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Novel fluorescent dyes based on oligopropylamines for the in vivo staining of eukaryotic unicellular algae.  


Weakly basic fluorescent dyes are used to visualize organelles within live cells due to their affinity to acidic subcellular organelles. In particular, they are used to stain the silica deposited in the silica deposition vesicles (SDVs) of diatoms during the course of their frustule synthesis. This study involved the synthesis of fluorescent dyes derived from oligopropylamines, compounds similar to those found in diatoms. The dyes were obtained by reacting oligopropylamines with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The reaction was realized using methylated oligopropylamines with two or three nitrogen atoms and yielded two novel fluorescent dyes: NBD-N2 and NBD-N3. The dyes appeared to be highly efficient in the in vivo staining of growing siliceous frustules of diatoms at concentrations at least 10 times lower than those required for staining with HCK-123. NBD-N3 also efficiently stained other subcellular vesicles of eukaryotic unicellular algae. NBD-N2 stained only growing diatom frustules, whereas NBD-N3 also stained various subcellular organelles of different eukaryotic unicellular algae. NBD-N2 and NBD-N3 were not removed from stained diatom frustules by drastic treatments with H(2)SO(4) and H(2)O(2). Fluorescent silica can also be obtained by its chemical precipitation in the presence of NBD-N2 and NBD-N3. PMID:20691656

Annenkov, V V; Danilovtseva, E N; Zelinskiy, S N; Basharina, T N; Safonova, T A; Korneva, E S; Likhoshway, Y V; Grachev, M A



Staining and photography for chromosome banding with the fluorescent dyes quinacrine mustard and Hoechst 33258  

Microsoft Academic Search

The techniques of chromosome banding are now used in many laboratories for precise chromo- some identification. Chromosome band morphology is recognized and enhanced by staining with Giemsa or fluorescent dyes. The Giemsa or G bands produced by pretreatment with trypsin, urea or acid-saline (ASG) are almost the same bands as the Q bands produced during staining with quinacrine mustard. The

Mary R. Bordelon



Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA  

PubMed Central

The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

Nyberg, Lena; Persson, Fredrik; Akerman, Bjorn; Westerlund, Fredrik



Fluorescent staining of gels.  


Certain transition metal complexes show intensive fluorescence when bound to proteins. They can be used to stain gels after electrophoresis with a sensitivity approaching that of silver staining, but in a much simpler and more reproducible procedure. Stains can be prepared easily and at a fraction of the cost of commercially available reagents.Hydrophobic dyes can be used to stain gels without fixing; they do not interfere with later blotting or electro-elution. PMID:22585519

Buxbaum, Engelbert



Detection of glycoproteins in polyacrylamide gels using Pro-Q Emerald 300 dye, a fluorescent periodate Schiff-base stain.  


Pro-Q Emerald 300 glycoprotein stain generates a bright-green fluorescent signal upon reacting with periodic acid-oxidized carbohydrate groups on proteins. With this dye, it is possible to detect proteins directly in the gel without the need to transfer them to a membrane. This dye is more sensitive than the standard periodic acid Schiff's base which uses acidic fuchsin dye. PMID:22585521

Mehta-D'souza, Padmaja



A Novel Staining Protocol for Multiparameter Assessment of Cell Heterogeneity in Phormidium Populations (Cyanobacteria) Employing Fluorescent Dyes  

PubMed Central

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, ‘dead cell’ nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4?,6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead.

Tashyreva, Daria; Elster, Josef; Billi, Daniela



A novel staining protocol for multiparameter assessment of cell heterogeneity in Phormidium populations (cyanobacteria) employing fluorescent dyes.  


Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, 'dead cell' nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4',6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead. PMID:23437052

Tashyreva, Daria; Elster, Josef; Billi, Daniela



Candida, fluorescent stain (image)  


This microscopic film shows a fluorescent stain of Candida. Candida is a yeast (fungus) that causes mild disease, but in immunocompromised individuals it may cause life-threatening illness. (Image ...


Exploring the dynamics of fluorescence staining of bacteria with cyanine dyes for the development of kinetic assays  

NASA Astrophysics Data System (ADS)

Bacterial infections continue to be one of the major health risks in the United States. The common occurrence of such infection is one of the major contributors to the high cost of health care and significant patient mortality. The work presented in this thesis describes spectroscopic studies that will contribute to the development of a fluorescent assay that may allow the rapid identification of bacterial species. Herein, the optical interactions between six bacterial species and a series of thiacyanine dyes are investigated. The interactions between the dyes and the bacterial species are hypothesized to be species-specific. For this thesis, two Gram-negative strains, Escherichia coli (E. coli) TOP10 and Enterobacter aerogenes; two Gram-positive bacterial strains, Bacillus sphaericus and Bacillus subtilis; and two Bacillus endospores, B. globigii and B. thuringiensis, were used to test the proposed hypothesis. A series of three thiacyanine dyes---3,3'-diethylthiacyanine iodide (THIA), 3,3'-diethylthiacarbocyanine iodide (THC) and thiazole orange (THO)---were used as fluorescent probes. The basis of our spectroscopic study was to explore the bacterium-induced interactions of the bacterial cells with the individual thiacyanine dyes or with a mixture of the three dyes. Steady-state absorption spectroscopy revealed that the different bacterial species altered the absorption properties of the dyes. Mixed-dye solutions gave unique absorption patterns for each bacteria tested, with competitive binding observed between the bacteria and spectrophotometric probes (thiacyanine dyes). Emission spectroscopy recorded changes in the emission spectra of THIA following the introduction of bacterial cells. Experimental results revealed that the emission enhancement of the dyes resulted from increases in the emission quantum yield of the thiacyanine dyes upon binding to the bacteria cellular components. The recorded emission enhancement data were fitted to an exponential (mono-exponential or bi-exponential) function, and time constants were extracted by regressing on the experimental data. The addition of the TWEEN surfactants decreased the rate at which the dyes interacted with the bacterial cells, which typically resulted in larger time constants derived from an exponential fit. ANOVA analysis of the time constants confirmed that the values of the time constants clustered in a narrow range and were independent of dye concentration and weakly dependent on cell density.

Thomas, Marlon Sheldon


Microencapsulated Fluorescent Dye Penetrant.  

National Technical Information Service (NTIS)

Microencapsulated fluorescent dye pentrant materials were evaluated for feasibility as a technique to detect cracks on metal surfaces when applied as a free flowing dry powder. Various flourescent dye solutions in addition to a commercial penetrant (Zyglo...

S. Allinikov



21 CFR 864.1850 - Dye and chemical solution stains.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food...HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and...



21 CFR 864.1850 - Dye and chemical solution stains.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food...HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and...



Chromosome characterization using single fluorescent dye  


Chromosomes are characterized by fluorescent emissions from a single fluorescent dye that is excited over two different wavelengths. A mixture containing chromosomes is stained with a single dye selected from the group consisting of TOTO and YOYO and the stained chromosomes are placed in a flow cytometer. The fluorescent dye is excited sequentially by a first light having a wavelength in the ultraviolet range to excite the TOTO or YOYO to fluoresce at a first intensity and by a second light having a wavelength effective to excite the TOTO or YOYO dye to fluoresce at a second intensity. Specific chromosomes may be identified and sorted by intensity relationships between the first and second fluorescence emissions.

Crissman, Harry A. (Los Alamos, NM); Hirons, Gregory T. (Irvine, CA)



A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy  

Microsoft Academic Search

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by




Nile red: a selective fluorescent stain for intracellular lipid droplets  

Microsoft Academic Search

We report that the dye nile red, 9-diethylamino-5H-benzo(a)phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading.




Observation of Soft Contact Lens Disinfection with Fluorescent Metabolic Stains  

PubMed Central

A rapid fluorescent staining method using a tetrazolium dye and propidium iodide for the in-use assessment of disinfection of Pseudomonas aeruginosa biofilms on soft contact lenses showed that 11 to 13% of cells on lenses remained actively respiring and recoverable by culture methods after 30 min of exposure to 3% hydrogen peroxide.

Gavin, J.; Button, N. F.; Watson-Craik, I. A.; Logan, N. A.



Nile red: a selective fluorescent stain for intracellular lipid droplets  

PubMed Central

We report that the dye nile red, 9-diethylamino-5H- benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red- stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.



21 CFR 864.1850 - Dye and chemical solution stains.  

Code of Federal Regulations, 2013 CFR

...CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains...hematology. (b) Classification. Class I (general controls). These devices are exempt from the premarket...



[Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine].  


Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:6203878

Petschel, K; Naujok, A; Kempter, P; Seiffert, W; Zimmermann, H W



Storable, thermally activated, near-infrared chemiluminescent dyes and dye-stained microparticles for optical imaging  

PubMed Central

Optical molecular imaging employs relatively harmless, low-energy light and technically straightforward instrumentation. Self-illuminating, chemiluminescent systems are especially attractive since they have inherently high signal contrast due to the lack of background emission. Currently, chemiluminescence imaging involves short-lived molecular species that are not stored but instead generated in situ, and they typically emit visible light, which does not penetrate far through heterogeneous biological media. Here, we describe a new paradigm for optical molecular imaging using squaraine rotaxane endoperoxides (SREPs), interlocked fluorescent and chemiluminescent dye molecules that have a squaraine chromophore encapsulated inside a macrocycle endoperoxide. SREPs can be stored indefinitely at temperatures below ?20 °C, but upon warming to body temperature they undergo a unimolecular chemical reaction and emit near infrared light that can pass through a living mouse. Dye-stained microparticles are easily prepared for in vivo near-infrared optical imaging using commercial imaging stations.

Baumes, Jeffrey M.; Gassensmith, Jeremiah J.; Giblin, Jay; Lee, Jung-Jae; White, Alexander G.; Culligan, William J.; Leevy, W. Matthew; Kuno, Masaru; Smith, Bradley D.



Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.  


The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols. PMID:10075818

Tuma, R S; Beaudet, M P; Jin, X; Jones, L J; Cheung, C Y; Yue, S; Singer, V L



An innovative brilliant blue FCF method for fluorescent staining of fungi and bacteria.  


Most natural and synthetic dyes currently used for microbial fluorescent staining are toxic or carcinogenic and are harmful to animals, humans and the environment. A food dye for microbial staining, brilliant blue FCF, was used as an alternative to lactofuchsin and lactophenol blue. Brilliant blue FCF shows pronounced microbial cell fluorescence staining of an array of pathogenic/toxigenic (Fusarium granunearum 3- and 15-acetyldeoxynivalenol chemotypes, and Escherichia coli O157:H7) and beneficial fungi and bacteria (Trichoderma harzianum and Bacillus subtilis). Brilliant blue FCF has no toxic effects on the microbes tested and is inexpensive. PMID:20560873

Chau, H W; Goh, Y K; Si, B C; Vujanovic, Vladimir



Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells  

Microsoft Academic Search

Synopsis  Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340\\/394

D. Marques; F. W. D. Rost



Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes  

PubMed Central

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1–10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.

Zhao, Zhenjun; Aliwarga, Yulina; Willcox, Mark DP



Image analysis of dye stained patterns in soils  

NASA Astrophysics Data System (ADS)

Quality of surface water and groundwater is directly affected by flow processes in the unsaturated zone. In general, it is difficult to measure or model water flow. Indeed, parametrization of hydrological models is problematic and often no unique solution exists. To visualise flow patterns in soils directly dye tracer studies can be done. These experiments provide images of stained soil profiles and their evaluation demands knowledge in hydrology as well as in image analysis and statistics. First, these photographs are converted to binary images classifying the pixels in dye stained and non-stained ones. Then, some feature extraction is necessary to discern relevant hydrological information. In our study we propose to use several index functions to extract different (ideally complementary) features. We associate each image row with a feature vector (i.e. a certain number of image function values) and use these features to cluster the image rows to identify similar image areas. Because images of stained profiles might have different reasonable clusterings, we calculate multiple consensus clusterings. An expert can explore these different solutions and base his/her interpretation of predominant flow mechanisms on quantitative (objective) criteria. The complete workflow from reading-in binary images to final clusterings has been implemented in the free R system, a language and environment for statistical computing. The calculation of image indices is part of our own package Indigo, manipulation of binary images, clustering and visualization of results are done using either build-in facilities in R, additional R packages or the LATEX system.

Bogner, Christina; Trancón y Widemann, Baltasar; Lange, Holger



Sizing of single fluorescently stained DNA fragments by scanning microscopy  

PubMed Central

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan



Novel application of low pH-dependent fluorescent dyes to examine colitis  

PubMed Central

Background Endoscopy capable of fluorescence observation provides histological information on gastrointestinal lesions. We explored the novel application of low pH-dependent fluorescent dyes for fluorescence observation of crypt structure and inflammatory cell infiltration in the colon. Methods Low pH-dependent fluorescent dyes were applied to the colonic mucosa of normal mice for observation under fluorescence stereomicroscopy system. We also examined mouse models of colitis, which were induced by trinitrobenzenesulfonic acid, dextran sulfate sodium or interleukin-10 deficiency. Results Topical application of low pH-dependent fluorescent dyes revealed crypts as ring-shaped fluorescent stains by visualizing the mucin granules of goblet cells. Because of the minimal fluorescence intensity of the low pH-dependent fluorescent dyes in phosphate-buffered saline, it was not necessary to wash the mucosa before the fluorescence observation. 4-Nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was quicker to achieve complete staining (three minutes) than LysoSensor Green DND-153 and DND-189 (20 minutes). In each type of colitis, NBD-PZ revealed the destruction of the crypts as the disappearance of the ring-shaped fluorescent stains and the infiltration of inflammatory cells as the aggregation of punctate fluorescent stains through visualization of lysosomes. Conclusions Low pH-dependent fluorescent dyes, especially NBD-PZ, are suitable for topical application to the colonic mucosa and have characteristics that allow for the histological examination of colitis.



Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)  

NASA Astrophysics Data System (ADS)

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila



Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: Optical measurement of membrane potential  

Microsoft Academic Search

Summary The absorption, fluorescence, dichroism, and birefringence of stained squid axons were measured during action potentials and voltage clamp steps in an effort to find large optical signals that could be used to monitor membrane potential. Changes in all four optical properties were found that were linearly related to membrane potential and, with several new dyes, the signal-to-noise ratios were

W. N. Ross; B. M. Salzberg; L. B. Cohen; A. Grinvald; H. V. Davila; A. S. Waggoner; C. H. Wang



Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta.  


Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. PMID:22802389

Card, Stuart D; Tapper, Brian A; Lloyd-West, Catherine; Wright, Kathryn M



A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.  


A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity. PMID:1692790

Ali, R; Sayeed, S A



Plasmon-enhanced fluorescence of dye molecules  

NASA Astrophysics Data System (ADS)

Enhanced fluorescence from Rhodamine 6G (R6G) mixed with gold colloids has been observed. We used fluorescence microscopy to correlate the fluorescence intensity of the dyes with the localized surface plasmon resonance (LSPR) spectra of the gold nanoparticles to which they are attached. Spectroscopic studies show that a 2.8-fold amplification of the fluorescence signal in presence of colloidal Au nanoparticles (GNP) was observed in resonance with plasmons. Such fluorophore-metal complex presents a unique opportunity for developing a new class of contrast agents for optical imaging and fluorescence based sensing, having a great potential for applications in the fields of medical diagnostics and biotechnology.

Iosin, Monica; Baldeck, Patrice; Astilean, Simion



Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels  

PubMed Central

The rates of dissociation of three non-intercalative unsymmetrical cyanine dyes, BEBO, BETO and BOXTO from mixed-sequence DNA have been studied with the DNA either free in solution or in confining porous agarose gels. The properties of the new dyes were compared to the related intercalating dyes BO, BO-PRO, TO-PRO and YO-PRO. With DNA in solution, BEBO dissociates more slowly than the monovalent BO and interestingly also more slowly than the divalent dye BO-PRO. Similarly, both BETO and BOXTO exhibit considerably slower dissociation than TO-PRO. The new dyes show biexponential dissociation kinetics in mixed-sequence DNA. The average rate of dissociation increases with increasing ionic strength, but the salt dependence of the dissociation is weaker than for the corresponding intercalating dye. The rate of dye-dissociation decreases by a factor of about 105 in the gel. The rates for the dyes generally follow the pattern that we observe with the DNA in free solution, however a more accentuated stabilization was seen for intercalators than for groove-bound dyes. The results show that, in particular, BOXTO is a promising candidate as a preferentially groove-bound DNA-stain with a large enhancement of the fluorescence quantum yield upon binding to DNA, and which exhibits slow and salt-insensitive dissociation compared to corresponding intercalative dyes.

Eriksson, Maja; Karlsson, H. Jonas; Westman, Gunnar; ?kerman, Bjorn



The Relative Location of the Dye Staining Endpoint Indicated With Polypropylene Glycol-Based Caries Dye versus Conventional Propylene Glycol-Based Caries Dye  

PubMed Central

Objectives This study determined the difference in the location of the caries dye staining endpoint of 1% Acid Red dye in propylene glycol versus that of 1% Acid Red dye in polypropylene glycol. Methods Freshly extracted permanent molar crowns with primary occlusal carious lesions were chisel-split axially to expose the lesion in cross-section on both halves. One half was stained with propylene glycol-based dye and the other with polypropylene glycol-based dye. For the control group, both halves were stained with propylene glycol-based dye. The dye staining front was marked on digital images of the stained split surfaces, and the images were aligned using reference notches. The distance between the marked staining front lines was measured in five locations, and the measurement protocol was repeated. Weighted averages and a 95% confidence interval for the distance between marked staining front lines were calculated for the control and experimental groups. Results The weighted average distance for the experimental group (0.298 mm, 95% confidence interval 0.240 mm – 0.357 mm) was about four times that of the control group (0.070 mm, 95% confidence interval 0.051 mm – 0.089 mm). Generally, the marked staining line for the polypropylene glycol-based dye specimens was located shallow (occlusal) to the propylene glycol-based staining line (range ?0.12 mm to 0.66 mm). Conclusions The staining endpoint of 1% Acid Red dye in polypropylene glycol is shallower than that of 1% Acid Red dye in propylene glycol. The method is useful for comparing staining endpoints of caries dye formulations. (Eur J Dent 2008;2:29–36)

Boston, Daniel W; Jefferies, Steven R; Gaughan, John P



Caffeine Interaction with Fluorescent Calcium Indicator Dyes  

Microsoft Academic Search

We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding at the indicator chelation sites. Binding affinities for calcium of

M. Muschol; B. R. Dasgupta; B. M. Salzberg



Use of Fluorescent Dyes for Readily Recognizing Sperm Damage  

PubMed Central

Sperm is produced by the testis and mature in the epididymis. For having a successful conception, the fertilizing sperm should have functional competent membranes, intact acrosome, functional mitochondria and an intact haploid genome. The effects of genetic and environmental factors result in sperm vulnerability to damage in the process of spermatogenesis and maturation. In recent years, the feasibility of detecting sperm damage is enhanced through the advances in technologies like fluoscerent staining techniques assisted with fluorescence microscope, flow cytometry and computer analysis systems. Fluoscerent staining techniques involve the use of fluorescent dyes, either directly or indirectly for binding them with some ingredients of sperm and evaluating the damage of the structure or function of the sperm, i.e. membrane, acrosome, mitochondria, chromosome or DNA.

Farah, Omar Ibrahim; Cuiling, Li; Jiaojiao, Wang; Huiping, Zhang



Counterion-dye staining for DNA in electrophoresed gels using indoine blue and methyl orange.  


In this study, we describe a sensitive staining method for DNA in agarose and polyacrylamide gels using organic visible dyes, indoine blue (IB) and methyl orange (MO). The counterion-dye staining method uses two oppositely charged dyes to form a hydrophobic ion pair complex in the staining solution. A decrease in the number of free forms of dyes in staining solution can enhance the selectivity of binding between the dye and DNA, and can reduce nonspecific background staining. As a result, the sensitivity of counterion-dye staining was significantly improved compared with other dye-based staining. This method uses a staining solution consisting of 0.008% IB, 0.002% MO, 10% ethanol and 0.2 M sodium acetate at pH 4.7, and can detect 5 ng of lambda DNA/HindIII within 60 min in agarose gels and 10 ng of PhiX174 DNA/HaeIII within 20 min in polyacrylamide gels. PMID:16568503

Hwang, Sun-Young; Jin, Li-Tai; Yoo, Gyurng-Soo; Choi, Jung-Kap



Fluorescence OPA FROG of NIR Dyes  

NASA Astrophysics Data System (ADS)

In standard applications, optical parametric amplification (OPA) is accomplished using a white light continuum as the seed. This presentation will describe a design for an ultrafast fluorescence-OPA-FROG (Frequency Resolved Optical Gating) experiment and its utility for measuring fluorescence dynamics on an ultrashort timescale. This technique has several attractive features compared to current state-of-the-art fluorescence upconversion because it has the potential to amplify weak fluorescence, detection occurs at the wavelength of the fluorescence signal in the visible or near IR spectral region, and the phase-matching condition is kpump = ksignal + kidler. We will demonstrate time gating and effective amplification of the fluorescence of common near infrared dyes, IR 125, IR 132, and IR 140 in DMSO.

Woodward, Colleen; Levinger, Nancy



Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining  

Microsoft Academic Search

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are

Keiko Goto; T. Akematsu; H. Shimazu; T. Sugiyama



Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy  

Microsoft Academic Search

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence

L. F. Borges; S. R. Taboga; P. S. Gutierrez



Fluorescence emission spectra of calcofluor stained yeast cell suspensions: heuristic assessment of basis spectra for their linear unmixing.  


