift and no major spectral overlap with commonly used chromophores such as fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE). PerCP was conjugated directly to various mouse monoclonal antibodies (mAb) specific for human leukocyte markers or to avidin for use with biotinylated-mAb, and used to perform three color single-laser flow cytometry. The efficacy of this method was demonstrated by analyzing the heterogeneity of thymus T lineage subsets and B lymphocyte subsets in blood. CD4-CD8-, CD4+CD8+ and CD4+CD8- or CD4-CD8+ subsets differ in their expression of cell-cell interaction markers including CD18, CD28, CD44 and Leu 8, and activation/subset markers CD45RO, CD45RA and CD26. Some CD5+ peripheral blood B cells, unlike CD5-B cells, expressed CD45RO or high levels of CD54 (ICAM-1) suggesting the CD5+ B cell population contains activated lymphocytes. The availability of such an accessible method for three color analysis will make it possible to do routine three color monitoring of immunologic diseases such as AIDS, and autoimmune or periodontal diseases.

Antibodies, Monoclonal↗
PubMed2004

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays.

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.

Fluorescent Dyes↗
PubMed1987

[Quantitative evaluation of the uptake capacity of intact thrombocytes using fluorescent dyes].

A new approach to quantitative determination of fluorescent dye uptake by intact cells is suggested. Fluorescent amine acridine orange selectively accumulating in 5HT granules of platelets has been used. Fluorescence signal analysis allows the estimation of a relative granule volume and the ratios of acridine orange transfer over cytoplasmic and granule membranes. The following results were obtained in human and rabbit platelets: a relative granule size was 14 +/- 1 % and 29 +/- 2 % of the total cell volume, intra-granule to extra-granule dye concentration ratios were 2260 +/- 382 and 30000 +/- 5550, while intra-cytoplasm to extra-cytoplasm concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.

Acridine Orange↗
PubMed2005

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability.

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.

Base Sequence↗
PubMed1988

Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours.

The DNA-binding fluorescent dye Hoechst 33342 (H33342) has been used in a series of investigations of the vascular parameters of two murine tumours. This dye has been shown, to have a short half-life in the circulation (T1/2 less than 2 min), but is stably bound for at least 2 h after it enters cells. It can be used in morphometric studies on frozen sections to determine the effective vascular volume, the capillary fraction and the size distribution of blood vessels in each tumour. These latter two parameters cannot be deduced from the less labour intensive techniques using radioactive isotopes. The effective vascular volume perfused in 1 min by H33342 was compared with the volume perfused in 30 min with 51Cr labelled erythrocytes. Similar volumes were estimated with the two techniques in a murine carcinoma and in a sarcoma. Both techniques showed that the vascular volume decreased in larger tumours. The H33342 analysis of vessel size showed the decrease in capillary vessels in the carcinomas was even greater, falling from 70% in small tumours to 20% in larger tumours. The deteriorating vascular network in larger tumours is associated with an increasing fraction of necrotic tissue. Experiments in which the isotopes and dye were co-injected suggest that at 40 mgkg-1 the dye may rapidly lead to a partial shutdown of the tumour vascular bed. This is less marked with 20 mg kg-1. In spite of this effect there is in general a close correlation between the volumes perfused by labelled red blood cells and the fluorescent dye.

Adenocarcinoma↗
PubMed1995

Topographical projections from the medial septum-diagonal band complex to the hippocampus: a retrograde tracing study with multiple fluorescent dyes in rats.

Direct projections from the medial septum-diagonal band complex (MS-DB) to the hippocampus were studied in rats using the retrograde labeling method with fluorescent dyes. In the first series of experiments, the distribution patterns of retrogradely labeled cells in the MS-DB were examined in sagittal sections, following simultaneous injection of two different fluorescent dyes separately into the ventral hippocampus (VHPC) and the dorsal hippocampus (DHPC). It was found that the DHPC received fiber terminals mainly from the rostral half of the MS-vertical limb of the diagonal band (VDB) and the core part of the horizontal limb of the diagonal band (HDB), whereas the VHPC received fiber terminals largely from the caudal half part of the MS-VDB and the marginal part of the HDB. The marginal part appears to form a 'C' and not a complete circle around the core part. In the second series of experiments, the distribution patterns of retrogradely labeled cells in the MS-DB were investigated in sagittal sections, following simultaneous injection of three or two different fluorescent dyes separately into the subfields of the DHPC. The CA1 received fiber terminals mainly from the HDB and VDB, while the hilus received fiber terminals mainly from the MS. The CA2-CA3 received fiber terminals almost equally from the MS, VDB and HDB. The present results reveal distinctive topographical organizations of the septohippocampal connections.

Animals↗
PubMed2003

Application of the novel fluorescent dye Beljian red to the differentiation of Giardia cysts.

Beljian red (BR) is a novel long Stokes shift fluorescent dye that fluoresces orange when illuminated with UV or blue light. Due to its long Stokes shift, and the fact that it is excitable at 488 nm, BR has particular utility in multi-colour applications with short Stokes shift fluorophores such as fluorescein. Here we have demonstrated that BR can be used to discriminate Giardia cysts seeded into water samples from those naturally present in the sample. We show that the dye does not interfere with other staining methods such as DAPI, and is compatible with mAb-FITC staining in a multi-colour fluorescence technique. This should be useful in determining the specific recovery of protozoan parasites from environmental samples.

Animals↗
PubMed1978

Probes of transmembrane potentials in platelets: changes in cyanine dye fluorescence in response to aggregation stimuli.

A noncovalent fluorescent probe that responded to changes in transmembrane potential was used to study the response of washed human platelets to aggregating agents. Concentration-dependent changes in the fluorescence were observed in response to ADP and to thrombin. No such changes were observed in response to collagen fibrils. Thus there was an indication that platelet membrane potential changed in response to aggregating stimuli, supporting the hypothesis that the mechanisms of platelet aggregation resembled the mechanisms of other systems that show stimulus-response coupling (e.g., muscle, adrenal chromaffin cells). The different responses to specific agents indicate that the agents may trigger platelet aggregation through different mechanisms.

Adenosine Diphosphate↗