Fluorescence emission spectra of yeast cell suspensions stained with calcofluor have recently been identified as promising markers of variations in the quality of yeast cell wall. It is shown in this paper how the raw fluorescence spectra of calcofluor can be transformed to reliable spectral signatures of cell wall quality, which are independent of actual dye-to-cell concentrations of examined cell suspensions. Moreover, the presented approach makes it possible to assess basis fluorescence spectra that allows for the spectral unmixing of raw fluorescence spectra in terms of respective fluorescence contributions of calcofluor solvated in the suspension medium and bound to yeast cell walls. PMID:22538834

Plášek, Jaromír; Dostál, Marek; Gášková, Dana



Evaluation of some fluorescent dyes for water tracing  

Microsoft Academic Search

Eight fluorescent dyes were compared in the laboratory and in field experiments to assess their utility in quantitative tracing work. The properties considered included sensitivity and minimum detectability, the effect of water chemistry on dye fluorescence, photochemical and biological decay rates, adsorption losses on equipment and sediments, toxicity to man and aquatic organisms, and cost. Orange dyes are more useful

P. L. Smart; I. M. S. Laidlaw



FluoroMyelin(TM) Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin  

PubMed Central

FluoroMyelin™ Red is a commercially available water-soluble fluorescent dye that has selectivity for myelin. This dye is marketed for the visualization of myelin in brain cryosections, though it is also used widely to stain myelin in chemically fixed tissue. Here we have investigated the suitability of FluoroMyelin™ Red as a vital stain for live imaging of myelin in myelinating co-cultures of Schwann cells and dorsal root ganglion neurons. We show that addition of FluoroMyelin™ Red to the culture medium results in selective staining of myelin sheaths, with an optimal staining time of 2 hours, and has no apparent adverse effect on the neurons, their axons, or the myelinating cells at the light microscopic level. The fluorescence is bright and photostable, permitting long-term time-lapse imaging. After rinsing the cultures with medium lacking FluoroMyelin™ Red, the dye diffuses out of the myelin with a half life of about 130 minutes resulting in negligible fluorescence remaining after 18–24 hours. In addition, the large Stokes shift exhibited by FluoroMyelin™ Red makes it possible to readily distinguish it from popular and widely used green and red fluorescent probes such as GFP and mCherry. Thus FluoroMyelin™ Red is a useful reagent for live fluorescence imaging studies on myelinated axons.

Monsma, Paula C.; Brown, Anthony



Fluorescent Dyes for Luminescent Solar Concentrators.  

National Technical Information Service (NTIS)

Dyes were developed, for application in luminescent solar concentrators. Most suitable are perylen dyes and perylimid dyes, boron complexes of naphtholactam dyes and polycarbocyclic dyes. These compounds cover the whole color range from yellow to blue. In...

R. Iden G. Seybold A. Stange H. Eilingsfeld



An easy method for cutting and fluorescent staining of thin roots  

PubMed Central

Background and Aims Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here. Methods To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light. Key Results Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots. Conclusions This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.

Zelko, Ivan; Lux, Alexander; Sterckeman, Thibault; Martinka, Michal; Kollarova, Karin; Liskova, Desana



Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue.  


A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research. PMID:21850222

Dong, Wei-Hua; Wang, Tian-Yun; Wang, Fang; Zhang, Jun-He



Hair ignition by dye laser for port-wine stain: risk factors evaluated.  


Flashlamp-pumped pulsed dye laser is the preferred treatment for port-wine stain. Vascular hemoglobin and epidermal melanin are competing sites for dye laser absorption and damage. The case presented illustrates the potential hazard of ignition induced by dye laser treatment on the face of a patient receiving inhalation anesthesia. A 6-year-old girl with almost black hair was treated for a port-wine stain covering most of the right half of her face. She was treated with dye laser under general anesthesia administered by mask. A laser pulse close to the upper part of the eyebrow induced a blaze and the eyebrow was instantly destroyed by the fire. Regrowth of the eyebrow was complete after a few months. Hair specimens of various colors were exposed experimentally to dye laser irradiation in room and oxygen-saturated atmospheres. Risk factors of ignition are high laser dosage, a high oxygen level, repeated pulses and dark colored hair. PMID:11357290

Molin, L; Hallgren, S



Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye  

SciTech Connect

We have prepared uniform silica nanoparticles (NPs) doped with a two-photon absorbing zwitterionic hemicyanine dye by reverse microemulsion method. Obvious solvatochromism on the absorption spectra of dye-doped NPs indicates that solvents can partly penetrate into the silica matrix and then affect the ground and excited state of dye molecules. For dye-doped NP suspensions, both one-photon and two-photon excited fluorescence are much stronger and recorded at shorter wavelength compared to those of free dye solutions with comparative overall dye concentration. This behavior is possibly attributed to the restricted twisted intramolecular charge transfer (TICT), which reduces fluorescence quenching when dye molecules are trapped in the silica matrix. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells with low cytotoxicity. - Graphical abstract: Water-soluble silica NPs doped with a two-photon absorbing zwitterionic hemicyanine dye were prepared. They were found of enhanced one-photon and two-photon excited fluorescence compared to free dye solutions. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells.

Wu Weibing [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); School of Light-Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037 (China); Liu Chang [Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096 (China); Wang Mingliang, E-mail: [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Huang Wei [Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096 (China); Zhou Shengrui; Jiang Wei; Sun Yueming [School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189 (China); Cui Yiping; Xu Chunxinag [Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096 (China)



A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.  


We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain. PMID:20230772

Cong, Wei-Tao; Zhu, Zhong-Xin; He, Hong-Zhang; Jin, Yan; Jiang, Cheng-Xi; Choi, Jung-Kap; Jin, Li-Tai; Li, Xiao-Kun



Identification of active fluorescence stained bacteria by Raman spectroscopy  

NASA Astrophysics Data System (ADS)

Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen



Synthesize dye-bioconjugates to visualize cancer cells using fluorescence microscopy  

NASA Astrophysics Data System (ADS)

The clinical diagnosis of most cancers is based on evaluation of histology microscopic slide to view the size and shape of cellular nuclei, and morphological structure of tissue. To achieve this goal in vivo and in deep tissue, near infrared (NIR) dyes-bovine serum albumin (BSA) and immunoglobulin G (IgG) conjugates were synthesized. The spectral study show that the absorption and fluorescence of the dye-conjugates are in the "tissue optical window" between 650 nm and 1100 nm. The internalization and pinocytosis of the synthesized compound were investigated in cell level using fluorescence microscopy to obtain the optimal concentration and staining time scale.

Pu, Yang; Tang, Rui; Xue, Jianpeng; Wang, W. B.; Xu, Baogang; Shen, Duanwen; Bloch, Sharon; Zhou, Mingzhou; Achilefu, S.; Alfano, R. R.



Efficacy of Dye-Stained Enteral Formula in Detecting Pulmonary Aspiration  

PubMed Central

Study objective To determine the extent to which a mixture of human gastric juice and enteral formula stained with two concentrations of FD&C Blue No. 1 food dye (0.8 and 1.5 mL/L) is visible in suctioned tracheobronchial secretions following three forced small-volume pulmonary aspirations over a 6-h period in an animal model. Design Experimental 2 × 3 repeated measures. Setting Animal laboratory and an acute care hospital. Participants Ninety New Zealand white rabbits weighing approximately 3 kg each, and 90 acutely ill adults who furnished gastric juice. Interventions A mixture of human gastric juice and enteral formula stained with 0.8 or 1.5 mL of dye per liter was instilled intratracheally over a 30-min period into anesthetized intubated animals at baseline, 2 h, and 4 h. A total of 0.4 mL/kg of the mixture was instilled at each session. Ninety minutes after each instillation, suctioned secretions were examined for visible dye and blood. Measurements and results Dye was visible in 46.3% of the secretions (125 of 270). The concentration of dye had no significant effect on dye visibility. Blood that was present in 114 of 270 of the secretions (42.2%) interfered with dye visibility in all but two secretions. For reasons unknown, even in the absence of blood, dye visibility decreased from 90.2% (55 of 61 secretions) after the first aspiration event to only 61% (25 of 41 secretions) after the third aspiration event. Conclusions Findings from this animal model study do not support the use of the dye method to detect repeated small-volume aspirations. For clinicians who choose to use the dye method in selected situations, it appears that a dye concentration of 0.8 mL/L may be as effective in detecting aspiration as a 1.5 mL/L concentration.

Metheny, Norma A.; Dahms, Thomas E.; Stewart, Barbara J.; Stone, Kathleen S.; Edwards, Sharon J.; Defer, Julie E.; Clouse, Ray E.



DAPI staining and fluorescence microscopy techniques for phytoplasmas.  


The 4',6-diamidino-2-phenylindole (DAPI) stain technique is a simple method that was developed for confirming the presence of phytoplasmas in hand-cut or freezing microtome sections of infected tissues. DAPI binds AT-rich DNA preferentially, so that phytoplasmas, localized among phloem cells, can be visualized in a fluorescence microscope. The procedure is quick, easy to use, inexpensive, and can be used as a preliminary or quantitative method to detect or quantify phytoplasma-like bodies in infected plants. PMID:22987410

Andrade, Nancy M; Arismendi, Nolberto L



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

Microsoft Academic Search

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

G. Genicot; J. L. M. R. Leroy; A. Van Soom; I. Donnay



Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection  

Microsoft Academic Search

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used.

Michael A Marino; Joseph M Devaney; P. Ann Davis; James E Girard



Novel fluorescent dyes for single DNA molecule techniques.  


To answer the demands of scientific and medical imaging issues, the family of nucleic acid fluorescent dyes is constantly enlarging. Most of the developed dyes reveal high qualities in bulk solution assays but are inefficient to produce a strong and sufficiently stable signal to enable the application of single-molecule techniques. Therefore, we tested 12 novel monomeric and homodimeric cyanine dyes for potential single DNA molecule imaging. Although their qualities in bulk solutions have already been described, nothing was known about their behavior on a single-molecule level. All 12 dyes demonstrated strong emission when intercalated into single DNA molecules and stretched on a silanized surface, which makes them the perfect choice for fluorescent microscopy imaging. A comparison of their fluorescence intensity and photostability with the most applicable dyes in single-molecule techniques, fluorescent dyes YOYO-1 and POPO-3, was carried out. They all exhibited a strong signal, comparable to that of YOYO-1. However, in contrast to YOYO-1, which is visualized under a green filter only, their emission permits red filter visualization. As their photostability highly exceeds that of similar spectrum POPO-3 dye, the studied dyes stand out as the best choice for a broad range of solid surface single-molecule applications when yellow to red DNA backbone fluorescence is needed. PMID:23415397

Zarkov, Alexander; Vasilev, Aleksey; Deligeorgiev, Todor; Stoynov, Stoyno; Nedelcheva-Veleva, Marina


Assessment of islet cell viability using fluorescent dyes  

Microsoft Academic Search

Summary  A rapid fluorometric method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide

H. L. Bank



Evaluation of a fluorescent lectin-based staining technique for some acidophilic mining bacteria  

SciTech Connect

A fluorescence-labeled wheat germ agglutinin staining technique was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.

Fife, D.J.; Bruhn, D.F.; Miller, K.S.; Stoner, D.L.



Plasmonic nanohybrid with ultrasmall Ag nanoparticles and fluorescent dyes.  


We investigate a hybrid nanocomposite combining fluorescent dyes and ultrasmall (<3 nm) silver nanocrystals in a block copolymer micelle. Although the metal nanoparticles are significantly smaller than the electromagnetic skin depth, we observe a modification of the exciton lifetime and the nonradiative energy transfer among the dyes. This behavior is absent in a control experiment with dyes whose energetic levels are far from the plasmonic resonance, establishing the plasmonic nature of the interaction. PMID:21534536

Rainò, Gabriele; Stöferle, Thilo; Park, Chanhee; Kim, Ho-Cheol; Topuria, Teya; Rice, Philip M; Chin, In-Joo; Miller, Robert D; Mahrt, Rainer F



Van Gieson's picrofuchsin. The staining mechanisms for collagen and cytoplasm, and an examination of the dye diffusion rate model of differential staining  

Microsoft Academic Search

The staining mechanism of van Gieson's picrofuchsin was studied by use of simple protein model systems and tissue sections, and by spectrophotometry and dialysis experiments. At the endpoint of the staining reaction (equilibrium) cytoplasm is yellow. Dye dilution experiments demonstrated that the highest affinity in the tissue section — picrofuchsin system is between binding sites in cytoplasmic protein and acid

Poul Prentø



Atomic Fluorescence Using a Tunable Flashlamp Pumped Dye Laser.  

National Technical Information Service (NTIS)

Determination of analytical calibration curves for the atomic fluorescence of sodium vapor is described. A tunable flashlamp pumped Rhodamine 6G dye laser was used as the excitation source. The sample cell consisted of an evacuated quartz tube containing ...

H. L. Brod



Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use  

PubMed Central

Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm.

Tenuta, Tiziana; Monopoli, Marco P.; Kim, JongAh; Salvati, Anna; Dawson, Kenneth A.; Sandin, Peter; Lynch, Iseult




EPA Science Inventory

A preliminary study of the manufacture of Stilbene dyes and fluorescent brightening agents was conducted to determine if process waste streams might contain hazardous material. The study first identifies the dyes and pigments that belong to this segment of the industry, the amoun...


Spectrally resolved visualization of fluorescent dyes permeating into skin  

NASA Astrophysics Data System (ADS)

We present a spectrally resolved confocal imaging approach to qualitatively asses the overall uptake and the penetration depth of fluorescent dyes into biological tissue. We use a confocal microscope with a spectral resolution of 5 nm to measure porcine skin tissue after performing a Franz-Diffusion experiment with a submicron emulsion enriched with the fluorescent dye Nile Red. The evaluation uses linear unmixing of the dye and the tissue autofluorescence spectra. The results are combined with a manual segmentation of the skin's epidermis and dermis layers to assess the penetration behavior additionally to the overall uptake. The diffusion experiments, performed for 3h and 24h, show a 3-fold increased dye uptake in the epidermis and dermis for the 24h samples. As the method is based on spectral information it does not face the problem of superimposed dye and tissue spectra and therefore is more precise compared to intensity based evaluation methods.

Maeder, Ulf; Bergmann, Thorsten; Beer, Sebastian; Burg, Jan Michael; Schmidts, Thomas; Runkel, Frank; Fiebich, Martin



Staining of intracellular granules in fresh epiphyseal cartilage by cationic dyes  

Microsoft Academic Search

Hand-cut sections of fresh epiphyseal cartilage from young rats were stained at pH 4.5 in 0.01% solutions of various cationic dyes of the thiazine, oxazine, azine, triphenylmethane, acridine, and phthallocyanin classes. The intracellular ?-and ?-metachromatic granules, previously demonstrated in fresh tissues with toluidine blue, were also demonstrated well with azure A, methylene blue, and brilliant cresyl blue. The granules were

Albert Hirschman; Dorothy M. McCabe



Fluorescent aggregates Cyan 40 and Thiazole Orange dyes in solution  

NASA Astrophysics Data System (ADS)

Absorption and fluorescence electronic spectra of cyanine dyes Cyan 40 and Thiazole Orange (TO) in water and in binary mixtures: water + ethanol, water + DMF, water + dioxane and chloroform + hexane were studied. It was revealed, that increase of concentration of Cyan 40 and TO dyes in water and chloroform + hexane binary mixture promotes the formation of fluorescent H-aggregates. Meanwhile, in contrary to the well-known fluorescent aggregates, electronic absorption band of aggregated molecules is shifted to the shorter wavelengths and fluorescence band is shifted to the longer wavelengths relative to the bands of the monomer molecules. Observed spectral changes are explained on the basis of Davidov's theory of molecular excitons as splitting of the electron-excited states due to the aggregation of dye molecules.

Nizomov, Negmat; Kurtaliev, Eldar N.; Rahimov, Sherzod I.



Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Using Coomassie Blue  

Microsoft Academic Search

A fixation-free and fast protein-staining method for sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye

Wei-Hua Dong; Tian-Yun Wang; Fang Wang; Jun-He Zhang



Validation of a dye stain assay for vaginally inserted HEC-filled microbicide applicators  

PubMed Central

Background The reliability and validity of self-reports of vaginal microbicide use are questionable given the explicit understanding that participants are expected to comply with study protocols. Our objective was to optimize the Population Council's previously validated dye stain assay (DSA) and related procedures, and establish predictive values for the DSA's ability to identify vaginally inserted single-use, low-density polyethylene microbicide applicators filled with hydroxyethylcellulose gel. Methods Applicators, inserted by 252 female sex workers enrolled in a microbicide feasibility study in Southern India, served as positive controls for optimization and validation experiments. Prior to validation, optimal dye concentration and staining time were ascertained. Three validation experiments were conducted to determine sensitivity, specificity, negative predictive values and positive predictive values. Results The dye concentration of 0.05% (w/v) FD&C Blue No. 1 Granular Food Dye and staining time of five seconds were determined to be optimal and were used for the three validation experiments. There were a total of 1,848 possible applicator readings across validation experiments; 1,703 (92.2%) applicator readings were correct. On average, the DSA performed with 90.6% sensitivity, 93.9% specificity, and had a negative predictive value of 93.8% and a positive predictive value of 91.0%. No statistically significant differences between experiments were noted. Conclusions The DSA was optimized and successfully validated for use with single-use, low-density polyethylene applicators filled with hydroxyethylcellulose (HEC) gel. We recommend including the DSA in future microbicide trials involving vaginal gels in order to identify participants who have low adherence to dosing regimens. In doing so, we can develop strategies to improve adherence as well as investigate the association between product use and efficacy.

Katzen, Lauren L.; Fernandez-Romero, Jose A.; Sarna, Avina; Murugavel, Kailapuri G.; Gawarecki, Daniel; Zydowsky, Thomas M.; Mensch, Barbara S.



Assessment of antifungal effects of a novel compound from Burkholderia cepacia against Fusarium solani by fluorescent staining  

Microsoft Academic Search

A novel compound CF66I produced by Burkholeria cepacia was investigated for its antifungal effects against Fusarium solani by three different fluorescent dyes. Dual staining with propidium iodide (PI) and fluorescein diacetate (FDA) demonstrated\\u000a high doses of CF66I (120.0 ?g ml?1) killed the fungi by acting primarily on the cell membrane. However, at fungistatic concentration (20.0 ?g ml?1) of this compound, microscopic observations revealed swelling hyphae with

Xin Li; Chun-Shan Quan; Hui-Ying Yu; Jian-Hua Wang; Sheng-Di Fan



Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores  


Novel fluorescent heterodimeric DNA-staining energy transfer dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts. Kits and individual compounds are provided, where the kits find use for simultaneous detection of a variety of moieties, particularly using a single narrow wavelength irradiation source. The individual compounds are characterized by high donor quenching and high affinity to dsDNA as a result of optimizing the length of the linking group separating the two chromophores.

Glazer, Alexander N. (Orinda, CA); Benson, Scott C. (Oakland, CA)



Optical signals from early embryonic chick heart stained with potential sensitive dyes: evidence for electrical activity.  

PubMed Central

1. Using an optical method for monitoring membrane potential, spontaneous electrical activity in the very early embryonic chick heart at the 7-9 somite stages was measured. 2. Spontaneous absorption signals from the 7-8 somite embryonic chick hearts stained with a potential sensitive merocyanine-oxazolone dye were demonstrated. The signals were observed also when a merocyanine-rhodanine dye was used. These signals were identified as spontaneous electrical activity in the embryonic heart cells. 3. The action spectrum in absorption of the merocyanine-oxazolone dye was triphasic in early embryonic chick heart with an increase in transmittance from 750 to 700, a decrease from 700 to 600, and an increase from 600 to 525 nm. 4. The magnitude of the signal was about 10(-3) of the resting intensity at 675 nm, with the merocyanine-oxazolone dye. The spontaneous absorption signals had a signal-to-noise ratio of about 10, respectively. 5. The absorption signals were markedly depressed by a higher external K+-concentration, however, were not affected by tetrodotoxin (TTX). 6. The results indicate that spontaneous electrical activity is generated at the 7-9 somite developmental stage before the initiation of heartbeat. Images Fig. 6

Fujii, S; Hirota, A; Kamino, K



Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria  

PubMed Central

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.

Fife, Dee Jay; Bruhn, Debby F.; Miller, Karen S.; Stoner, Daphne L.



Early detection of breast cancer: a molecular optical imaging approach using novel estrogen conjugate fluorescent dye  

NASA Astrophysics Data System (ADS)

Estrogen induced proliferation of mutant cells is widely understood to be the one of major risk determining factor in the development of breast cancer. Hence determination of the Estrogen Receptor[ER] status is of paramount importance if cancer pathogenesis is to be detected and rectified at an early stage. Near Infrared Fluorescence [NIRf] Molecular Optical Imaging is emerging as a powerful tool to monitor bio-molecular changes in living subjects. We discuss pre-clinical results in our efforts to develop an optical imaging diagnostic modality for the early detection of breast cancer. We have successfully carried out the synthesis and characterization of a novel target-specific NIRf dye conjugate aimed at measuring Estrogen Receptor[ER] status. The conjugate was synthesized by ester formation between 17-? estradiol and a hydrophilic derivative of Indocyanine Green (ICG) cyanine dye, bis-1,1-(4-sulfobutyl) indotricarbocyanine-5-carboxylic acid, sodium salt. In-vitro studies regarding specific binding and endocytocis of the dye performed on ER+ve [MCF-7] and control [MDA-MB-231] adenocarcinoma breast cancer cell lines clearly indicated nuclear localization of the dye for MCF-7 as compared to plasma level staining for MDA-MB-231. Furthermore, MCF-7 cells showed ~4.5-fold increase in fluorescence signal intensity compared to MDA-MB-231. A 3-D mesh model mimicking the human breast placed in a parallel-plate DOT Scanner is created to examine the in-vivo efficacy of the dye before proceeding with clinical trials. Photon migration and florescence flux intensity is modeled using the finite-element method with the coefficients (quantum yield, molar extinction co-efficient etc.) pertaining to the dye as obtained from photo-physical and in-vitro studies. We conclude by stating that this lipophilic dye can be potentially used as a target specific exogenous contrast agent in molecular optical imaging for early detection of breast cancer.

Bhattacharjee, Shubhadeep; Jose, Iven



Disposable nitrate-selective optical sensor based on fluorescent dye  

Technology Transfer Automated Retrieval System (TEKTRAN)

A simple, disposable thin-film optical nitrate sensor was developed. The sensor was fabricated by applying a nitrate-selective polymer membrane on the surface of a thin polyester film. The membrane was composed of polyvinylchloride (PVC), plasticizer, fluorescent dye, and nitrate-selective ionophore...


Caged-fluorescent-dye-based studies of turbulent scalar mixing  

Microsoft Academic Search

The initialization of a flow field with distinct and spatially segregated scalar components represents a significant experimental difficulty. Many theoretical modeling efforts in turbulent mixing, however, seek to describe the temporal evolution of a scalar concentration field that begins with this type of idealized initial conditions experimentally. This technique uses photoactivatable (caged) fluorescence dyes dissolved in the flow medium. Caged

James Guilkey; Kyle Gee; Joseph Klewicki; Patrick R. McMurtry



Diolistics: incorporating fluorescent dyes into biological samples using a gene gun  

Microsoft Academic Search

The hand-held gene gun provides a rapid and efficient method of incorporating fluorescent dyes into cells, a technique that is becoming known as diolistics. Trans- porting fluorescent dyes into cells has, in the past, used predominantly injection or chemical methods. The use of the gene gun, combined with the new generation of fluorescent dyes, circumvents some of the problems of

John A. O’Brien; Sarah C. R. Lummis



Visible fluorescent detection of proteins in polyacrylamide gels without staining  

Microsoft Academic Search

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform

Carol L Ladner; Jing Yang; Raymond J Turner; Robert A Edwards



Fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counter ion-dyes, Coomassie brilliant blue R-250 and neutral red.  


A fast and sensitive protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using both an acidic dye, Coomassie Brilliant Blue R-250 (CBBR) and a basic dye, Neutral Red (NR) is described. It is based on a counter ion-dye staining technique that employs oppositely charged two dyes to form an ion-pair complex. The selective binding of the free dye molecules to proteins in an acidic solution enhances the staining effect of CBBR on protein bands, and also reduces gel background. It is a rapid staining procedure, involving fixing and staining steps with short destaining that are completed in about 1 h. As the result, it showed two to fourfold increase in sensitivity comparing with CBBR staining. The stained protein bands can be visualized at the same time of staining. PMID:12433209

Choi, Jung-Kap; Yoo, Gyurng-Soo



Neoplasm diagnostics based on fluorescence of polymethine dyes  

NASA Astrophysics Data System (ADS)

Investigated polymethine dye TICS has near IR bands of fluorescence and absorption within the transparency region of biological tissues. It can be detected up to 1.5 cm from the surface of the skin. The intensity of a fluorescence signal of TICS is linear for doses up to 2 mg/kg in both tumor and muscle tissue. The ratio of an intensity of light induced fluorescence in tumor tissue to one in muscle tissue is up to 3.6 for rapidly growing tumors. The retention time of TICS is 7 days in all tissues. TICS can be used in the detection of tumor boundaries and tumor internal structure.

Samtsov, Michael P.; Voropay, Eugene S.; Chalov, Vadim N.; Zhavrid, Edvard A.



Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.  

PubMed Central

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B. Images

Ostle, A G; Holt, J G



A berberine-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue  

Microsoft Academic Search

Summary A fluorescent staining procedure to detect suberin, lignin and callose in plants has been developed. This procedure greatly improves on previous methods for visualizing Casparian bands in root exodermal and endodermal cells, and performs equally well on a variety of other plant tissues. Berberine was selected as the most suitable replacement forChelidonium majus root extract after comparing the staining

Mark C. Brundrett; Daryl E. Enstone; Carol A. Peterson



Thioflavin S fluorescent and congo red anisotropic stainings in the histologic demonstration of amyloid  

Microsoft Academic Search

Two methods employed for the histological detection of amyloid, the recently developed Thioflavin S fluorescent microscopic procedure and the Congo red anisotropic staining were compared. It was observed that in senile alterations of the brain, heart, and pancreas and in secondary amyloidosis both methods demonstrate the same structures. Dichroism of Congo red stained structures was also studied and it was

G. Kelényi



Fast fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by palmatine.  


A fast and sensitive protein fluorescent detection method in SDS-PAGE using the natural product palmatine is described. Palmatine is an alkaloid found in various plants exhibiting a broad spectrum of antibiotic activity in humans. The sensitivity of palmatine staining is similar to those of the SYPRO Red, SYPRO Tangerine, and SYPRO Orange protein gel stains - about 4 ng per protein band. This detection sensitivity is comparable to colloidal CBB staining. Since proteins stained with palmatine do not need destaining, the staining procedure can be easily shortened and completed in about 30 min. Stained proteins can be photographed using a UV transilluminator. The results of the present study suggest that the palmatine staining is sensitive, rapid, low cost, and safe for a broad application to the research of protein. PMID:18081205

Cong, Wei-Tao; Jin, Li-Tai; Hwang, Sun-Young; Choi, Jung-Kap



Dictyostelium extracellular vesicles containing hoechst 33342 transfer the dye into the nuclei of living cells: a fluorescence study.  


Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476-87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed. PMID:18074206

Tatischeff, Irène; Lavialle, Françoise; Pigaglio-Deshayes, Sophie; Péchoux-Longin, Christine; Chinsky, Laurent; Alfsen, Annette



Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens.  

PubMed Central

Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy. Images

Ng, V L; Yajko, D M; McPhaul, L W; Gartner, I; Byford, B; Goodman, C D; Nassos, P S; Sanders, C A; Howes, E L; Leoung, G



A simple fluorescence technique to stain the plasma membrane of human neutrophils  

Microsoft Academic Search

Three different fluorochromes were tested for their ability to label the plasma membrane proteins of neutrophils without labelling intracellular structures. A fluorescence quenching technique was used to differentiate between extra- and intracellularly localized fluorescence. Fluorescamin and fluoresceinisothiocyanate were shown to stain intracellular structures as well as the plasma membranes of the cells. Another fluorochrome, Evans Blue, is proposed since this

J. Hedl; C. Dahlgren; I. Rundquist



Prenecrotic Contracture Damage in Cardiomyocytes: Photochemical Fluorochrome Staining and Fluorescent Microscopy of the Myocardium  

Microsoft Academic Search

Prenecrotic contracture changes in cardiomyocytes during ischemic and metabolic alteration of the myocardium are detected at the photooptic level by photochemical fluorochrome staining and examination in fluorescent light. Comparison of the fluorescent and polarization microscopic pictures of damaged cardiomyocytes showed relationships between optically active and isotropic constituents of the cell myofibrillar system. Contracture changes are determined by local mass redistribution

L. M. Nepomnyashchikh; V. G. Zimmermann



Synthesis, spectroscopic characteristic of novel fluorescent dyes of pyrazoline compounds  

NASA Astrophysics Data System (ADS)

Four novel fluorescence dyes of the pyrazoline were synthesized and fully characterized by means of 1H, 13C NMR, and HRMS. The optical, electrochemical properties were also investigated. Solvent effect on the fluorescence characteristics of the four compounds indicates that the emission wavelength was red-shifted with the increase of solvent polarity. As we expected, the results indicated that these compounds exhibited high quantum yields. Quantum chemical calculations were used to obtain optimized ground-state geometry, spatial distributions of the HOMO, LUMO levels of the compounds.

Wang, Hai-Ying; Zhang, Xiao-Xiao; Shi, Jing-Jing; Chen, Gang; Xu, Xiao-Ping; Ji, Shun-Jun


Fluorescence lifetime imaging with near-infrared dyes  

NASA Astrophysics Data System (ADS)

Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they are bound to. Unfortunately, neither the lasers nor the detectors of commonly used confocal and multiphoton laser scanning microscopes allow for excitation and detection of NIR fluorescence. We therefore upgraded existing confocal TCSPC FLIM systems with NIR lasers and NIR sensitive detectors. In multiphoton systems we used the Ti:Sa laser as a one-photon excitation source in combination with an NIR-sensitive detector in the confocal beam path. We tested a number of NIR dyes in biological tissue. Some of them showed clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information on the tissue constitution and on local biochemical parameters.

Becker, Wolfgang; Shcheslavskiy, Vladislav



Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes  

USGS Publications Warehouse

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P = 0.004) and thawing treatments (P = 0.04), and mitochondrial membrane potential (P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P = 0.0258) and with mitochondrial membrane potential (P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced. ?? 2006 Elsevier Inc. All rights reserved.

Paniagua-Chavez, C. G.; Jenkins, J.; Segovia, M.; Tiersch, T. R.



Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria  

Microsoft Academic Search

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that

Scott Forster; Jason R. Snape; Hilary M. Lappin-Scott; Jonathan Porter



Acridine orange fluorescence techniques as alternatives to traditional Giemsa staining for the diagnosis of malaria in developing countries  

Microsoft Academic Search

Traditional Giemsa-stained thick blood films were compared with 2 fluorescence microscopy techniques, acridine orange (AO) staining of thin blood films and the quantitative buffy coat (QBC™) method, for the microscopical diagnosis of malaria. Of 200 samples examined, 141 were positive by Giemsa staining, 146 by AO and 137 by QBC™. Overall sensitivities for the 2 fluorescence techniques compared to Giemsa

B. S. Lowe; N. K. Jeffa; L. New; C. Pedersen; K. Engbaek; K. Marsh



Effects of a nanoscale silica matrix on the fluorescence quantum yield of encapsulated dye molecules  

NASA Astrophysics Data System (ADS)

The effects that nanometer-sized matrices have on the properties of molecules encapsulated within the nanomatrix are not fully understood. In this work, dye-doped silica nanoparticles were employed as a model for studying the effects of a nanomatrix on the fluorescence quantum yield of encapsulated dye molecules. Two types of dye molecules were selected based on their different responses to the surrounding media. Several factors that affect fluorescence quantum yields were investigated, including aggregation of dye molecules, diffusion of atmospheric oxygen, concentration of dye molecules, and size of the nanomatrix. The results showed that the silica nanomatrix has a varied effect on the fluorescence quantum yield of encapsulated dye molecules, including enhancement, quenching and insignificant changes. Both the properties of dye molecules and the conditions of the nanomatrix played important roles in these effects. Finally, a physical model was proposed to explain the varied nanomatrix effects on the fluorescence quantum yield of encapsulated dye molecules.

Liang, Song; Shephard, Kali; Pierce, David T.; Zhao, Julia Xiaojun



The Use of Fluorescent Antibody Staining in the Diagnosis of Rabies  

PubMed Central

Brain material from 750 domestic and wild animals submitted to this laboratory for rabies diagnosis was studied by the following three methods: a) microscopic examination of Williams' stained impressions, b) mouse inoculation test, and c) microscopic examination of impressions stained with fluorescein-tagged antibodies. The purpose of this investigation was to compare the sensitivity of the fluorescent antibody technique with that of two classical methods, when applied to the routine diagnosis of rabies. From the results obtained by one or the other method of study, 175 specimens were diagnosed as positive. Of these, only 58 (33 per cent) were detected by the examination of Williams' stained impressions. On the other hand, two rabid cases were missed by the mouse inoculation test, and four by the fluorescent antibody technique. Without being completely reliable, the last two methods proved to be almost equally sensitive and much more so than the examination of Williams' stained impressions. ImagesFigure 1.Fig. 2.Fig. 3.

Beauregard, M.; Boulanger, P.; Webster, W. A.



Phosphoprotein staining for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using fluorescent reagent morin hydrate.  


A fluorescence-based stain with 3,5,7,2',4'-pentahydroxyflavone (morin hydrate, MH) was designed to stain phosphoproteins in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Al(3+) was applied as a "fixed bridge," providing an efficient energy transfer channel between phosphoprotein and MH, to produce a strong fluorescent complex for the determination of phosphoprotein. As little as 62.5ng of ?-casein (7 or 8 phosphates) and ?-casein (5 phosphates), 125ng of ovalbumin (2 phosphates), and ?-casein (1 phosphate) could be visualized with a wide linear dynamic range. In comparison with conventional methods, MH stain is a time-saving method that takes just 90min. It also has good compatibility with routine protein stainings such as Coomassie Brilliant Blue R (CBBR) and SYPRO Ruby for total protein analysis. PMID:23274386

Wang, Xu; Hwang, Sun-Young; Cong, Wei-Tao; Jin, Li-Tai; Choi, Jung-Kap



Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.  


Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols. PMID:1692672

Jay, G D; Culp, D J; Jahnke, M R



Chromatin staining of Drosophila testes.  


This protocol describes chromatin staining of Drosophila testes. To visualize DNA, preparations fixed using methanol-acetone, paraformaldehyde, or formaldehyde can be stained with several DNA-binding dyes. If the slides are to be examined with a fluorescence microscope equipped with filters that permit ultraviolet (UV) excitation, suitable dyes for DNA staining are Hoechst 33258 or 4',6-diamidino-2-phenylindole (DAPI). If the slides are to be analyzed with a confocal microscope not equipped with a UV laser, DNA can be stained with either propidium iodide or TOTO-3 iodide. PMID:22854562

Bonaccorsi, Silvia; Giansanti, Maria G; Cenci, Giovanni; Gatti, Maurizio



Ethidium bromide: a fast fluorescent staining procedure for the detection of symbiotic partnership of flagellates and prokaryotes.  


The hindgut of 'lower' termites harbors a dense population of flagellates and bacteria. The flagellates possess ecto- and endosymbiotic prokaryotes. Most of them are hardly visible in the phase contrast microscope. Staining with the DNA-intercalating agent ethidium bromide visualizes the nuclei of the flagellates as well as the ecto- and endosymbiotic bacteria as red objects. Furthermore, it is possible to distinguish between endosymbiotic methanogens and other bacteria. Following UV excitation, the blue-green autofluorescence of the methanogenic bacteria eclipses the red fluorescence light of the intercalated ethidium bromide. The dye facilitates the observation of symbiotic bacteria and helps identify the number, shape, localization, and dividing status of the nuclei. Thus, it is a powerful tool for the examination of microorganisms in complex habitats, which are rich in strongly autofluorescent material, like wood. PMID:10192044

Fröhlich, J; König, H



Synthetic routes to fluorescent dyes exhibiting large Stokes shifts.  


Derivatives of isomeric 2-(hydroxytolyl)-4,6-dimethylamino-1,3,5-triazines have been synthesized in high yields in a controlled manner using a multistep reaction sequence. Iodination of either 2-(1'-hydroxy-6'-methylphen-2'-yl)- or 2-(1'-hydroxy-4'-methylphen-2'-yl)-4,6-dimethylamino-1,3,5-triazine with ICl provides species differing in the positioning of the iodo group relative to the hydroxyl which readily undergo Suzuki, Sonogashira, and Heck reactions under Pd(0) catalysis. Thus, thienyl, bisthienyl, and 3,4-ethylenedioxythienyl groups have been directly grafted, while unsubstituted polycyclic aromatics such as pyrene and perylene have been linked via alkyne bridges, as have ethynyldifluoroborondipyrromethane (BODIPY) dyes prepared in situ. The presence of a hydrogen bond in the ground state involving the hydroxyl substituent has been established by proton NMR and several X-ray structure determinations. All of the new dyes with a simple substituent (phenyl, thienyl) exhibited a pronounced green fluorescence resulting from an intramolecular proton transfer in the excited state (ESIPT) which produces a large Stokes shift (>10,000 cm(-1)). With other dyes, the fluorescence of the keto form responsible for the ESIPT process could be used as the input energy in efficient intramolecular energy transfer processes. Replacing perylene with pyrene allowed reversal of the direction of energy transfer from the polyaromatic module to the keto form. PMID:22834858

Rihn, Sandra; Retailleau, Pascal; De Nicola, Antoinette; Ulrich, Gilles; Ziessel, Raymond



Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues.  


Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures and host-plant tissues with a minimum of sample preparation: Blankophor and KOH-aniline blue fluorescent stainings. Blankophor (50 ppm in water or 15% KOH) staining was used to study the zoospore encystement on the leaf surface after release from sporangia. The aniline blue dye (0.05% in 0.067 M K(2)HPO(4), pH 9-9.5, after hot KOH whitening) was used to observe the invasive structures inside host tissues that lead to the production of sporangiophores and infectious sporangia. We tested modifications of some parameters of the procedures to determine the most appropriate for high throughput analyses adapted to our pathosystem and equipment facilities. PMID:17107808

Díez-Navajas, Ana María; Greif, Charles; Poutaraud, Anne; Merdinoglu, Didier



Anatomical differences in response to treatment of port-wine stains by the pulsed dye laser  

NASA Astrophysics Data System (ADS)

Two-hundred and fifty-seven patients (136 adults and 121 children) with port-wine stains of the head and neck were treated with the flashlamp-pumped pulsed dye laser. The head and neck was subdivided into 8 anatomical regions (forehead/temple, periorbital, medial cheek, nose, upper cutaneous lip, lateral cheek, chin and neck) which were independently evaluated for response. Response to treatment was found to be associated with the anatomical location of the lesion; in both adults and children the mid-facial region (medial cheek, nose and upper cutaneous lip) responded less favorably to treatment than the other regions of the head and neck (periorbital, forehead/temple, lateral cheek, neck and chin). In adults and children, mean percent lesional lightening of the mid-facial regions was 70.7% compared to 82.3% of the other regions of the head and neck with an estimated difference of 11.6% (95% confidence interval: 8.7% - 14.6%). The mean number of treatments for adults was 3.7, while this number in children was 3.9. All side effects were transient, and included cutaneous depressions, hypopigmentation and hyperpigmentation.

Renfro, Lisa; Geronemus, Roy G.



Laser velocimetry with fluorescent dye-doped polystyrene microspheres.  


Simultaneous Mie scattering and laser-induced fluorescence (LIF) signals are obtained from individual polystyrene latex microspheres dispersed in an air flow. Microspheres less than 1 ?m mean diameter were doped with two organic fluorescent dyes, Rhodamine B (RhB) and dichlorofluorescein (DCF), intended either to provide improved particle-based flow velocimetry in the vicinity of surfaces or to provide scalar flow information (e.g., marking one of two fluid streams). Both dyes exhibit measureable fluorescence signals that are on the order of 10(-3) to 10(-4) times weaker than the simultaneously measured Mie signals. It is determined that at the conditions measured, 95.5% of RhB LIF signals and 32.2% of DCF signals provide valid laser-Doppler velocimetry measurements compared with the Mie scattering validation rate with 6.5 W of 532 nm excitation, while RhB excited with 1.0 W incident laser power still exhibits 95.4% valid velocimetry signals from the LIF channel. The results suggest that the method is applicable to wind tunnel measurements near walls where laser flare can be a limiting factor and monodisperse particles are essential. PMID:23595429

Lowe, K Todd; Maisto, Pietro; Byun, Gwibo; Simpson, Roger L; Verkamp, Max; Danehy, Paul M; Tiemsin, Pacita I; Wohl, Christopher J



Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments.  


The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation. PMID:21108866

Levitus, Marcia; Ranjit, Suman



Taenia taeniaeformis: effectiveness of staining oncospheres is related to both temperature of treatment and molecular weight of dyes utilized.  


Methods to determine viability of taeniid oncospheres following treatments with potential lethality have practical application in efforts to control transmission. Here we investigated several methods, in lieu of infectivity studies, to assess oncosphere viability and determine lethal temperature treatment regimens. In the first experiment, a standard treatment to exshell oncospheres with 0.5% hypochlorite was assessed for influence on oncosphere recovery of Taenia taeniaeformis eggs. Recovery of eggs and exshelled oncospheres decreased with increasing time in hypochlorite, which indicated that hypochlorite can damage eggs and oncospheres, translating into potential overestimation of lethality of experimental treatments. Losses in hypochlorite were accentuated when eggs were pretreated at 75 degrees C, but not lower temperatures, including 65 degrees C, indicating a sharp threshhold between 65 degrees C and 75 degrees C where eggs and oncospheres became hypersensitive to subsequent hypochlorite treatment. To further investigate this change in relation to temperature, non-vital (acridine orange, AO) and vital (propidium iodide, PI; trypan blue, TB) dyes were used to assess staining of oncospheres (exshelled or not) under conditions ranging from room temperature up to 95 degrees C. The behaviors of dyes as related to internal staining of oncospheres were described using non-linear regression and a sigmoid four-parametric model to determine the inflection point (T50). Each of the dyes differed significantly in T50 estimates, e.g. AO (69.22+/-0.53), PI (73.89+/-0.52) and TB (79.43+/-0.45). For these dyes, the T50 increased in relation to the increasing molecular weight of the dyes. Collectively, the results suggested that barriers to chemical permeability exist in eggs that breakdown incrementally with increasing temperatures above 65 degrees C. This staining behavior and the likelihood that the temperatures involved are above a lethal threshhold clarify a basic limitation in the use of vital dyes to assess oncosphere viability. The results may be relevant to other Taenia spp. PMID:18063313

Chapalamadugu, Kalyan C; Busboom, Jan R; Nelson, Mark L; Hancock, Dale D; Tang, Juming; Jasmer, Douglas P



Control of the fluorescence of dye-antibody conjugates by (2-hydroxypropyl)-?-cyclodextrin in fluorescence microscopy and flow cytometry.  


When proteins are conjugated to fluorescent organic dyes, fluorescence emission of the dye molecules is usually decreased, sometimes up to 50-70%. This quenching phenomenon has been acknowledged for decades, but as yet, there are no simple, practical methods to control the fluorescence of dyes conjugated to proteins, especially for dyes conjugated to immunoglobulins. Here, we report that the addition of (2-hydroxypropyl)-?-cyclodextrin (HP?CD) to dye-antibody conjugates can increase fluorescence up to 2.5-fold in cell imaging and flow analysis. This method may be an effective way to increase the sensitivity of detection of fluorescent organic labels used in immunology, histochemistry, and cell biology. PMID:21846137

Guryev, Oleg; Abrams, Barny; Lomas, Chip; Nasraty, Qudrat; Park, Emily; Dubrovsky, Tim



Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition  

PubMed Central

Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques.

Avens, Heather J.; Berron, Brad J.; May, Allison M.; Voigt, Katerina R.; Seedorf, Gregory J.; Balasubramaniam, Vivek; Bowman, Christopher N.



Analysis of single blood cells for CSF diagnostics via a combination of fluorescence staining and micro-Raman spectroscopy.  


This contribution provides a new approach for single blood cell analysis in cerebrospinal fluid (CSF) with the possibility of utilizing simultaneously on the same sample the unique capabilities of the two methods fluorescence staining and Raman spectroscopy. By doing so this technique enables the potential of accurate and rapid cell identification in order to determine cell parameters immediately (e.g. the study of the level of activation or phagocytosis activity of single blood cells). Fluorescence labeling of blood cells offers the great possibility of differentiating easily between the subtypes of white blood cells, while Raman spectroscopy reveals molecular fingerprint information with a spatial resolution down to the diffraction limit. Compared to an unstained cell, the presented results nicely demonstrate that the selected fluorescence dye does not influence the Raman spectrum of a labeled blood cell notably. By the combined application of Raman spectroscopy and statistical data analysis a distinction between white blood cell substructures could be performed. Since several blood cell types also differ in the amount of their cell components, differentiation between several blood cell types is also possible when one blood cell is described in the database by several Raman spectra according their presented sub-microscopic structures. This capability with the possibility of accurate and rapid blood cell identification in cerebrospinal fluid is extremely promising for implementation in clinical diagnostics. PMID:18810290

Harz, Michaela; Kiehntopf, Michael; Stöckel, Stephan; Rösch, Petra; Deufel, Thomas; Popp, Jürgen



Differential Fluorescent Staining of Human Chromosomes with Daunomycin and Adriamycin-The D-Bands  

Microsoft Academic Search

Human chromosome preparations were treated with a group of anthracycline antibiotics. Well-defined, orange-red fluorescent bands were observed on chromosomes after the slide was stained with daunomycin and adriamycin but not with nogalamycin. The characteristic differential bands appeared to be similar to the banding patterns obtained by the quinacrine techniques. Interaction of these antibiotics with DNA could provide information on the

C. C. Lin; J. H. van de Sande



A coordination complex system for generic, ultrafast, and sensitive multimode fluorescent staining of biomolecules.  


Gel electrophoresis staining methodologies documented thus far are largely utilized in a biomolecule context-dependent manner. We report herein the development of a generic, ultrafast, and sensitive multimode fluorescent system for the efficient identification of DNA, RNA, and proteins. Interaction between a positively charged, planar ligand-based coordination complex with partner biomolecule leads to aggregation-induced fluorescence quenching and allows for the image contrast generation within one minute. Alternatively, successive reactions of the biomolecule-loaded gel with cation and ligand, in either order of sequence, provide an equally effective staining efficacy. Image contrast reversal is accomplished through a facile washing or photobleaching procedure. The versatility in the applicable target species and signal generation modes provides a hint at the design of novel staining structures and potentially enables the high-throughput readout of biomolecules. PMID:22145885

Liu, Xingqiang; Li, Lingjuan; Sun, Jingjing; Yan, Yishu; Shu, Xin; Liu, Baoqing; Sha, Wei; Feng, Hui; Sun, Sha; Zhu, Jin



A double filtering method for measuring the translational velocity of fluorescently stained cells  

SciTech Connect

The authors propose a double filtering method to measure translational velocity for tracking fluorescently stained cells. This method employs two diffraction gratings installed in the infinity space through which the parallel pencil beam of the fluorescence passes. With this method, the change in light intensity whose period is proportional to the translational velocity of the sample can be obtained at the imaging surface. By using a sample that has a random distribution of fluorescence intensity, the authors verified that translational velocity measurements could be achieved using the proposed method.

Yasokawa, Toshiki; Ishimaru, Ichirou; Kuriyama, Shigeki; Masaki, Tsutomu; Takegawa, Kaoru; Tanaka, Naotaka [Faculty of Engineering, Kagawa University, 2217-20 Hayashi-cho, Takamatsu, Kagawa 761-0396 (Japan); Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793 (Japan); Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795 (Japan)



Rapid image-based cytometry for comparison of fluorescent viability staining methods.  


The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation. PMID:22718197

Chan, Leo L; Wilkinson, Alisha R; Paradis, Benjamin D; Lai, Ning



Toward ultra-stable fluorescent dyes for single-molecule spectroscopy  

NASA Astrophysics Data System (ADS)

The wide-spread use of fluorescent dyes in molecular diagnostics and fluorescence microscopy together with new developments such as single-molecule fluorescence spectroscopy provide researchers from various disciplines with an ever expanding toolbox. Single-molecule fluorescence spectroscopy relies to a large extent on extraordinary bright and photostable organic fluorescent dyes such as rhodamine- or cyanine- derivatives. While in the last decade singlemolecule equipment and methodology have significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields), instable emission ("blinking") and photobleaching become more and more the bottleneck of further development and spreading of single-molecule fluorescence studies. In recent years, agents and recipes have been developed to increase the photostability of conventional fluorescent dyes. Here, we investigate some of these strategies at the single-molecule level. In particular, we focus on the dye selection criteria for multi-color applications. We investigate fluorescent dyes from the rhodamine, carborhodamine, cyanine, and oxazine family and show that within one dye class the photophysical properties are very similar but that dyes from different classes show strikingly different properties. These findings facilitate dye selection and provide improved chemical environment for demanding fluorescence microscopic applications.

Kasper, Robert; Heilemann, Mike; Tinnefeld, Philip; Sauer, Markus



Topography and response timing of intact cerebellum stained with absorbance voltage-sensitive dye.  


Physiological activity of the turtle cerebellar cortex (Cb), maintained in vitro, was recorded during microstimulation of inferior olive (IO). Previous single-electrode responses to such stimulation showed similar latencies across a limited region of Cb, yet those recordings lacked spatial and temporal resolution and the recording depth was variable. The topography and timing of those responses were reexamined using photodiode optical recordings. Because turtle Cb is thin and unfoliated, its entire surface can be stained by a voltage-sensitive dye and transilluminated to measure changes in its local absorbance. Microstimulation of the IO evoked widespread depolarization from the rostral to the caudal edge of the contralateral Cb. The time course of responses measured at a single photodiode matched that of single-microelectrode responses in the corresponding Cb locus. The largest and most readily evoked response was a sagittal band centered about 0.7 mm from the midline. Focal white-matter (WM) microstimulation on the ventricular surface also activated sagittal bands, whereas stimulation of adjacent granule cells evoked a radial patch of activation. In contrast, molecular-layer (ML) microstimulation evoked transverse beams of activation, centered on the rostrocaudal stimulus position, which traveled bidirectionally across the midline to the lateral edges of the Cb. A timing analysis demonstrated that both IO and WM microstimulation evoked responses with a nearly simultaneous onset along a sagittal band, whereas ML microstimulation evoked a slowly propagating wave traveling about 25 cm/s. The response similarity to IO and WM microstimulation suggests that the responses to WM microstimulation are dominated by activation of its climbing fibers. The Cb's role in the generation of precise motor control may result from these temporal and topographic differences in orthogonally oriented pathways. Optical recordings of the turtle's thin flat Cb can provide insights into that role. PMID:19004999

Brown, Michael E; Ariel, Michael



Topography and Response Timing of Intact Cerebellum Stained With Absorbance Voltage-Sensitive Dye  

PubMed Central

Physiological activity of the turtle cerebellar cortex (Cb), maintained in vitro, was recorded during microstimulation of inferior olive (IO). Previous single-electrode responses to such stimulation showed similar latencies across a limited region of Cb, yet those recordings lacked spatial and temporal resolution and the recording depth was variable. The topography and timing of those responses were reexamined using photodiode optical recordings. Because turtle Cb is thin and unfoliated, its entire surface can be stained by a voltage-sensitive dye and transilluminated to measure changes in its local absorbance. Microstimulation of the IO evoked widespread depolarization from the rostral to the caudal edge of the contralateral Cb. The time course of responses measured at a single photodiode matched that of single-microelectrode responses in the corresponding Cb locus. The largest and most readily evoked response was a sagittal band centered about 0.7 mm from the midline. Focal white-matter (WM) microstimulation on the ventricular surface also activated sagittal bands, whereas stimulation of adjacent granule cells evoked a radial patch of activation. In contrast, molecular-layer (ML) microstimulation evoked transverse beams of activation, centered on the rostrocaudal stimulus position, which traveled bidirectionally across the midline to the lateral edges of the Cb. A timing analysis demonstrated that both IO and WM microstimulation evoked responses with a nearly simultaneous onset along a sagittal band, whereas ML microstimulation evoked a slowly propagating wave traveling about 25 cm/s. The response similarity to IO and WM microstimulation suggests that the responses to WM microstimulation are dominated by activation of its climbing fibers. The Cb's role in the generation of precise motor control may result from these temporal and topographic differences in orthogonally oriented pathways. Optical recordings of the turtle's thin flat Cb can provide insights into that role.

Brown, Michael E.; Ariel, Michael



Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments  

PubMed Central

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-105 CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

Fuller, Mark E.; Streger, Sheryl H.; Rothmel, Randi K.; Mailloux, Brian J.; Hall, James A.; Onstott, Tullis C.; Fredrickson, James K.; Balkwill, David L.; DeFlaun, Mary F.



pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?  

PubMed Central

Chemically synthesized near-infrared (NIR) aza-BODIPY dyes displayed OFF/ON fluorescence at acidic pH (pKa = 6.2-6.6) through the suppression of photoinduced electron transfer (PET) and/or internal charge transfer (ICT) process. The apparent pKas of the dyes were shifted well above physiological pH in hydrophobic microenvironment, which led to “turned-on” fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin (BSA) also activated the fluorescence, though to a much less extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with signal/background ratio as high as ?10 in no-wash live cell imaging. The dye 1 labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly due to their different cellular uptake and intracellular activation capabilities. After separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum (ER) showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pH (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes were switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultra high fluorescence contrast ratio, persistent fluorescent signal, and minimum biological interference of dye 1 make it an ideal choice for live cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescent properties of pH-sensitive dyes should be carefully examined before used as pH indicators.

Zhang, Xiao-Xiang; Wang, Zhe; Yue, Xuyi; Ma, Ying; Kiesewetter, Dale O.; Chen, Xiaoyuan



Comparison of intense pulsed light (IPL) and pulsed dye laser (PDL) in port-wine stain treatment  

Microsoft Academic Search

ObjectiveThe pulsed dye laser (PDL) is the gold standard for the treatment of port-wine stains (PWS). However, intense pulsed light sources (IPLS) have often been used recently for PWS treatment not only due to their broader wavelength span which covers more than one absorption peak of hemoglobin, but also their larger spot size, and less purpura formation. An open 4-year

Michael Drosner; Jürgen Ellwanger; Kristina Schöttle; Markus Stockmeier; Florian Gatty; Georg Hellbrügge; Kåre Christiansen



Seasonal changes in fluorescence intensity of kiwifruit nuclei stained with propidium iodide and measured by flow cytometry  

Microsoft Academic Search

Nuclei were extracted from kiwifruit (Actinidia deliciosa) axillary buds and shoot apices at intervals during two growing seasons. Nuclei from fruit tissues were also examined. Extracted nuclei were stained with propidium iodide, which intercalates into double stranded nucleic acids, and the intensity of the staining reaction (fluorescence channel number) was measured by flow cytometry.Fluorescence intensity (mean channel number) of 2C

M. E. Hopping



Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.  

PubMed Central

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique. Images

Apel, W A; Dugan, P R; Filppi, J A; Rheins, M S



Imaging of interferences between cellular particles and the fluorescent dye Merocyanine-540 during normoxia and anoxia  

NASA Astrophysics Data System (ADS)

Subcellular particles (mitochondria, cellular pigments etc.) are mainly responsible for light scattering in living tissues in relation to the functional state. 2-D images clarify the respective tissue status during normoxia or anoxia such as the redox state of cytochromes. We realized tissue imaging of twelve isolated perfused rat livers stained with Merocyanine-540 during normoxia, hypoxia and anoxia. Merocyanine-spectra have shown a maximum fluorescence peak at 596 +/- 2 nm. The optical response increased under desoxygenation and decreased under reoxygenation and might be correlated to electrical potential alterations. Furthermore, we record oscillations with frequencies over 7/sec (420/min) which might be correlated to intracellular processes. The additional use of dyes for tissue imaging gives us the opportunity of new insights into organ function.

Mahlke, Christine; Dammann, Marc; Steinbach, Valerie; Stingl, Maria-Theresa; Kessler, Manfred D.



Fluorescence intensity changes associated with contractile activation in frog muscle stained with Nile Blue A.  

PubMed Central

1. Extrinsic fluorescence intensity changes were studied in frog semitendinosus muscles stained with Nile Blue A in response to electrical stimulation. Muscles were stretched and put into hypertonic solutions to prevent movement. The muscles were illuminated at 90 degrees to their long axis with a narrow beam of light at a central wave-length of 6250 . Fluorescence emission was measured at 90 degrees to the exciting light using a filter which absorbed light of wave-lengths shorter than 6400 . 2. In response to a single stimulus the fluorescence intensity increases briefly. The fluorescence response is propagated at a constant velocity of about 1.5 m/sec. The average ratio of the maximum fluorescence intensity change to the resting fluorescence is 4.5 times 10-3 for supramaximal shocks. The fluorescence intensity change starts early in the falling phase of the action potential. 3. The fluorescence intensity change increases when nitrate replaces chloride and decreases when D2O replaces H2O. The rates of rise and fall of the fluorescence change was unaffected by nitrate replacement of chloride but are slowed where D2O replaces H2O. The rates of rise and fall of the fluorescence change increase with increasing temperature for all solutions used. The peak fluorescence intensity change, however, goes through a maximum at about 17 degrees C for aqueous chloride and nitrate solutions in the range of 10-25 degrees C. With D2O solutions, the peak fluorescence intensity increases monotonically in this range of temperatures. 4. The fluorescence intensity change in response to trains of action potentials are not additive. 5. Depolarization of muscles treated with tetrodotoxin using triangular-shaped fluid electrodes produces an increase in fluorescence at about the same threshold values required to elicit tension in preparations that are not fully stretched. The fluorescence intensity change precedes in time tension development. Near threshold depolarizations, the delay in onset of the fluorescence response can be 80 msec or longer. Byond threshold, delays become shorter and peak responses larger. During maintained depolarization, after the peak response, fluorescence declines to a plateau value. 6. The results suggest that the fluorescence intensity changes are associated with excitation-contraction coupling, possibly with changes in the transmembrane potential of the sarcoplasmic reticulum.

Bezanilla, F; Horowicz, P



The mode of cell wall growth in selected archaea is similar to the general mode of cell wall growth in bacteria as revealed by fluorescent dye analysis.  


The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth in Archaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaea follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C. PMID:21169435

Wirth, Reinhard; Bellack, Annett; Bertl, Markus; Bilek, Yvonne; Heimerl, Thomas; Herzog, Bastian; Leisner, Madeleine; Probst, Alexander; Rachel, Reinhard; Sarbu, Christina; Schopf, Simone; Wanner, Gerhard



Imaging of subendocardial myocardial blood flow by dye-staining cardioscopy in patients with coronary artery disease.  


This study was carried out to image subendocardial myocardial blood flow (SMBF) by dye-staining cardioscopy (DSC) in patients with coronary artery disease.In patients with epicardial coronary artery disease, SMBF plays a direct and critical role in determining the extent and severity of cardiac function and symptoms. If SMBF could be clinically imaged instantaneously, the effects of medical and interventional treatment on it can be directly evaluated. However, there are no clinically available methods for direct and real-time imaging of SMBF. Twenty-three patients [6 with chest pain syndrome (CPS); 3 with vasospastic angina pectoris (VSA); 9 with angina pectoris due to organic coronary stenosis (AP); 5 with old myocardial infarction OMI)] underwent DSC of the left ventricle by selective intracoronary injection of 1 mL of 2.5% Evans blue dye solution (EB). Five patients with acute myocardial infarction (AMI) underwent DSC before and after coronary stent deployment. The endocardial surface was stained diffusely blue with EB indicating normal blood flow in patients with CPS; stained in a patchy fashion indicating patchy blood flow in patients with VSA; and stained in a patchy fashion or not stained indicating patchy or no blood flow in those with AP and OMI. Myocardial staining with EB was observed after coronary stent deployment in all patients with AMI, indicating restoration of the SMBF. It is evident that SMBF could be imaged by DSC. This imaging modality is useful for the evaluation of therapies and accurate guidance of transendocardial therapies of the ischemic myocardium. PMID:20966601

Uchida, Yasumi; Uchida, Yasuto; Sakurai, Takeshi; Kanai, Masahito; Sugiyama, Yukou



Class specific antibodies and fluorescent staining patterns in acute and chronic forms of schistosomiasis mansoni.  


Sera from 40 patients with acute (10) and chronic (30) forms of schistosomiasis mansoni were studied in order to correlate class specific circulating antibodies with fluorescent patterns developed in sections of both worms and liver granulomata. At the acute stage of the infection, IgA antibodies were present, IgM titers were high (about 10 times those found in the chronic stage), and IgG, IgM, IgA, and IgE antibodies were shown by focal fluorescent staining of worms and granulomata. At the chronic stage, IgA antibodies were absent and IgG antibodies showed mostly a diffuse staining pattern in both kinds of sections. The relevance of these observations to diagnosis in clinical cases or epidemiological surveys is discussed. PMID:378002

Kanamura, H Y; Hoshino-Shimizu, S; Camargo, M E; da Silva, L C



Acetylcholine-Synthesizing Amacrine Cells: Identification and Selective Staining by Using Radioautography and Fluorescent Markers  

Microsoft Academic Search

The fluorescent DNA stain 4,6,diamidino-2-phenylindole (DAPI) was applied to the cut axons of the rabbit optic tract, from which it was retrogradely transported to the retinal ganglion cell bodies. The labelled retinas were isolated from the eye and maintained in vitro in the presence of [3H]choline. They were then quick-frozen, freeze-dried, vacuum-embedded, and radioautographed on dry emulsion for identification of

R. H. Masland; J. W. Mills; S. A. Hayden



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. PMID:14579352

Pastukhov, Alexander V; Ropson, Ira J



Fluorescent TiO 2 powders prepared using a new perylene diimide dye: Applications in latent fingermark detection  

Microsoft Academic Search

A new, highly fluorescent dye was synthesised using oleylamine combined with a perylene dianhydride compound. The new dye was characterised by 1H NMR, UV–vis spectroscopy and fluorescence spectroscopy as well as quantum yield. The dye was absorbed onto titanium dioxide nanoparticles for use as a fingerprint detection powder. The new fluorescent powder was applied to latent fingermarks deposited onto different

Mi Jung Choi; Tanya Smoother; Aiden A. Martin; Andrew M. McDonagh; Philip J. Maynard; Chris Lennard; Claude Roux



Syntheses and fluorescent properties of 2,5-diamino-3,6-dicyanopyrazine dyes  

Microsoft Academic Search

2,5-Diamino-3,6-dicyanopyrazine (2), a new fluorescent chromophore for functional dye materials, was synthesized by an oxidative coupling reaction of 2,3-diamino-3-(phenylthio)acrylonitrile (1). Compound 2 has a symmetrical structure and a strong intramolecular charge-transfer chromophoric system. It shows strong yellowish-green fluorescence in solution, and thus has good potential as a synthetic intermediate for fluorescent dye chromophores. Alkylation of the amino groups of 2

Kazuko Shirai; Atsushi Yanagisawa; Hiroshi Takahashi; Koushi Fukunishi; Masaru Matsuoka



A rapid staining procedure for arbuscules of living arbuscular mycorrhizas using neutral red as acidotropic dye  

Microsoft Academic Search

Arbuscules are the most conspicuous structures of arbuscular mycorrhizas. Due to their large surface area, they are regarded\\u000a as putative sites of nutrient exchange between host and symbiont. A new staining technique for arbuscules is presented in\\u000a which arbuscules are selectively stained by acidotropic (i.e. based on the ion-trap mechanism) accumulation of neutral red\\u000a (activity stain). Criteria to distinguish acidotropic

Martin Guttenberger



The use of live fluorescence staining techniques in surgery: a review.  


ABSTRACT Intraoperative fluorescence may allow improvements to existing surgical procedures or offer scope for new operations. Despite articles describing its use dating back more than a decade, its emergence as a commonly used adjunct is still anticipated. While awareness and availability of special equipment may limit the uptake of these techniques, intraoperative fluorescence could represent a key innovation in the future of surgery. Further awareness of techniques and more clinical trials are needed to promote a wide base of clinical expertise from which further innovations can be made. This literature review begins with a discussion of the physics and chemistry of fluorophores and the properties needed for use in clinical practice. Uses in the majority of surgical specialties will be considered and the current literature addressed. Common uses include delineating hollow visci such as blood vessels or demonstrating pathology such as tumors. Fluorescent stains used have been safe, effective, and often easier to use than the established methods. Finally, novel materials such as antibodies and nanoparticles will be mentioned as new developments on the horizon of intraoperative fluorescent staining. PMID:23617319

Roberts, Harry W; Donati-Bourne, Jack F; Wilson, Victoria Lf; Wilton, Joanne C



The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes  

Microsoft Academic Search

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of

Steffen Härtel; Svitlana Tykhonova; Marcus Haas; Horst A. Diehl



Staining diatoms with rhodamine dyes: control of emission colour in photonic biocomposites  

PubMed Central

The incorporation of rhodamine dyes in the cell wall of diatoms Coscinodiscus granii and Coscinodiscus wailesii for the production of luminescent hybrid nanostructures is investigated. By systematic variation of the substitution pattern of the rhodamine core, we found that carbonic acids are considerably better suited than esters because of their physiological compatibility. The amino substitution pattern that controls the optical properties of the chromophore has no critical influence on dye uptake and incorporation, thus a variety of biocomposites with different emission maxima can be prepared. Applications in biomineralization studies as well as in materials science are envisioned.

Kucki, Melanie; Fuhrmann-Lieker, Thomas



The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa.  


The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa. PMID:15566965

Flajshans, Martin; Cosson, Jacky; Rodina, Marek; Linhart, Otomar



Short communication Inhibition of multidrug resistance transporters in the diatom Thalassiosira rotula facilitates dye staining  

Microsoft Academic Search

Cells are protected by multidrug resistance transporters, which remove potentially harmful chemicals entering the cells from the environment or originating endogenously from the cellular metabolism. Multidrug resistance transporters have not been investigated so far in marine eukary- otic algae like diatoms. We investigated the uptake of a calcium-sensitive dye, Fura 2 acetoxymethylester (AM), by the marine diatom Thalas- siosira rotula

Cordula Scherer; Karen Wiltshire; Ulf Bickmeyer


Dynamical visualization of "coffee stain phenomenon" in droplets of polymer solution via fluorescent microscopy.  


In the drying process of polymer solution droplets, we propose an experimental procedure for visualizing the solute concentration profile by combining the fluorescent microscopy with the lateral profile observation. We have conducted a dynamical observation of the transport process of the solute polymer toward the edge that causes the "coffee stain phenomenon". We have found that the polymer concentration increases sharply near the edge, while it remains almost constant in the central region until the last stage of drying. The method is useful to understand the dynamical process that occurs near the contact line. PMID:18844390

Kajiya, Tadashi; Kaneko, Daisaku; Doi, Masao



Fluorescent DNA Nanotags Featuring Covalently Attached Intercalating Dyes: Synthesis, Antibody Conjugation and Intracellular Imaging  

PubMed Central

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, due to the fact that the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal due to the large shift in emission wavelength allowed by energy transfer.

Stadler, Andrea L.; Santos, Junriz Delos; Stensrud, Elizabeth S.; Dembska, Anna; Silva, Gloria L.; Liu, Shengpeng; Shank, Nathaniel I.; Kunttas-Tatli, Ezgi; Sobers, Courtney J.; Gramlich, Philipp M. E.; Carell, Thomas; Peteanu, Linda A.; McCartney, Brooke M.; Armitage, Bruce A.



Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections.  


X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis. PMID:24121824

Levitsky, Konstantin L; Toledo-Aral, Juan José; López-Barneo, José; Villadiego, Javier



Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections  

PubMed Central

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images. This method, combined with immunofluorescence, makes it possible to unequivocally identify X-gal-labelled cells in tissue sections, emerging as a valuable tool in gene expression and cell tracing analysis.

Levitsky, Konstantin L.; Toledo-Aral, Juan Jose; Lopez-Barneo, Jose; Villadiego, Javier



Fluorescence dye as novel label molecule for quantitative SERS investigations of an antibiotic  

Microsoft Academic Search

Within this contribution, the proof-of-principle for a new concept for indirect surface-enhanced Raman spectroscopy (SERS)\\u000a detection is presented. The fluorescence dye FR-530 is applied as a label molecule for the antibiotic erythromycin. The antibiotic\\u000a binds directly to the label molecule. Changes within the SERS spectrum of the fluorescence dye appearing with the presence\\u000a of the antibiotic are utilized for the

Anne März; Sabine Trupp; Petra Rösch; Gerhard J. Mohr; Jürgen Popp


Cytofluorometric Detection of Rodent Malaria Parasites Using Red-Excited Fluorescent Dyes  

PubMed Central

Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy.

Gerena, Y.; Gonzalez-Pons, M.; Serrano, A. E.



Time-resolved laser-induced fluorescence study on dyes used in DNA sequencing  

SciTech Connect

Research on the time-resolved fluorescence of fluorescein isothiocyanate, NBD, tetramethylrhodamine isothiocyanate, and Texas Red - the dyes used for fluorescence-based DNA sequencing - is described. Mean fluorescence lifetiems in both aqueous buffer solution and 5.3%T, 4.8%C polyacrylamide gel were determined as a function of excitation wave-lengths at 337, 470, and 550 nm and were found to be 3.5, 1.1, 2.5, and 4.3 ns; the detection limits are 10, 200, 200 and 200 amol for FITC, NBD, TEMR, and T. Red, respectively. Comparisons of fluorescence parameters between the conjugated dyes and the free dyes are also reported. Results on the optimization of the excitation source wavelengths to improve sensitivity and reduce background scattering in polyacrylamide gel are also reported. Time-resolved fluorescence was successfully applied to resolve spectral overlapping of emissions in both solution and in polyacrylamide gel. 12 refs., 6 figs., 1 tab.

Chang, Kaisyang; Force, R.K. (Univ. of Rhode Island, Kingston (United States))



Synthesis of pH-activatable red fluorescent BODIPY dyes with distinct functionalities.  


A series of tunable pH-dependent BODIPY dyes were synthesized and further functionalized in a Knoevenagel condensation reaction with various aldehydes. In this fashion, monofunctional dyes containing an alkyne, azide, or carboxylic acid (masked as its methyl ester) as ligation sites as well as asymmetrical bifunctional dyes were obtained, without compromising their pH-dependency. In addition, fluorescence excitation and emission maxima for these dyes were shown to be significantly red-shifted in comparison to their tetramethyl precursors. PMID:21942639

Hoogendoorn, Sascha; Blom, Annet E M; Willems, Lianne I; van der Marel, Gijsbert A; Overkleeft, Herman S



Promising fluorescent dye for solar energy conversion based on a perylene perinone.  


We describe the synthesis of a dye based on a perylene perinone and evaluate its potential as the functional material for use in the luminescent solar concentrator (LSC). The dye extends the absorption wavelength of LSCs using the perylene-based dye Lumogen Red 305 by more than ~50 nm, translating into the collection of potentially 25% more photons at a reasonable fluorescent quantum yield and photostability. When the new perinone is used in a two-waveguide LSC in conjunction with Red 305, the integrated edge emission of the total LSC system may be increased more than 24% when compared to the Red 305 dye alone. PMID:21221140

Debije, Michael G; Verbunt, Paul P C; Nadkarni, Pradeep J; Velate, Suresh; Bhaumik, Kankan; Nedumbamana, Sankaran; Rowan, Brenda C; Richards, Bryce S; Hoeks, Theo L



Inhibition of multidrug resistance transporters in the diatom Thalassiosira rotula facilitates dye staining.  


Cells are protected by multidrug resistance transporters, which remove potentially harmful chemicals entering the cells from the environment or originating endogenously from the cellular metabolism. Multidrug resistance transporters have not been investigated so far in marine eukaryotic algae like diatoms. We investigated the uptake of a calcium-sensitive dye, Fura 2 acetoxymethylester (AM), by the marine diatom Thalassiosira rotula in the presence and absence of substances known to inhibit multidrug resistance transporters (ATP-binding cassette transporters, ABC). Three inhibitors known to block transporters in living organisms were tested in the marine diatom T. rotula. We applied verapamil, which blocks multidrug resistance P-glycoprotein (MDR1), probenecid as an inhibitor of organic anion transport and the specific inhibitor of multidrug resistance-associated protein (MRP), MK571, obtaining positive results with the highly specific MK571. This leads to the assumption that the cells of T. rotula possess MRP transporters. Marine diatom cells can now be loaded by incubation with a calcium-sensitive dye, which facilitates measurements of cellular calcium signals without using methods risking injury of the cell membrane. This opens an avenue for investigation on diatom calcium signalling and perhaps how they process environmental signals. PMID:18036827

Scherer, Cordula; Wiltshire, Karen; Bickmeyer, Ulf



Tumor hypoxia fluorescence imaging using 2-nitroimidazole bis-carboxylic acid indocyanine dye conjugate  

NASA Astrophysics Data System (ADS)

We present tumor hypoxia mapping by diffuse optical fluorescence tomography. A novel 2-nitroimidazole bis-carboxylic acid indocyanine dye conjugate has been developed for tumor-targeted hypoxia fluorescence imaging. The hypoxia probe has been evaluated in-vitro using 4T1 tumor cell lines and in-vivo tumor targeting in mice. In-vivo tumor targeting in six mice demonstrated that a measured half-life of 2-nitroimidazole-indocyanine dye wash out in the tumor was significantly longer (112+/-32.37 minutes) than that of bis-carboxylic acid indocyanine dye (69.75+/-14.01 minutes). The bis-carboxylic acid indocyanine dye was completely washed out from the tumor site within 3-5 hours post-injection, but 2-nitroimidazole-ICG remained for up to 21 hours in the tumor site. Near infrared fluorescence images of mice tumors showed a 2.6-fold contrast of dye uptake with hypoxic conjugate injection (7.46+/-1.68 ?M) compared to that with indocyanine dye injection (2.9+/-0.60 ?M). The in-vitro cell studies were performed to assess fluorescence labeling comparing hypoxia to normoxia conditions. A fluorescence emission ratio of 2.5-fold was found between the cells treated with the 2-nitroimidazole-indocyanine dye and incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed when compared to cells treated with unconjugated indocyanine dye alone. Fluorescence images acquired using a Li-COR scanner from harvested tumor samples support the in vivo monitoring and imaging results.

Biswal, Nrusingh C.; Pavlik, Christopher; Smith, Michael B.; Aguirre, Andres; Zanganeh, Saeid; Xu, Yan; Kuhn, Liisa T.; Claffey, Kevin P.; Zhu, Quing



Molecular encapsulation of fluorescent dyes affords efficient narrow-band dye laser operation in water.  


A water-based narrow-band high-efficiency dye laser was designed by means of a supramolecular host-guest chemical approach. The lasing characteristics of rhodamine B and sulforhodamine B (Kiton Red S) dyes in aqueous solution with the macrocyclic host cucurbit[7]uril (CB7) as additive were investigated in a narrow-band dye laser setup. Significant improvements in both photostability and thermo-optical properties of the aqueous CB7-complexed dye systems were observed as compared to the uncomplexed dyes in ethanol solution. The tuning curves for the new dye-CB7-water systems were constructed by measuring the laser output at different wavelengths, which showed similar peak efficiencies and red-shifted gains compared to the ethanolic solutions of the dyes, while dye laser operation revealed comparable pump threshold energies and slope efficiencies. The combined results render the dye-CB7-water system an attractive active medium for high-repetition rate dye laser operation. PMID:20839271

Mohanty, Jyotirmayee; Jagtap, Krishna; Ray, Alok K; Nau, Werner M; Pal, Haridas



Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects  

Microsoft Academic Search

The radiative and nonradiative decay rates of lissamine dye molecules, chemically attached to differently sized gold nanoparticles, are investigated by means of time-resolved fluorescence experiments. A pronounced fluorescence quenching is observed already for the smallest nanoparticles of 1 nm radius. The quenching is caused not only by an increased nonradiative rate but, equally important, by a drastic decrease in the

E. Dulkeith; A. C. Morteani; T. Niedereichholz; T. A. Klar; J. Feldmann; S. A. Levi; F. C. van Veggel; D. N. Reinhoudt; M. Möller; D. I. Gittins



Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes  

PubMed Central

We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to 10-fold quenching of the fluorescence upon hybridization was observed when the dye moiety was attached close to the 3? end and the 3?-terminal base was either dG or dC. No quenching upon hybridization was observed when the dye was positioned within the same sequence context but close to the 5? end. The presence of a dG overhang quenches the fluorescence less efficiently than a blunt end dG-dC or dC-dG base pair. When located internally in the double strand, the dG-dC base pair does not affect the fluorescence of the nearby dye. Guanosine in a single-stranded oligonucleotide quenches the fluorescence of nearby dye by <2-fold. Upon duplex formation, this quenching is eliminated and the fluorescence increases. This increase can only be detected when the fluorophore is located at least 6 nt from the terminal dG-dC base pair. The change of fluorescence polarization upon duplex formation inversely correlates with the change of intensity. Fluorescein conjugated to a single-stranded oligonucleotide or a duplex undergoes a bi-exponential decay with ?4 and ?1 ns lifetimes.

Nazarenko, Irina; Pires, Rick; Lowe, Brian; Obaidy, Mohamad; Rashtchian, Ayoub



Fluorescence of a photosensitizer based on an indotricarbocyanine dye in photochemotherapy  

NASA Astrophysics Data System (ADS)

We present the results for studies of the spectral luminescence properties of a symmetric indotricarbocyanine dye (PD1) in HeLa tumor cells and animal tissues in vivo during a photochemotherapy session and after the end of the session. We have established that when the dye is exposed to light in tumor tissues, changes occur in the position and half-width of the dye fluorescence spectra, while in a culture of HeLa cells its spectral characteristics are constant. Based on analysis of the effect of overlap between the absorption spectra of endogenous biomolecules and the fluorescence spectra of the dye plus comparison of the experimental data with numerical modeling results, we have concluded that the observed changes in the fluorescence spectra of PD1 in vivo are due to a change in the ratio of the different forms of hemoglobin in the tumor tissue. We have shown that the spectral characteristics of PD1, fluorescing in the near IR range, correlate with the depth of tumor tissue necrosis achieved on exposure to light. We have established that tumor tissue necrosis occurs down to a depth of 2 cm in the case of all strains studied: S-45, SM-1, and W-256, where as a result of exposure to light, we observe an increase in the half-width and a short-wavelength shift of the fluorescence spectrum of the dye PD1, and also the intensity of its fluorescence does not recover.

Samtsov, M. P.; Voropay, E. S.; Liashenka, L. S.; Melnikau, D. G.; Kapleusky, K. N.; Lugovskij, A. P.



Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection  

PubMed Central

We report the design, synthesis and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and that were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed of Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively low FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.

Marti, Angel A.; Li, Xiaoxu; Jockusch, Steffen; Stevens, Nathan; Li, Zengmin; Raveendra, Bindu; Kalachikov, Sergey; Morozova, Irina; Russo, James J.; Akins, Daniel L.



Fluorescent dye conjugates of rabbit arylsulfatase A as a biological tracer for protein endocytosis.  


Fluorescent dye conjugates of arylsulfatase A (ASA) from rabbit liver were prepared at pH 9.0 in 0.1 M sodium bicarbonate buffer. The modification of amino or sulphadryl groups by dichlorotriazinylamino-fluorescein or Lucifer yellow fluorescent dyes did not alter the characteristic features of the enzyme molecule such as enzyme activity, dimerization of the protein molecule at pH 4.5 and anomalous kinetics of the native enzyme. The fluorescence intensity of the Lucifer yellow enzyme conjugates were quenched when the pH of the protein solution was changed from pH 7.5 to 4.5. Therefore, the Lucifer yellow enzyme conjugate can be used to study the kinetics of pH-dependent association and dissociation of the ASA. Availability of such fluorescent dyes conjugates of ASA or other lysosomal enzyme may be used as a biological tracer to study the receptor dependent endocytosis of enzyme molecules. PMID:23636651

Hassan, Md Imtaiyaz; Waheed, Abdul; Ahmad, Faizan; Van Etten, Robert L



Groove-binding unsymmetrical cyanine dyes for staining of DNA: syntheses and characterization of the DNA-binding  

Microsoft Academic Search

Two new crescent-shaped unsymmetrical cyanine dyes have been synthesised and their interactions with DNA have been investigated by different spec- troscopic methods. These dyes are analogues to a minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a benzothiazole substituent. To investigate how

H. Jonas Karlsson; Maja Eriksson; Erik Perzon; Bjorn Akerman; Per Lincoln; Gunnar Westman



Evaluation of Polymethine Dyes as Potential Probes for Near Infrared Fluorescence Imaging of Tumors: Part - 1  

PubMed Central

Near-infrared (NIR) organic dyes have become important for many biomedical applications, including in vivo optical imaging. Conjugation of NIR fluorescent dyes to photosensitizing molecules (photosensitizers) holds strong potential for NIR fluorescence image guided photodynamic therapy (PDT) of cancer. Therefore, we were interested in investigating the photophysical properties, in vivo tumor-affinity and fluorescence imaging potential of a series of heterocyclic polymethine dyes, which could then be conjugated to certain PDT agents. For our present study, we selected a series of symmetrical polymethine dyes containing a variety of bis-N-substituted indole or benzindole moieties linked by linear conjugation with and without a fused substituted cyclohexene ring. The N-alkyl side chain at the C-terminal position was functionalized with sulfonic, carboxylic acid, methyl ester or hydroxyl groups. Although, among the parent cyanine dyes investigated, the commercially available, cyanine dye IR783 (3) (bis-indole-N-butylsulfonate)-polymethine dye with a cyclic chloro-cyclohexene moiety showed best fluorescence-imaging ability, based on its spectral properties (?Abs=782 nm, ?Fl=810 nm, ? = 261,000 M-1cm-1, ?Fl?0.08) and tumor affinity. In addition to 3, parent dyes IR820 and Cypate (6) were also selected and subjected to further modifications by introducing desired functional groups, which could enable further conjugation of the cyanine dyes to an effective photosensitizer HPPH developed in our laboratory. The synthesis and biological studies (tumor-imaging and PDT) of the resulting bifunctional conjugates are discussed in succeeding paper (Part-2 of this study).

James, Nadine S.; Chen, Yihui; Joshi, Penny; Ohulchanskyy, Tymish Y.; Ethirajan, Manivannan; Henary, Maged; Strekowsk, Lucjan; Pandey, Ravindra K



Base-content dependence of emission enhancements, quantumyields, and lifetimes of cyanine dyes bound to double-strand DNA: Photophysical properties of monomeric and bichromophoric DNA stains  

SciTech Connect

This paper reports fluorescence quantum yield, emission enhancement, and emission lifetime measurements for 10 cyanine dyes complexed to calf thymus DNA (CT-DNA), (dAdT){sub 10}, and (dGdC){sub 6} duplexes. Six of the dyes are linked bichromophores with four cationic charges per molecule, and four are monomers with two cationic charges per molecule. All of the dyes exhibit either bi- or triexponential emission decay kinetics reflecting different dye/ds DNA modes of binding, and the average radiative lifetime for the bichromophores bound to ds DNA is 5.1{+-}0.8 ns. These results are consistent with expectations that binding-induced restriction of torsion about the central methine bridge is responsible for the large emission enhancements of these dyes. Scrutiny of the lengths of average emission lifetime for these 10 dyes on (dAdT){sub 10} and (dGdC){sub 6} duplexes finds that they do not vary as expected if electron transfer (ET) emission quenching were an important process. There are also differences in emission quantum yield between dyes with pyridinium and quinolinium structural components when bound to (dAdT){sub 10} and (dGdC){sub 6} duplexes. These differences are very distinct for the monomeric dyes where pyridinium dyes have 4-fold greater emission yields on (dAdT){sub 10} duplexes and quinolinium dyes have 2-fold greater emission yields on (dGdC){sub 6} duplexes. 68 refs., 7 figs., 6 tabs.

Netzel, T.L.; Nafisi, K.; Zhao, M. [Georgia State Univ., Atlanta, GA (United States); Lenhard, J.R. [Eastman Kodak Co., Rochester, NY (United States); Johnson, I. [Molecular Probes, Inc., Eugene, OR (United States)



Efficient entrapment of dye in hollow silica nanoparticles: direct evidence using fluorescence spectroscopy.  


Using hollow silica nanoparticles we demonstrate a simple and highly efficient way of removing hydrophilic dye (Rhodamine B) from water by encapsulation within these hollow spheres. The hollow silica spheres were obtained by using a surfactant templated procedure. Using fluorescence spectroscopy, we also show the evidence of the dye being absorbed within the hollow core of the silica shell (which is crucial for many applications) and differentiate from the adsorption of dye on the surface of the silica shell. It was found that that up to 94 % of the hydrophilic dye could be entrapped using these hollow shells within 72 h of exposure. Fluorescence spectroscopy shows a red shift in the dye encapsulated in the hollow silica which is due to aggregation of the dye and enables us to follow quantitatively the uptake of the dye molecules by the silica shells with time. The evidence for the encapsulation of the dye in these hollow spheres was reinforced by carrying out a comparative study, using solid silica particles. PMID:23846303

Baruah, Arabinda; Kumar, Sandeep; Vaidya, Sonalika; Ganguli, Ashok K



Argon-pumped tunable dye laser therapy for facial port-wine stain hemangiomas in adults--a new technique using small spot size and minimal power  

SciTech Connect

A low power, argon-pumped tunable dye laser was used to deliver yellow light of 577 nm. Individual blood vessels within port-wine stain hemangiomas were treated with a 0.1-mm beam of light using 8 X magnification. This technique permits excellent resolution of facial and nuchal port-wine stain hemangiomas in adults without the adverse complications of textural change, permanent pigmentation abnormality, or hypertrophic scarring.

Scheibner, A.; Wheeland, R.G.



Green Tea Catechins Quench the Fluorescence of Bacteria-Conjugated Alexa Fluor Dyes  

PubMed Central

Accumulating evidence suggests that Green tea polyphenolic catechins, especially the (-)-epigallocatechin gallate (EGCG), can be cross-linked to many proteins, and confer a wide range of anti-bacterial activities possibly by damaging microbial cytoplasmic lipids and proteins. At the doses that conferred protection against lethal polymicrobial infection (induced by cecal ligation and puncture), EGCG significantly reduced bacterial loads particularly in the liver and lung. To elucidate its bactericidal mechanisms, we determined whether EGCG affected the fluorescence intensities of bacteria-conjugated Alexa Fluor 488 or 594 dyes. When mixed with unconjugated Alexa Fluor 488 or 594 dyes, EGCG or analogs did not affect the fluorescence intensity of these dyes. In a sharp contrast, EGCG and some analogs (e.g., Catechin Gallate, CG), markedly reduced the fluorescence intensity of Gram-positive Staphylococcus aureus-conjugated Alexa 594 and Gram-negative Escherichia coli-conjugated Alexa 488. Interestingly, co-treatment with ethanol impaired the EGCG-mediated fluorescence quenching of the G+ S. aureus, but not of the G- E. coli-conjugated Alexa Flour dyes. In light of the notion that Alexa Fluor dyes can be quenched by aromatic amino acids, it is plausible that EGCG exerts anti-microbial activities possibly by altering microbial protein conformations and functions. This possibility can now be explored by screening other fluorescence-quenching agents for possible antimicrobial activities.

Zhao, Lin; Li, Wei; Zhu, Shu; Tsai, Sheena; Li, Jianhua; Tracey, Kevin J.; Wang, Ping; Fan, Saijun; Sama, Andrew E.; Wang, Haichao



Room temperature single-photon Source:Single-dye molecule fluorescence in Liquid Crystal host  

Microsoft Academic Search

We report on new approaches toward an implementation of an efficient, room temperature, deterministically polarized, single-photon source (SPS) on demand-a key hardware element for quantum information and quantum communication. Operation of a room temperature SPS is demonstrated via photon antibunching in the fluorescence from single terrylene-dye molecules embedded in a cholesteric liquid crystal host. Using oxygen-depleted liquid crystal hosts, dye-bleaching

Svetlana G. Lukishova; Ansgar W. Schmid; Andrew J. McNamara; Robert W. Boyd; Carlos R. Stroud



Determination of torasemide by fluorescence quenching method with some dihalogenated fluorescein dyes as probes  

NASA Astrophysics Data System (ADS)

A novel fluorescence quenching method for the determination of torasemide (TOR) with some dihalogenated fluorescein dyes as fluorescence probes was developed. In acidulous medium, TOR could interact with some dihalogenated fluorescein dyes such as dichlorofluorescein (DCF), dibromofluorescein (DBF) and diiodofluorescein (DIF) to form binary complexes, which could lead to fluorescence quenching of above dihalogenated fluorescein dyes. The maximum fluorescence emission wavelengths were located at 532 nm (TOR-DCF), 535 nm (TOR-DBF) and 554 nm (TOR-DIF). The relative fluorescence intensities (?F = F0 - F) were proportional to the concentration of TOR in certain ranges. The detection limits were 4.8 ng mL-1 for TOR-DCF system, 9.8 ng mL-1 for TOR-DBF system and 35.1 ng mL-1 for TOR-DIF system. The optimum reaction conditions, influencing factors were studied; and the effect of coexisting substances was investigated owing to the highest sensitivity of TOR-DCF system. In addition, the reaction mechanism, composition and structure of the complex were discussed by quantum chemical calculation and Job's method. The fluorescence quenching of dihalogenated fluorescein dyes by TOR was a static quenching process judging from the effect of temperature and the Stern-Volmer plots. The method was satisfactorily applied to the determination of TOR in tablets and human urine samples.

Cui, Zhiping; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Hu, Xiaoli; Tian, Jing



Fluorescent staining of resting spores of Polymyxa betae as a fungal vector of rhizomania disease of sugar beet in soil  

Microsoft Academic Search

When the resting spores of Polymyxa betae were pretreated with 2% sodium dodecyl sulfate (SDS) and then stained with various fluorochromes including 3,3?-dihexyloxacarbocyanine\\u000a iodide [DiOC6(3)], calcofluor, and a fluorescein isothiocyanate (FITC)-conjugated lectin, such as wheat germ lectin (WGA)\\u000a or caster bean lectin, most spores fluoresced brightly. FITC-WGA mainly stained the cell surface, while DiOC6(3) stained the\\u000a cytoplasm. After pretreatment with

Mitsuru Sayama; Yoji Momota; Shigehito Takenaka



The role of rare earth oxide nanoparticles in suppressing the photobleaching of fluorescent organic dyes  

NASA Astrophysics Data System (ADS)

Organic dyes are widely used for both industrial as well as in scientific applications such as the fluorescent tagging of materials. However the process of photobleaching can rapidly degrade dye fluorescence rendering the material non-functional. Thus exploring novel methods for preventing photobleaching can have widespread benefits. In this work we show that the addition of minute quantities of rare earth (RE) oxide nanoparticles can significantly suppress the photobleaching of dyes. The fluorescence of Rhodamine and AlexaFluor dyes was measured as a function of time with and without the addition of CeO2 and La2O3 nanoparticle additives (two RE oxides that contain an oxygen vacancy based defect structure), as well as with FeO nanoparticles (which has an oxygen excess stoichiometry). We find that the rare earth oxides significantly prolonged the lifetimes of the dyes. The results allow us to develop a model based upon the presence of oxygen vacancies defects that allow the RE oxides to act as oxygen scavengers. This enables the RE oxide particles to effectively remove reactive oxygen free radicals generated in the dye solutions during the photoabsorption process.

Guha, Anubhav; Basu, Anindita



Comparison of Chemicon SimulFluor Direct Fluorescent Antibody Staining with Cell Culture and Shell Vial Direct Immunoperoxidase Staining for Detection of Herpes Simplex Virus and with Cytospin Direct Immunofluorescence Staining for Detection of Varicella-Zoster Virus  

Microsoft Academic Search

A new rapid direct immunofluorescence assay, the SimulFluor direct fluorescent-antibody (DFA) assay, which can simultaneously detect herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), was evaluated in comparison with our current standard procedures of (i) shell vial direct immuno- peroxidase (shell vial IP) staining and cell culture for detection of HSV and (ii) cytospin




Evaluation of a Rapid Fluorescent Staining Method for Detection of Mycobacteria in Clinical Specimens ?  

PubMed Central

Rapid detection of mycobacterial disease is essential. Using multiple specimen types and concentrations of mycobacteria, we compared two commercial auramine O stains. The more rapid stain permitted consistent acid-fast bacillus quantitation and exhibited less debris staining, and the staining procedure required less time (?2 min) to perform. These results suggest that the rapid stain may be more cost-effective and efficient for use in clinical laboratories.

Hendry, Cindy; Dionne, Kim; Hedgepeth, Annie; Carroll, Karen; Parrish, Nicole



Quantitative fluorescence spectrophotometry of acridine orange-stained unfixed cells. Potential for automated detection of human uterine cancer.  


After staining with acridine orange (AO), the nuclei of unfixed cells from the human female genital tract exhibited the same fluorescence behavior previously observed for human and murine leukocytes and mouse ascites tumor cells. With staining conditions chosen to assure saturation of the green-fluorescing AO-nucleic acid complex in normal cells, corrected fluorescence emission spectra were recorded from the entire nucleus of 341 cells taken from 32 normal and 28 abnormal patients. Intensity of the recorded spectra was expressed in phosphor particle units, a fixed arbitrary unit of fluorescence intensity, to display intensity differences among the spectra from the various cell types. In all abnormal samples, one or more cells were found with 530-nm nuclear fluorescence intensity considerably greater than the maximum intensity recorded from normal cells. Determination of the adequacy of 530-nm nuclear fluorescence intensity as a criterion for cancer detection requires additional investigation. Additional criteria, if needed, may be supplied by the metachromasy of AO-stained unfixed cells. PMID:56389

Golden, J F; West, S S; Echols, C K; Shingleton, H M



Lasing and fluorescence properties of dye-doped xerogel  

NASA Astrophysics Data System (ADS)

Dye-doped SiO2-xerogels are prepared for application as laser active medium. An optical surface and a sealing of the samples are achieved by covering them with silicon oil and an optical glass window. All optical properties were compatible with the assumption that the active chromophores are dissolved in the liquid which fills the pores of the xerogel. A replacement of the dye molecules destroyed by the pumping radiation via diffusion was observed. For stilbene 3 a diffusion coefficient of D = 0.35 × 10-9 m2/s was determined. Thus a durable operation of the laser with low repetition rates is possible.

Weissbeck, A.; Langhoff, H.; Beck, A.



Rapid, Sensitive Detection of Proteins in Minigels With Fluorescent Dyes  

Microsoft Academic Search

\\u000a The advent of polyacrylamide gel electrophoresis (PAGE) in the minigel format (i.e., gel dimensions in the range of 6 × 9\\u000a cm or 8 × 8 cm × 0.75 to 1.5 mm thick), the widespread use of precast minigels, and the commercialization of colloidal Coomassie®\\u000a Blue G-250 staining formulations have raised expectations for rapid, sensitive postelectrophoresis staining methods. The proprietary

Thomas H. Steinberg; Courtenay R. Hart; Wayne F. Patton


The mutual influence of two different dyes on their sensitized fluorescence (cofluorescence) in nanoparticles from complexes  

NASA Astrophysics Data System (ADS)

We have studied the fluorescence sensitization and quenching for pairs of different dyes simultaneously incorporated into nanoparticles from complexes M(diketone)3phen, where M(III) is La(III), Lu(III), or Sc(III); diketone is p-phenylbenzoyltrifluoroacetone (PhBTA) or naphthoyltrifluoroacetone (NTA); and phen is 1,10-phenanthroline. We have shown that, upon formation of nanoparticles in the solution in the presence of two dyes the concentrations of which are either comparable with or lower than the concentration of nanoparticles (<20 nM), the intensities of the sensitized fluorescence of dyes in nanoparticles in binary solutions and in solutions of either of the dyes coincide. We have found that the intensity of sensitized fluorescence of small (<20 nM) concentrations of rhodamine 6G (R6G) or Nile blue (NB) increases by an order of magnitude upon simultaneous introduction into nanoparticles of 1 ?M of coumarin 30 (C30), while the intensity of fluorescence of C30 sensitized by complexes decreases by an order of magnitude. The same effect is observed as 1 ?M of R6G are introduced into nanoparticles with NB ([NB] ? 20 nM). The increase in the fluorescence of dye molecules upon their incorporation from the solution into nanoparticles from complexes is noticeably lower than that expected from the proposed ratio of concentrations of complexes and dyes in nanoparticles. Analysis of the obtained data indicates that the introduction of large concentrations of C30 or R6G dyes into nanoparticles makes it possible to prevent large energy losses due to impurities or upon transition to a triplet state that arises during the migration of the excitation energy over S 1 levels of complexes. Energy accumulated by these dyes is efficiently transferred to another dye that is present in the solution at lower concentrations and that has a lower-lying S 1 level, which makes it possible to increase its fluorescence by an order of magnitude upon its incorporation into nanoparticles.

Mironov, L. Yu.; Sveshnikova, E. B.; Ermolaev, V. L.



Relationship between stallion sperm motility and viability as detected by two fluorescence staining techniques using flow cytometry  

Microsoft Academic Search

Relationships between sperm motility parameters and viability were evaluated using two fluorescent staining techniques in fresh extended semen (fresh and after 24h storage at 5°C) that had various concentrations of dead sperm added to simulate different levels of viable and nonviable sperm. Both protocols incorporated SYBR-14 and propidium iodide (PI) while the second protocol added the mitochondrial probe JC-1. The

C. C Love; J. A Thompson; S. P Brinsko; S. L Rigby; T. L Blanchard; V. K Lowry; D. D Varner



Polarization and symmetry of electronic transitions in long fluorescence lifetime triangulenium dyes.  


To fully exploit the capabilities of fluorescence probes in modern experiments, where advanced instrumentation is used to probe complex environments, other photophysical properties than emission color and emission intensity are monitored. Each dye property can be addressed individually as well as collectively to provide in-depth information unavailable from the standard intensity measurements. Dyes with long emission lifetimes and strongly polarized transitions enable the monitoring of lifetime changes as well as emission polarization (anisotropy). Thus experiments can be designed to follow slow dynamics. The UV and visible electronic transitions of a series of red-emitting dyes based on the triangulenium motif are investigated. We resolve overlapping features in the spectra and assign the orientation of the transition moments to the molecular axes. The result is the complete Jablonski diagram for the UV and visible spectral region. The symmetries of the studied dyes are shown to have a large influence on the optical response, and they are clearly separated into two groups of symmetry by their photophysical properties. The C(2v) symmetric dyes, azadioxatriangulenium (ADOTA(+)) and diazaoxatriangulenium (DAOTA(+)), have high emission anisotropies, fluorescence lifetimes around 20 ns, and fluorescence quantum yields of ?50%. The trioxatriangulenium (TOTA(+)) and triazatriangulenium (TATA(+)) dyes-nominally of D(3h) symmetry-have fluorescence lifetimes around 10 ns lifetimes and fluorescence quantum yields of 10-15%. However, the D(3h) symmetry is shown to be lowered to a point group, where the axes transform uniquely such that the degeneracy of the E' states is lifted. PMID:23391292

Thyrhaug, Erling; Sørensen, Thomas Just; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W



Tuning the nature of the fluorescent state: a substituted polycondensed dye as a case study.  


An extensive spectroscopic analysis is presented of an elongated polycondensed dye with a donor-acceptor substitution. The charge-transfer (CT) state, polarized along the long molecular axis, is close in energy to a local excitation (LE) of the polycondensed system, roughly polarized along the short molecular axis, which makes this system particularly suitable to investigate the subtle LE/CT interplay. An essential-state model is presented that quantitatively reproduces absorption and fluorescence spectra, as well as fluorescence emission and excitation anisotropy spectra collected in solvents of different polarity and viscosity, which sets a sound basis for the understanding of how solvent polarity and solvent relaxation affect the nature of low-lying excitations. The markedly different fluorescence emission and excitation anisotropy spectra measured in glassy and liquid polar solvents unambiguously demonstrate the major role played by solvent relaxation in the definition of fluorescence properties of the dye. PMID:23180631

Sissa, Cristina; Calabrese, Valentina; Cavazzini, Marco; Grisanti, Luca; Terenziani, Francesca; Quici, Silvio; Painelli, Anna



Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye  

Microsoft Academic Search

Curcumin, the main yellow bioactive component of turmeric, has recently acquired attention by chemists due its wide range of potential biological applications as an antioxidant, an anti-inflammatory, and an anti-carcinogenic agent. This molecule fluoresces weakly and poorly soluble in water. In this detailed study of curcumin in thirteen different solvents, both the absorption and fluorescence spectra of curcumin was found

Digambara Patra; Christelle Barakat



The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa  

Microsoft Academic Search

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by

Martin Flajšhans; Jacky Cosson; Marek Rodina; Otomar Linhart



Fingerprint visualization enhancement by deposition of columnar thin films and fluorescent dye treatment.  


Enhanced visualization of latent fingerprints on two non-porous surfaces, smooth glass slides and highly reflecting rough aluminum sheets, is obtained by depositing columnar thin films (CTFs) of calcium fluoride (CaF2) and silica (SiO2) by physical vapor deposition at large oblique angles. Due to the vapor flux getting shadowed by the physical residues in the fingerprints, the CTFs are deposited only on the upraised ridges, resulting in highly enhancing the visibility of the fingerprint. The visualization of these fingerprints with deposited CTFs is further enhanced by subsequently treating them with a fluorescent dye and fluorescence imaging. A specific amino-acid reagent (1,2-indanedione) and non-specific laser dye (Rhodamine 6G), both allowed enhanced visualization of the CTFs grown on the fingerprints, due to the localization and entrenchment of the dye within the CTF regions. PMID:23597736

Dutta, Jhuma; Ramakrishna, S A; Mekkaoui Alaoui, I



Immobilized fluorescent dyes for sensitive pH measurements on enamel surfaces with fiber optics  

NASA Astrophysics Data System (ADS)

Information on the pH directly on surfaces of dental enamel is an important aspect in research on tooth decay. As an alternative to pH-electrodes our approach to the problem is the optical determination of pH by pH sensitive fluorescent dyes immobilized to tooth surfaces. In this study a model for measuring pH either on aminated cellulose substrates or on enamel (in vitro) with a fluorescein type dye is presented. The experimental realization is a fiber optic sensor with a nitrogen-pumped dye laser system and photodiode for the detection of the emitted fluorescence light. The surface pH values in the range between 4 and 7 were derived from the ratios of the excitation bands at 490 nm and 460 nm.

Rumphorst, A.; Seeger, Stefan; Duschner, H.



Improved fluorescent (calcium indicator) dye uptake in brain slices by blocking multidrug resistance transporters.  


ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that, also known as multidrug resistance proteins, transport a wide variety of substrates across biological membranes in an energy-dependent manner. Recently it has been shown that members of this protein family interfere with fluorescent (calcium indicator) dye uptake in taste buds of rat and in cells in the olfactory epithelium of larval Xenopus laevis, including olfactory receptor neurons. It has, however, not been resolved whether this effect only serves to extrude xenobiotics in sensory taste and olfactory cells, or alternatively, whether it is a more general feature of many central nervous system neurons. In the latter case blocking these transporters would improve fluorescent dye uptake in general. Here we show, by means of cell imaging, that also neurons of the olfactory bulb express multidrug resistance transporters, whereby a marked inhomogeneity among cells in the main and accessory olfactory bulb was observed. Blocking these transporters improved the net uptake of fluorescent dyes not only in cell somata of the olfactory bulb, but especially in fine neuronal structures such as individual dendrites or olfactory glomeruli, which consist of a tangle of tiny neuronal processes. We therefore suggest that the expression of multidrug resistance proteins may be common in cells of the central nervous system, and that the application of specific transport inhibitors could generally improve fluorescent dye uptake in brain slices, thereby improving calcium imaging conditions. PMID:17767961

Manzini, Ivan; Schweer, Tina-Saskia; Schild, Detlev



The orientation of transition moments of dye molecules used in fluorescence studies of muscle systems  

Microsoft Academic Search

Fluorescence and phosphorescence depolarization techniques can provide information on orientational order and rotational motion of crossbridges in muscle fibres. However the depolarization experiment monitors the orientation and motion of the crossbridges indirectly. The changes in depolarization arise from a change in the orientation of the transition dipoles of the dye attached to the crossbridge. In order to extract the physiologically

U. A. van der Heide; B. Orbons; H. C. Gerritsen; Y. K. Levine



Saturation of the fluorescence of dye solutions exposed to pulsed laser excitation  

Microsoft Academic Search

The fluorescence saturation curves of dye molecules were calculated for optically thin layers of solutions exposed to pulsed laser excitation. Intercombination conversion of molecules from an excited singlet state to a triplet state was taken into account for various distributions of the photon flux density of the exciting radiation as a function of time and in the beam cross section.

S Ya Dzhasim; N Ya Serov; V V Fadeev; A M Chekalyuk



An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes  

Microsoft Academic Search

A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver

S. E. Bloom; C. Goodpasture



Bacterial Viability and Antibiotic Susceptibility Testing with SYTOX Green Nucleic Acid Stain  

Microsoft Academic Search

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a >500-fold enhancement




Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay.  


Leukocyte differential count is one of the most frequently ordered clinical tests in hospitals. This paper reports a point-of-care test for the leukocyte count by using a microflow cytometer and a fluorescent dye assay. The dye assay relied on fluorescent detection alone to count leukocytes in blood and to identify leukocyte subtypes. By combining the fluorescent assay with a sheathless microflow design, the proposed method achieved a minimal sample volume by eliminating excessive dilution and sheath flow. In this paper, a four-part leukocyte differential count including lymphocyte, monocyte, neutrophil and eosinophil was demonstrated, and the whole test consumed only a small amount of blood (5 ?L) and reagents (68 ?L in total). The merits of minimal sample volume, long reagent shelf life and portable instrument made this method optimal for point-of-care applications. PMID:23389050

Shi, Wendian; Guo, Luke; Kasdan, Harvey; Tai, Yu-Chong



An optical nanocavity incorporating a fluorescent organic dye having a high quality factor.  


We have fabricated an L3 optical nanocavity operating at visible wavelengths that is coated with a thin-film of a fluorescent molecular-dye. The cavity was directly fabricated into a pre-etched, free-standing silicon-nitride (SiN) membrane and had a quality factor of Q = 2650. This relatively high Q-factor approaches the theoretical limit that can be expected from an L3 nanocavity using silicon nitride as a dielectric material and is achieved as a result of the solvent-free cavity-fabrication protocol that we have developed. We show that the fluorescence from a red-emitting fluorescent dye coated onto the cavity surface undergoes strong emission intensity enhancement at a series of discrete wavelengths corresponding to the cavity modes. Three dimensional finite difference time domain (FDTD) calculations are used to predict the mode structure of the cavities with excellent agreement demonstrated between theory and experiment. PMID:20499907

Adawi, Ali M; Murshidy, Mohamed M; Fry, Paul W; Lidzey, David G



Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye  

NASA Astrophysics Data System (ADS)

Curcumin, the main yellow bioactive component of turmeric, has recently acquired attention by chemists due its wide range of potential biological applications as an antioxidant, an anti-inflammatory, and an anti-carcinogenic agent. This molecule fluoresces weakly and poorly soluble in water. In this detailed study of curcumin in thirteen different solvents, both the absorption and fluorescence spectra of curcumin was found to be broad, however, a narrower and simple synchronous fluorescence spectrum of curcumin was obtained at ? ? = 10-20 nm. Lippert-Mataga plot of curcumin in different solvents illustrated two sets of linearity which is consistent with the plot of Stokes' shift vs. the ET30. When Stokes's shift in wavenumber scale was replaced by synchronous fluorescence maximum in nanometer scale, the solvent polarity dependency measured by ?SFSmax vs. Lippert-Mataga plot or ET30 values offered similar trends as measured via Stokes' shift for protic and aprotic solvents for curcumin. Better linear correlation of ?SFSmax vs. ?* scale of solvent polarity was found compared to ?absmax or ?emmax or Stokes' shift measurements. In Stokes' shift measurement both absorption/excitation as well as emission (fluorescence) spectra are required to compute the Stokes' shift in wavenumber scale, but measurement could be done in a very fast and simple way by taking a single scan of SFS avoiding calculation and obtain information about polarity of the solvent. Curcumin decay properties in all the solvents could be fitted well to a double-exponential decay function.

Patra, Digambara; Barakat, Christelle



Phosphoproteome profile of human liver Chang's cell based on 2-DE with fluorescence staining and MALDI-TOF/TOF-MS.  


Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro-Q Diamond dye, for the high-throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2-DE, then sequentially stained with Pro-Q Diamond and Coomassie Blue G-250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI-TOF/TOF-MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2-DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection. PMID:17987627

Liu, Jinfeng; Cai, Yun; Wang, Jinglan; Zhou, Qi; Yang, Bing; Lu, Zhuang; Jiao, Liyan; Zhang, Dongyang; Sui, Shaohui; Jiang, Ying; Ying, Wantao; Qian, Xiaohong



Large Stokes shift fluorescent dyes based on a highly substituted terephthalic acid core.  


The synthesis of dyes based on a highly substituted terephthalic acid core is described, starting from readily available 2,5-dihydroxy-terephthalic acid diethyl ester. The dyes are highly colored, soluble in organic solvents and reasonably fluorescent in solution and in the solid state. The maxima for absorption and emission are around 402 and 502 nm, respectively. The fluorophores are readily cyclized to generate compounds which comprise the basic 6,13-dihydroxy-chromeno[2,3-b]xanthene-7,14-dione unit. These new derivatives are nonfluorescent. PMID:22381136

Benniston, Andrew C; Winstanley, Thomas P L; Lemmetyinen, Helge; Tkachenko, Nikolai V; Harrington, Ross W; Wills, Corinne



Phosphine-Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy  

PubMed Central

We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine TCEP (tris(2-carboxyethyl)phosphine). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with ultraviolet light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for superresolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.

Vaughan, Joshua C.; Dempsey, Graham T.; Sun, Eileen; Zhuang, Xiaowei



Modulation of quantum dot photoemission based on fluorescence resonance energy transfer to a photochromic dye acceptor  

NASA Astrophysics Data System (ADS)

We demonstrate the use of a photochromic dye to achieve fluorescence resonance energy transfer (FRET) modulation between a QD donor and the dye acceptor brought in close proximity in a selfassembled QD-protein-dye conjugate. The E. coli maltose binding protein (MBP) appended on its C-terminal with an oligohistidine attachment domain, immobilized onto CdSe-ZnS core-shell QDs was labeled with a sulfo-N-hydroxysuccinimide activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]). Two different dye-to-MBP-protein ratios of 1:1 and 5:1 were used. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using irradiation with white light (>500 nm) or with UV light (~365 nm), respectively. QDs surrounded by ~20 MBP-BIPS with a dye to protein ratio of 1 showed ~25% loss in their photoemission with consecutive repeated switches, while QDs surrounded by ~20 MBP-BIPS with BIPS to MBP ratio of 5 produced a substantially more pronounced rate of FRET where the QD emission was quenched by ~60%. This result suggests the possibility of using QD-protein conjugates to assemble reversible FRET nanoassemblies where the QD emission can be controlled by changing the properties of the acceptors dyes bound to the protein.

Medintz, Igor L.; Clapp, Aaron R.; Trammel, Scott A.; Mattoussi, Hedi M.



Fluorescence properties of near-infrared tricarbocyanine dyes bound to double-stranded DNA  

NASA Astrophysics Data System (ADS)

The non-covalent binding and spectroscopic properties of several near-infrared tricarbocyanine dyes to sonicated calf-thymus DNA is reported. The dyes investigated were diethylthiatricarbocyanine iodide (DTTCI), diethyloxatricarbocyanine iodide (DOTCI) and 1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide (HDITCI), which are cationic and possess absorption maxima at 772 nm, 695 nm and 750 nm, respectively, in DMSO. In buffered aqueous solutions, these dyes demonstrated extensive ground state aggregation as seen by gross changes in the NIR absorption spectra. In the presence of double-stranded DNA (dsDNA), the absorption peak in the NIR was restored, indicative of dye-deaggregation. Inspection of the fluorescence emission spectra revealed enhancement ratios of bound-to-free dye ranging from 4.5 for DOTCI to 128 for DTTCI. Scatchard analyses of the dye-dsDNA complexes showed two linear regions, suggestive of at least two possible binding sites on the DNA. For DTTCI, the binding constants were 2.88 X 104 M-1 and 4.78 X 103 M-1.

Soper, Steven A.; Davidson, Yolanda Y.; Gunn, Bonnie M.



Solvatochromism, multiphoton fluorescence, and resonance energy transfer in a new octupolar dye-pair  

NASA Astrophysics Data System (ADS)

A new octupolar molecule (E, E, E)-2,4,6-Tris [2-(4-N, N-diphenylaminophenyl) vinyl] pyridine (DPATSP) has been synthesized and studied by steady state and time-resolved fluorescence in condensed phase. The large ?-conjugation along the chain has been found to be responsible for large solvent-induced shift of fluorescence. Pumping with fs pulses at 790 nm have been found to induce the two-photon fluorescence. The two-photon absorption (2PA) cross section has been measured by the two photon induced fluorescence (2PIF) method. The N-methyl derivative of DPATSP shows a red shift in the absorption spectrum. The overlap of the fluorescence spectrum of DPATSP with the absorption spectrum of the DPATSP-Me makes a Förster resonance energy transfer (FRET) system. The FRET efficiency has been studied by embedding this dye pair in a polymer matrix.

Namboodiri, C. K. R.; Bisht, P. B.; Mukkamala, R.; Chandra, B.; Aidhen, I. S.



Cross-shore Exchange on a Rip-channeled Beach Using Fluorescent Dye  

NASA Astrophysics Data System (ADS)

The cross-shore exchange of material between the surf zone and inner-shelf was examined during seven fluorescent dye tracer deployments. Field observations were obtained at a beach in Sand City, Monterey Bay, CA, which is comprised of shore-connected shoals with incised, quasi-periodic, O(125m), rip channels, during June, 2010. Typical deployment conditions consisted of low tides and offshore waves with Hs of 0.6 to 1.8m, Tmo of 8.1 to 9.4s, and approaching the shore with normal incidence, resulting in persistent rip current events. Fluorescent Rhodamine dye was released at a point inside the surf zone and the distribution over time and space was observed. An array of stationary fluorometers was deployed at the offshore edge of the surf zone and at the lateral boundaries encompassing two rip current systems. Persons equipped with a fluorometer and GPS walked around in the surf zone study region to increase the spatial coverage. Near-surface dye concentration was also measured using fluorometers mounted on three moving, GPS-equipped vessels outside of the surf zone to examine the distribution and offshore extent of the dye plumes. Surfzone drifters were deployed simultaneously during each dye deployment to spatially map the flow field. In order to examine the vertical structure of the cross-shore exchange, multiple vertical casts of co-located fluorometer and CTD measurements were conducted in dye plumes outside of the surf zone. Qualitative results give spatial estimates of the particle pathways and show alongshore variability of the dye expulsions, with dye exiting the surf zone as plumes through the rip currents only, and extending up to three surfzone widths offshore. Dye plumes outside of the surf zone were found to be coincident with the thermocline, and were depth-uniform and encompassed greater than half, or all, of the water column. Quantitative measures of the decay rate of the dye in the surf zone will provide an estimate of the surf zone flushing time. This effort was supported by the National Science Foundation and the Office of Naval Research.

Brown, J.; Macmahan, J. H.; Reniers, A. J.



Dye fluorescence enhancement and quenching by gold nanoparticles: Direct near-field microscopic observation of shape dependence  

Microsoft Academic Search

We investigated the enhancement and quenching of dye fluorescence by single gold nanoparticles using an aperture-type scanning near-field optical microscope to evaluate the shape dependence. Enhancement or reduction of fluorescence was found to be strongly dependent on the shape of the particle. Gold nanoplates showed significant fluorescence enhancement, as did aggregated nanoparticles for the most part. On the other hand,

Noriko Nishizawa Horimoto; Kohei Imura; Hiromi Okamoto



Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein  

NASA Astrophysics Data System (ADS)

Biological molecules together with available labeling chemistries provide an ideal setting to investigate the interaction between two closely spaced dye molecules. The photo-excitation of a donor molecule can be non-radiatively transferred to a near-by acceptor molecule via the induced-dipole-induced-dipole interaction in a distance-dependent manner. In this work, we further elaborate on single-molecule fluorescence resonance energy transfer measurements between two dye molecules attached to a single protein - staphylococcal nuclease molecules [T. Ha, A.Y. Ting, J. Liang, W.B. Caldwell, A.A. Deniz, D.S. Chemla, P.G. Schultz, S. Weiss, Proc. Natl. Acad. Sci. USA 96 (1999) 893-898]. Temporal fluctuations in the energy transfer signal include: (1) reversible transitions to dark states; (2) irreversible photodestruction; (3) intersystem crossing to and from the triplet state; (4) spectral fluctuations; (5) rotational dynamics of the dyes; and (6) distance changes between the two dyes. To extract biologically relevant information from such measurements, an experimental strategy and data analysis schemes are developed. First, abrupt photophysical events, such as (1)-(3) are identified and removed from the data. The remaining slow, gradual fluctuations in the energy transfer signal are due to spectral shifts, rotational dynamics and distance changes of the dyes. Direct measurements of each dye's spectral fluctuation and rotational dynamics indicate that these, by themselves, cannot fully account for the observed energy transfer fluctuations. It is therefore concluded that inter-dye distance changes must be present as well. The distance and orientational dynamics are shown to be dependent on the binding of the active-site inhibitor (deoxythymidine diphosphate) to the protein. The inhibitor most probably affects the protein's stability and the dye-protein interaction, possibly by amplifying the motion of the linker arm between the fluorophore and the protein.

Ha, Taekjip; Ting, Alice Y.; Liang, Joy; Chemla, Daniel S.; Schultz, Peter G.; Weiss, Shimon; Deniz, Ashok A.



Kinetics of staining and bleaching  

Microsoft Academic Search

Kinetics of staining and stain removal have been studied with food dyes and natural colorants. Kinetics of staining indicate\\u000a that the staining agent is adsorbed on the fiber surface and diffuses into the interior of the fibers. Similarities exist\\u000a between staining and dyeing mechanisms of cotton, polyester and nylon fibers. Stain removal by nonoxidative detergency involves\\u000a diffusion of the staining

Erik Kissa; Jenny M. Dohner; Ward R. Gibson; Donna Strickman



Local viscosity analysis of triblock copolymer micelle with cyanine dyes as a fluorescent probe.  


The local viscosity of Pluronic F127 triblock copolymer micelles in water was determined with cyanine dyes as fluorescent probes. These dyes show very weak fluorescence at a low temperature, but show enhanced fluorescence at a temperature higher than the critical micellization temperature (T(cm)). This is because a viscous environment within the micelle suppresses the formation of a nonradiative twisted intramolecular charge transfer (TICT) excited state of the dyes. The good correlation between the fluorescence quantum yields of the dyes and the viscosity and the temperature of the media allows a determination of local viscosity of micelle based on the fluorescence quantum yields. The local viscosity of both core and corona regions of micelles increases at >T(cm) and shows a maximum at a temperature 7-9 °C higher than T(cm), and decreases at higher temperature due to the increased fluidity. The core viscosity is larger than that of the corona, and the corona viscosity increases toward the micelle center. The polymer concentration has different effects on the core and corona viscosity: the corona viscosity increases with a polymer concentration increase at the entire temperature range, whereas the core viscosity increases only at a low temperature. The corona viscosity increase is due to the condensation of a large number of polyethylene oxide (PEO) blocks. In contrast, the dehydration degree of polypropylene oxide (PPO) blocks in the core scarcely changes, and the core has a similar composition regardless of polymer concentration. The larger polymer concentration promotes a micelle formation at lower temperature where the fluidity increase is very weak, resulting in larger core viscosity. PMID:20942435

Shiraishi, Yasuhiro; Inoue, Takuya; Hirai, Takayuki



Sentinel lymph nodes detection with an imaging system using Patent Blue V dye as fluorescent tracer  

NASA Astrophysics Data System (ADS)

Sentinel lymph node biopsy is the gold standard to detect metastatic invasion from primary breast cancer. This method can help patients avoid full axillary chain dissection, thereby decreasing the risk of morbidity. We propose an alternative to the traditional isotopic method, to detect and map the sentinel lymph nodes. Indeed, Patent Blue V is the most widely used dye in clinical routine for the visual detection of sentinel lymph nodes. A Recent study has shown the possibility of increasing the fluorescence quantum yield of Patent Blue V, when it is bound to human serum albumin. In this study we present a preclinical fluorescence imaging system to detect sentinel lymph nodes labeled with this fluorescent tracer. The setup is composed of a black and white CCD camera and two laser sources. One excitation source with a laser emitting at 635 nm and a second laser at 785 nm to illuminate the region of interest. The prototype is operated via a laptop. Preliminary experiments permitted to determine the device sensitivity in the ?mol.L-1 range as regards the detection of PBV fluorescence signals. We also present a preclinical evaluation performed on Lewis rats, during which the fluorescence imaging setup detected the accumulation and fixation of the fluorescent dye on different nodes through the skin.

Tellier, F.; Steibel, J.; Chabrier, R.; Rodier, J. F.; Pourroy, G.; Poulet, P.



Longitudinal diffusion behavior of hemicyanine dyes across phospholipid vesicle membranes as studied by second-harmonic generation and fluorescence spectroscopies.  


The adsorption and longitudinal diffusion behaviors of a series of hemicyanine dyes to phospholipid vesicle membranes were studied by second-harmonic generation (SHG) and fluorescence spectroscopies. It was observed that the longitudinal diffusion of cationic hemicyanine dyes takes place immediately after the initial adsorption of these dyes to the outer surface of the vesicle membrane. In contrast, hardly any amount of a zwitterionic hemicyanine dye with a sulfonate group diffused across the vesicle membrane within the measurement time (<2000 s). Based on the difference in the time-course responses of SHG and fluorescence spectroscopies for all of the hemicyanine dyes tested, we propose that hydration of the sulfonate group is mainly responsible for the low diffusivity of the zwitterionic hemicyanine dye. PMID:16715263

Yamaguchi, Akira; Nakano, Masaki; Nochi, Kimihisa; Yamashita, Tomohisa; Morita, Kotaro; Teramae, Norio



Organic dye penetration quantification into a dental composite resin cured by LED system using fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

A major characteristic of LEDs systems is the lower heat emission related with the kind of light generation and spectral emission band. Material temperature during photoactivation can promote different photocuring performance. Organic dye penetration could be a trace to identify the efficacy of photocured composite resin. A new method using fluorescent spectroscopy through digital image evaluation was developed in this study. In order to understand if there is a real influence of material temperature during the photoactivation procedure of a dental restorative material, a hybrid composite resin (Z250, 3M-Espe, USA) and 3 light sources, halogen lamp (510 mW/cm2) and two LED systems 470+/-10nm (345 and 1000 mW/cm2) under different temperatures and intensities were used. One thousand and five hundred samples under different associations between light sources and temperatures (0, 25, 50, 75 and 100oC were tested and immediately kept in 6G rodamin dye solution. Dye penetration was evaluated through fluorescent spectroscopy recorded by digital image data. Pixels in gray scale showed the percentage penetration of organic dye into the composite resin mass. Time and temperature were statistically significant (p<0.05) through the ANOVA statistical test. The lowest penetration value was with 60 seconds and 25oC. Time and temperature are important factors to promote a homogeneous structure polymerized composite resin more than the light source type, halogen or LEDs system.

Lizarelli, Rosane de Fátima Zanirato; Silva, Maciel E., Jr.; Lins, Emery C. C. C.; Costa, Mardoqueu M.; Pelino, José Eduardo P.; Bagnato, Vanderlei S.



Sensitization of fluorescence of dye molecules in nanoparticles of metal complexes  

NASA Astrophysics Data System (ADS)

We studied the dependence of absorption and fluorescence spectra of complexes of Al, In, Sc, Y, and La with dibenzoylmethane and naphthoyltrifluoroacetone, as well as the dependence of sensitized fluorescence of dyes in nanoparticles of these complexes, in relation to the water pH, the ratio between ions and diketones, and the ion selection. We showed that the ability of complexes of ions to form nanoparticles that efficiently sensitize dye molecules incorporated into them is determined by stability constants of these ions with organic ligands and by their ability to compete with the formation of hydroxy complexes of these ions. We found that nanoparticles consist of diketonates of different compositions and that Nile red incorporated into nanoparticles is an indicator of the dependence of the composition of nanoparticles on the selection of the central ion of complexes and conditions of their formation. We revealed that complexes M(diketone)(OH)2 self-assemble into nanoparticles with an admixture of dye molecules and efficiently sensitize dyes upon excitation into absorption bands of complexes. We showed that, at concentrations of rhodamine 6G in water smaller than 50 nM, the use of a solution that contains 50 ?M of Al(III), In(III), or Sc(III) + 50 ?M of naphthoyltrifluoroacetone makes it possible to increase the sensitivity of the luminescence analysis by 20-fold for the presence of rhodamine 6G in an aqueous solution.

Dudar', S. S.; Sveshnikova, E. B.; Ermolaev, V. L.



Rapid quantification of polyhydroxyalkanoates (PHA) concentration in activated sludge with the fluorescent dye Nile blue A.  


The present study was conducted (1) to develop a rapid quantification method of polyhydroxyalkanoates (PHA) concentration in activated sludge by Nile blue A staining and fluorescence measurement and (2) to perform on-line monitoring of PHA concentrations in activated sludge. Activated sludge samples collected from laboratory scale sequencing batch reactors and full-scale wastewater treatment plants were stained with Nile blue A and their fluorescence intensities were determined. There was a high correlation (R2 > 0.97) between the fluorescence intensities of Nile blue A and PHA concentrations in activated sludge determined by gas chromatography. The Nile blue A staining and fluorescence measurement method allows us to determine PHA concentrations in activated sludge within only five minutes and up to 96 samples can be measured at once by using microplate reader. On-line monitoring of PHA concentrations in activated sludge was achieved by using a fluorometer equipped with a flow cell and the time point at which PHA concentration in activated sludge reached the maximum level could be identified. In addition, we examined the influence of pH, floc size and co-existing chemicals in activated sludge suspension on the fluorescence intensities of Nile blue A. PMID:22097056

Oshiki, M; Satoh, H; Mino, T



Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes  

PubMed Central

Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53?/? cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.

Allen, Joshua E.; Hart, Lori S.; Dicker, David T.; Wang, Wenge; El-Deiry, Wafik S.



Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.  


Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation. PMID:23535632

Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June



A close look at fluorescence quenching of organic dyes by tryptophan.  


Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M(-1), 2) static quenching with a bimolecular association constant K(s) of 61 M(-1), and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M(-1). The latter two are characterized as nonfluorescent complexes, formed with approximately 30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20-30 kJ mol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales. PMID:16224752

Doose, Sören; Neuweiler, Hannes; Sauer, Markus



New silica and polystyrene nanoparticles labeled with longwave absorbing and fluorescent chameleon dyes  

Microsoft Academic Search

We are presenting new fluorescent nanoparticles (NPs) made from silica or polystyrene. Such NPs are potentially useful for\\u000a purposes of cellular imaging and sensing. The NPs were surface-modified with amino groups, and longwave absorbing and emitting\\u000a dyes were then conjugated, via their reactive chloro atoms, to the NPs. The reactions proceed at temperatures of around 65 °C\\u000a and in predominantly aqueous

Sayed M. Saleh; Reham Ali; Otto S. Wolfbeis


Immobilized fluorescent dyes for sensitive pH measurements on enamel surfaces with fiber optics  

Microsoft Academic Search

Information on the pH directly on surfaces of dental enamel is an important aspect in research on tooth decay. As an alternative to pH-electrodes our approach to the problem is the optical determination of pH by pH sensitive fluorescent dyes immobilized to tooth surfaces. In this study a model for measuring pH either on aminated cellulose substrates or on enamel

A. Rumphorst; Stefan Seeger; H. Duschner



Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin  

Microsoft Academic Search

.   To study vacuolar chloride (Cl?) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl? uptake into isolated tonoplast vesicles was measured using the Cl?-sensitive fluorescent dye lucigenin (N,N?-dimethyl-9,9?-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm,\\u000a respectively, and showed a high sensitivity towards Cl?, with a Stern-Volmer constant of 173 m\\u000a \\u000a ?1

F. Wissing; J. A. C. Smith



Bands separation in fluorescence spectra of ketocyanine dyes: evidence for their complex formation with monohydric alcohols  

Microsoft Academic Search

In the studies of binary solvent systems containing non-polar (toluene) and polar proton-donating components (monohydric alcohols) using ketocyanine dyes of 2,5-di-benzylidene-cyclopentanone-1 type as solvent polarity probes, we found that in addition to common solvent polarity-dependent shifts of fluorescence spectra, at low alcohol concentrations there appear two new well-resolved spectral bands. They are attributed to the emission of hydrogen bonded complexes

V. G. Pivovarenko; A. V. Klueva; A. O. Doroshenko; A. P. Demchenko



Fluorescent monomers as building blocks for dye labeled polymers: synthesis and application in energy conversion, biolabeling and sensors.  


This review focuses on side-chain functionalized polymers derived from direct (co)polymerization of fluorescent dyes. This overview about polymerizable dyes includes 1,8-naphthalimides, fluoresceins, rhodamines, coumarins, azo-dyes, oxadiazoles, diverse aromatic dyes as well as selected other dyes that cannot be classified within these groups. The discussed dyes have been functionalized with a polymerizable unit in order to apply straight-forward polymerization procedures. Therefore, the center of attention is set to the optical properties of the polymerizable dyes and the applicable polymerization techniques. Furthermore, the various applications (i.e., in biomedicine and pharmacy, as thermo-responsive materials and energy transfer materials, for dispersion of carbon nanotubes and others) of each polymer are discussed. PMID:23482971

Breul, Alexander M; Hager, Martin D; Schubert, Ulrich S



Fluorescence quenching of dyes covalently attached to single-walled carbon nanotubes.  


The development of chromophore-carbon nanotube hybrids requires efficient and accurate methods to investigate their photophysical properties. Using the ability of the fluorescence labeling of surface species (FLOSS) technique to determine the density of covalently attached dyes to the surface of single-walled carbon nanotubes (SWCNTs), the luminescence of dye-SWCNT hybrids was quantitatively studied with two chromophores: dansyl hydrazine (DH) and panacyl bromide (PB). The fluorescence intensity of PB-SWCNT hybrids was reduced by 20-80% compared to that of free PB. A strong positive correlation between the degree of quenching and the residual metal impurity content in the SWCNT sample suggests that quenching of fluorescence of PB in PB-SWCNTs may be caused by the metal impurities and not by SWCNTs. On the contrary, the intensity of fluorescence of DH-SWCNT hybrids was reduced by almost 2 orders of magnitude compared to free DH, independent of the residual metal content in the SWCNT sample, suggesting that quenching of fluorescence in DH-SWCNT hybrids might occur via charge transfer from DH chromophores to SWCNTs, and revealing the potential of DH-SWCNT hybrids for solar light harvesting applications. PMID:21766814

Chiu, Cheuk Fai; Dementev, Nikolay; Borguet, Eric



Sub-diffusion decays in fluorescence correlation spectroscopy: dye photophysics or protein dynamics?  


Transitions between bright and dark fluorescent states of several rhodamine dyes were investigated by fluorescence correlation spectroscopy. We resolved two sub-diffusion exponential decays for free rhodamines in aqueous solutions, of which the slower component scales linearly with the viscosity of the solution. Correlation data for proteins and DNA labeled with tetramethylrhodamine were fitted with three to four exponential decays describing flickering dynamics on a time scale between 0.5 and 100 ?s. We investigated the nature of these processes by performing experiments under different experimental conditions and for different samples. On the basis of how their population and lifetime change with viscosity, the oxygen content of the solution, the laser irradiance, and the detection geometry, we assigned these states, in the order of increasing lifetimes, to a triplet state, a hybrid between twisted-intramolecular-charge-transfer state and a ground state lactonic state, a lactonic state, and a photoionized state, respectively. Our data suggests that none of the observed sub-diffusion correlation decays can be directly assigned to the intramolecular dynamics of the labeled biomolecules. However, we found evidence that the intrinsic conformational dynamics of the biomolecule appears in the correlation curves as a modulation of the photophysics of the dye label. This shows the importance of accurate control measurements and appropriate modeling of the dye photophysics in fluorescence correlation studies, and it cautions against direct assignments of dark-state relaxation times to folding kinetics in proteins and nucleic acids. PMID:23675915

Mazouchi, Amir; Bahram, Abdullah; Gradinaru, Claudiu C



Simultaneous optical tweezing and laser-induced fluorescence of DAPI stained chromosomes  

Microsoft Academic Search

In this paper, we describe methods to increase the functionality of conventional optical tweezer techniques through the use of a 410 nm laser diode to induce fluorescence in biological samples. For the first time, to our knowledge, we demonstrate the use of a violet diode laser to induce fluorescence in particles, which are simultaneously tweezed by an infrared laser. Using

A. E. Carruthers; T. K. Lake; L. Paterson; J. W. Allen; W. Sibbett; K. Dholakia



Dual appearance of fluorescein staining in vivo of diseased human corneal epithelium. A non-contact photomicrographic study.  


Adherence of fluorescein sodium dye to diseased epithelial cells, a hitherto unreported phenomenon, was captured in photomicrographs in severe herpes zoster and keratoconjunctivitis sicca keratopathies. It is notable that this phenomenon differs completely from the well known fluorescent property of the dye penetrating into defective corneal epithelium, and that the staining pattern shown by adherent fluorescein correlates well with the staining pattern shown by rose bengal dye. PMID:1371224

Tabery, H M



Dual appearance of fluorescein staining in vivo of diseased human corneal epithelium. A non-contact photomicrographic study.  

PubMed Central

Adherence of fluorescein sodium dye to diseased epithelial cells, a hitherto unreported phenomenon, was captured in photomicrographs in severe herpes zoster and keratoconjunctivitis sicca keratopathies. It is notable that this phenomenon differs completely from the well known fluorescent property of the dye penetrating into defective corneal epithelium, and that the staining pattern shown by adherent fluorescein correlates well with the staining pattern shown by rose bengal dye. Images

Tabery, H M



Development of an image processing support system based on fluorescent dye to prevent elderly people with dementia from wandering.  


The wandering of elderly people with dementia is a significant behavioral problem and is a heavy burden on caregivers in residential and nursing homes. Thus, warning systems have been developed to prevent elderly people with dementia from leaving the premises. Some of these systems use radio waves. However, systems based on radio waves present several practical problems. For instance, the transmitter must be carried and may become lost; in addition, the battery of the transmitter must be changed. To solve these problems, we developed a support system that prevents elderly people with dementia from wandering. The system employs image processing technology based on fluorescent dye. The composition of the support system can be described as follows: fluorescent dye is painted in a simple shape on the clothes of an elderly person. The fluorescent color becomes visible by irradiation with a long wavelength of ultraviolet light. In the present paper, the relationship between the color of the dye and the cloth was investigated. A 3D video camera was used to acquire a 3D image and detect the simple shape. As a preliminary experiment, 3 colors (red, green and blue) of fluorescent dye were applied to cloths of 9 different colors. All fluorescent colors were detected on 6 of the cloths, but red and blue dye could not be detected on the other 3 cloths. In contrast, green dye was detectable on all 9 of the cloths. Additionally, we determined whether green dye could be detected in an actual environment. A rectangular shaped patch of green fluorescent dye was painted on the shoulder area of a subject, from the scapula to the clavicle. As a result, the green dye was detected on all 9 different colored cloths. PMID:24111431

Nishigaki, Yutaka; Tanaka, Kentaro; Kim, Juhyon; Nakajima, Kazuki



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

PubMed Central

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. Images Figure 1

Smith, K. A.; Hill, S. A.; Begg, A. C.; Denekamp, J.



Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy  

PubMed Central

Abstract We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.

Murade, C.U.; Subramaniam, V.; Otto, C.; Bennink, Martin L.



Effect of confined radiation field on spontaneous-emission lifetime in vacuum-deposited fluorescent dye films  

NASA Astrophysics Data System (ADS)

Spontaneous emission lifetimes of fluorescent dye layers in front of a metallic mirror were observed using a picosecond time-resolved fluorescence measurement system. Multilayer thin-film samples composed of glass substrate/indium—tinoxide layer/organic spacer layer/emission dye layer/organic spacer layer/MgAg mirror were prepared with vacuum-vapor deposition. The distance between an MgAg mirror and an emission dye layer was varied from 25 to 180 nm. The emission lifetime of the fluorescent dye layer largely depended on the MgAg mirror—emission layer distance and assumed a maximum at around 150 nm. A promising application of organic multilayer structures for the study on optical microcavity was suggested.

Tsutsui, T.; Adachi, C.; Saito, S.; Watanabe, M.; Koishi, M.



Dynamic staining of bacteria at a single-cell level  

NASA Astrophysics Data System (ADS)

Bacterial infectious diseases remain one of the major health hazards nation- and worldwide. The expedience of detection and identification of bacterial pathogens determines how early the diagnosis is, and hence, what the treatment and the outcome of the illness would be. As we have previously reported, the dynamics of fluorescence staining provides venues for the development of expedient assays for detection and identification of bacterial species[1]. We measured the kinetics of bacterial staining with cyanine and thioflavin dyes and investigated their photophysical properties. We demonstrated that the pseudo first-order kinetic constants of the fluorescence staining processes have species specificity without contrition dependence. Combining the dynamics of staining with real-time fluorescence microscopy we characterized the fluorescence staining process at the single-cell level with improved sensitivity and contrast.

Nuñez, Vicente; Upadhyayula, Srigokul; Lin, Adam; Chau, Kenny; Vullev, Valentine I.



Side illumination fluorescence emission characteristics from a dye doped polymer optical fiber under two-photon excitation  

NASA Astrophysics Data System (ADS)

Two-photon excited (TPE) side illumination fluorescence studies in a Rh6G-RhB dye mixture doped polymer optical fiber (POF) and the effect of energy transfer on the attenuation coefficient is reported. The dye doped POF is pumped sideways using 800 nm, 70 fs laser pulses from a Ti:sapphire laser, and the TPE fluorescence emission is collected from the end of the fiber for different propagation distances. The fluorescence intensity of RhB doped POF is enhanced in the presence of Rh6G as a result of energy transfer from Rh6G to RhB. Because of the reabsorption and reemission process in dye molecules, an effective energy transfer is observed from the shorter wavelength part of the fluorescence spectrum to the longer wavelength part as the propagation distance is increased in dye doped POF. An energy transfer coefficient is found to be higher at shorter propagation distances compared to longer distances. A TPE fluorescence signal is used to characterize the optical attenuation coefficient in dye doped POF. The attenuation coefficient decreases at longer propagation distances due to the reabsorption and reemission process taking place within the dye doped fiber as the propagation distance is increased.

Sheeba, M.; Rajesh, M.; Mathew, S.; Nampoori, V. P. N.; Vallabhan, C. P. G.; Radhakrishnan, P.



Dictyostelium Extracellular Vesicles Containing Hoechst 33342 Transfer the Dye into the Nuclei of Living Cells: A Fluorescence Study  

Microsoft Academic Search

Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms\\u000a of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al.,\\u000a Cell Mol Life Sci, 54: 476–87, 1998). The question to be addressed in the present

Irène Tatischeff; Françoise Lavialle; Sophie Pigaglio-Deshayes; Christine Péchoux-Longin; Laurent Chinsky; Annette Alfsen



Features of the delayed fluorescence kinetics of exogenous fluorophores in biological tissues  

NASA Astrophysics Data System (ADS)

Kinetic regularities of the delayed fluorescence and phosphorescence of exogenous fluorophores (organic molecule dyes) in biological tissues were established. Differences in the delayed fluorescence kinetics of the healthy and pathogenic tissues stained with erythrosine in vitro are discussed.

Letuta, S. N.; Kuvandykova, A. F.; Pashkevich, S. N.; Saletskii, A. M.



Site-selective interactions: squaraine dye-serum albumin complexes with enhanced fluorescence and triplet yields.  


We report the effect of steric factors of a few squaraine dyes, bis(2,4,6-trihydroxyphenyl)squaraine (1), bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (2), and bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine (3), on their binding with human (HSA) and bovine (BSA) serum albumins employing photophysical, chiroptical, biophysical, and microscopic techniques. These dyes interact with serum albumins very efficiently and exhibit site selectivity, involving synergistic effects of hydrophobic, hydrogen bonding, and electrostatic interactions. The association constants of these complexes have been determined and are found to be 4.9 x 10(6) and 4.1 x 10(5) M(-1), respectively, for the dyes 2 and 3 with BSA, while HSA showed relatively higher association constants of 6.0 x 10(6) and 9.9 x 10(5) M(-1). Highly clear distinction in site-selective binding can be ascertained from time-resolved fluorescence, displacement cum fluorimetry, and circular dichroism (CD) studies. The increased affinity toward the major binding site (site II, domain III) over the relatively smaller binding site (site I, domain II) in the serum albumin with the increasing size of the heavy atoms present in 2 and 3 as compared to 1 indicates the importance of steric factors thereby confirming that the dye structure has a predominant role in deciding site selectivity. The distance between the energy donor and acceptor was calculated using Forster theory, which agrees well with the reported site 1 binding agent dansylamine. In contrast, no energy transfer was observed between tryptophan (Trp-214) present in domain II of the albumins and the dyes 2 and 3, indicating that these derivatives bind less efficiently at site I due to steric conatraints but preferentially bind at site II. Laser flash photolysis studies of the dyes 2 and 3 in the presence of HSA exhibited ca. 2.5-fold enhancements in the triplet lifetimes and quantum yields when compared to that obtained in buffer. The uniqueness of these dyes is that they show substituent size-dependent selectivity at site II of serum albumins and signal the event through "turn on" fluorescence intensity as well as enhanced triplet excited state lifetimes and quantum yields, thereby indicating their potential use as NIR noncovalent protein labeling and photodynamic therapeutic agents. PMID:20380473

Jisha, Vadakkancheril S; Arun, Kalliat T; Hariharan, Mahesh; Ramaiah, Danaboyina



Chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes.  


The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 ?m in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting. PMID:22968684

Kazakova, Lyubov I; Shabarchina, Lyudmila I; Anastasova, Salzitsa; Pavlov, Anton M; Vadgama, Pankaj; Skirtach, Andre G; Sukhorukov, Gleb B



Diffusion of organic dyes in a niosome immobilized on a glass surface using fluorescence correlation spectroscopy.  


Giant multilameller niosomes containing cholesterol and triton X-100 are studied using fluorescence correlation spectroscopy (FCS). Dynamic light scattering (DLS) data indicates formation of niosomes of broadly two different sizes (diameter)--~150 nm and ~1300 nm. This is confirmed by field emission scanning electron microscopy (FE-SEM) and confocal microscopy. The diffusion coefficient (D(t)) of three organic dyes in the niosome immobilized on a glass surface is studied using fluorescence correlation spectroscopy. On addition of the room temperature ionic liquids (RTIL) (1-methyl-3-pentylimidazolium bromide, [pmim][Br] and 1-methyl- 3-pentylimidazolium tetra-fluoroborate, [pmim][BF(4)]) the size of the niosome particles increases. The D(t) of all the organic dyes (DCM, C343 and C480) increases on addition of RTILs, indicating faster diffusion. The viscosity calculated from the D(t) of the three dyes exhibits weak probe dependence. Unlike lipid or catanionic vesicle, the D(t) values in a niosome exhibit very narrow distribution. This indicates that the niosomes are fairly homogeneous with small variation of viscosity. PMID:22692627

Ghosh, Shirsendu; Mandal, Amit Kumar; Das, Atanu Kumar; Mondal, Tridib; Bhattacharyya, Kankan



Synthesis of fluorescent dye-tagged nanomachines for single-molecule fluorescence spectroscopy.  


In an effort to elucidate the mechanism of movement of nanovehicles on nonconducting surfaces, the synthesis and optical properties of five fluorescently tagged nanocars are reported. The nanocars were specifically designed for studies by single-molecule fluorescence spectroscopy and bear a tetramethylrhodamine isothiocyanate fluorescent tag for excitation at 532 nm. The molecules were designed such that the arrangement of their molecular axles and p-carborane wheels relative to the chassis would be conducive to the control of directionality in the motion of these nanovehicles. PMID:20828172

Vives, Guillaume; Guerrero, Jason M; Godoy, Jazmin; Khatua, Saumyakanti; Wang, Yu-Pu; Kiappes, J L; Link, Stephan; Tour, James M



Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA stain  

PubMed Central

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo. Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Flegel, Kerry; Sun, Dan; Grushko, Olga; Ma, Yiqin; Buttitta, Laura



Fluorescence Properties and Staining Behavior of Monodansylpentane, a Structural Homologue of the Lysosomotropic Agent Monodansylcadaverine  

Microsoft Academic Search

SUMMARY We have recently shown that monodansylcadaverine labels autophagic vacu- oles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaver- ine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion- trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the auto-

Axel Niemann; Jennifer Baltes; Hans-Peter Elsässer


Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content  

SciTech Connect

Measurements and theoretical calculations of fluorescent emission from four samples of polystyrene microspheres (diameter 0.92, 1.63, 1.90 and 4.18 containing the same fluorescent dye show a general dependence upon particle size, emission angle, and polarization conditions. However, for the excitation and detection conditions used in flow cytometry, the relative fluorescent intensities measured for the four particle sizes are proportional to the dye content to +10% accuracy, independent of particle size. Accordingly, the central dogma of flow cytometry that fluorescence is proportional to cellular dye content is valid to this accuracy for these solid, highly refractive polymer particles. Most mammalian cells are much less refractive, therefore, should conform more closely to the central dogma.

Kerker, M.; Van Dilla, M.A.; Brunsting, A.; Kratohvil, J.P.; Hsu, P.; Wang, D.S.; Gray, J.W.; Langlois, R.G.



Laser induced singlet-oxygen-sensitised delayed fluorescence of dyes in aqueous solutions  

SciTech Connect

It is shown that water-soluble derivatives of phthalocyanines - poly(diethoxyphosphinylmethyl)substituted aluminium phthalocyanines - emit intense singlet-oxygen-sensitised delayed fluorescence upon laser-induced formation of singlet oxygen in air-saturated aqueous (D{sub 2}O) solutions. The delayed fluorescence is emitted by the dye molecules which accepted energy from two molecules of singlet oxygen. The quantum efficiency of delayed fluorescence in aerated D{sub 2}O of the chloroaluminium complex of octa(diethoxyphosphinylmethyl) phthalocyanine corresponds to the rate constant of population of excited dye molecules which is equal to (5.5 {+-} 3) x 10{sup 12} mole{sup -2} L{sup 2} s{sup -1}. This value is only an order of magnitude smaller than that for tetra(4-tert.-butyl)phthalocyanine earlier studied in aerated organic solvents. It is shown that these phthalocyanine derivatives can be used as highly sensitive luminescence indicators of singlet oxygen produced in aqueous solutions of different compounds upon laser excitation. (laser applications and other topics in quantum electronics)

Krasnovskii, A A; Bashtanov, M E; Drozdova, N N [A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow (Russian Federation); Yuzhakova, O A; Luk'yanets, Evgenii A [Russian Research Centre 'Research Institute of Organic Intermediates and Dyes', Moscow (Russian Federation)



Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content  

Microsoft Academic Search

Measurements and theoretical calculations of fluorescent emission from four samples of polystyrene microspheres (diameter 0.92, 1.63, 1.90 and 4.18 containing the same fluorescent dye show a general dependence upon particle size, emission angle, and polarization conditions. However, for the excitation and detection conditions used in flow cytometry, the relative fluorescent intensities measured for the four particle sizes are proportional

M. Kerker; M. A. Van Dilla; A. Brunsting; J. P. Kratohvil; P. Hsu; D. S. Wang; J. W. Gray; R. G. Langlois



Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections.  


The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). PMID:23281280

Gu, Jun; Fu, Chit Yaw; Ng, Beng Koon; Gulam Razul, Sirajudeen; Lim, Soo Kim



A red-emissive aminobenzopyrano-xanthene dye: elucidation of fluorescence emission mechanisms in solution and in the aggregate state.  


We have designed and synthesized a new class of rhodamine dyes with an extended ?-conjugated system and named them 3',3''-bis(oxospiroisobenzofuran)-3,7-bis(diethylamino)benzopyrano-xanthene (ABPX01) dyes. ABPX01 exhibits fluorescence emission in both dilute solution and the aggregate state, whereas conventional rhodamine dyes show aggregation-induced quenching (AIQ). The chemical species of ABPX01 in solution were determined by spectrophotometric measurements and density functional theory (DFT) calculations to study the relationship among chemical species, color, and fluorescence emission. ABPX01 has various forms: the spirolactone form (ABPX01(0)), which is colorless; and the monocationic form (ABPX01H(+)) and the dicationic form (ABPX01H(2)(2+)), which are colored. By orienting a pair of spirolactone benzene moieties differently, the stereoisomers of trans- and cis-ABPX01(0) were separated and their crystal structures determined. ABPX01H(2)(2+) was identified to be a red fluorescent species. Detailed spectroscopic and electron microscopic investigations led to the assumption that the ABPX01H(2)(2+) formed ion associates with Cl(-) as counter anions in HCl aqueous solution, and the nano- and submicrometer-sized colloidal aggregates of ABPX01 hydrochloride exhibit fluorescence emission. To further verify the aggregation-induced emission enhancement (AIEE) mechanism, ABPX01 hydrochloride was synthesized and its fluorescence was similarly checked in the powder state. AIEE in ABPX01 might be attributed to the synergistic combination of the restriction of dye-dye interaction induced dimer formation by sterically hindered ion associates and carboxylic benzene moieties, and the structural rigidity and intermolecular arrangement of the xanthene moiety. We expect that the design strategy of ABPX dyes will be extended to the development of a wide variety of functional organic-dye-based fluorophores (ODFs) with suitable fluorescence-emission controlled mechanisms for many useful applications in new electroluminescent devices. PMID:23288343

Kamino, Shinichiro; Muranaka, Atsuya; Murakami, Miho; Tatsumi, Asana; Nagaoka, Noriyuki; Shirasaki, Yoshinao; Watanabe, Keiko; Yoshida, Kengo; Horigome, Jun; Komeda, Seiji; Uchiyama, Masanobu; Enomoto, Shuichi



Critical tonicity determination of sperm using fluorescent staining and flow cytometry  

SciTech Connect

The use of cryopreserved, rather than fresh, mammalian semen for artificial insemination confers several important medical and/or economic advantages. However, current methods for cryopreservation of both human and bovine spermatozoa result in approximately only a 50% survival rate with thawing, obviously reducing the fertilizing capacity of the semen. A primary consideration during the cooling process is to avoid intracellular ice crystal formation with its lethal consequences to the cell. Current techniques achieve this by controlling the cooling rate. Computation of the time necessary for this dehydration, and hence, the cooling rate, is dependent upon knowledge of the water permeability coefficient (L{sub {rho}}) and its activation energy. The fluorophore, 6-carboxyfluoroscein diacetate (CFDA), which is nonfluorescent, readily crosses the intact plasma membrane. Intracellular esterases hydrolyze CFDA to 6-carboxyfluoroscein, a fluorescent, membrane-impermeable fluorophore. Consequently, spermatozoa with intact plasma membranes fluoresce bright green (Garner et. al., 1986), but those with disrupted membranes do not. Therefore, the purpose of this study was to use loss of CFDA fluorescence to determine the osmolality at which 50% of the spermatozoa will swell and lyse (critical tonicity, CT). These data will then be used to determine the L{sub {rho}} and its activation energy for sperm, thus increasing the knowledge available in cellular cryopreservation. 15 refs., 3 figs.

Noiles, E.E.; Ruffing, N.A.; Kleinhans, F.W.; Mark, L.A.; Watson, P.F.; Critser, J.K. (Methodist Hospital, Indianapolis, IN (USA)); Horstman, L. (Purdue Univ., Lafayette, IN (USA). School of Veterinary Medicine); Mazur, P. (Oak Ridge National Lab., TN (USA))



Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization.  


Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5 ?g of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67 g/l of ethanol) 8.7×10(4) CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5 days) of alcoholic fermentation. PMID:22819715

Branco, Patrícia; Monteiro, Margarida; Moura, Patrícia; Albergaria, Helena



Characterization of type I collagen fibril formation using thioflavin T fluorescent dye.  


Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin. There was a linear correlation between the fluorescence increase and the amount of atelocollagen within a collagen concentration range of 0-0.15 mg/ml at pH 6.5 with 50 microM thioflavin T. In contrast, neither actinidain-processed collagen that keeps monomeric nature nor heat-denatured collagen could cause the fluorescence increase of thioflavin T at all. The relationship between the fluorescence increase and thioflavin T concentration was fit to a theoretical binary binding curve. An apparent dissociation constant, K(d), and a maximal fluorescence increase, DeltaF(max), were calculated at various pHs. The values of K(d) and DeltaF(max) were dependent on pH (K(d) was 9.4 microM at pH 6.5). The present finding demonstrates that thioflavin T specifically binds to collagen fibrils and may be used as a sensitive tool for the study of collagen structure. PMID:19204013

Morimoto, Koichi; Kawabata, Kazuya; Kunii, Saori; Hamano, Kaori; Saito, Takuya; Tonomura, Ben'ichiro



Vital staining of specific monoamine-containing cells in the leech nervous system  

Microsoft Academic Search

Neutral red and several related dyes selectively stain certain cells in the ventral nerve cord of the leech. These cells are identical with those that can be shown by the FalckHillarp fluorescence technique to contain serotonin or a catecholamine; evidence suggests that the catecholamine is dopamine. Although the mechanism of staining remains unknown, it does not depend on active uptake

Ann E. Stuart; A. J. Hudspeth; Zach W. Hall



Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence  

Microsoft Academic Search

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation

P. Kask; P. Piksarv; Ü. Mets



Fluorescence of organic dyes in lipid membranes: Site of solubilization and effects of viscosity and refractive index on lifetimes  

Microsoft Academic Search

The fluorescence decay of several organic dye molecules intercalated in egg phosphatidylcholine lipid membrane vesicles is\\u000a consistent with the existence of two or three prominent lifetime components rather than a single continuous distribution of\\u000a lifetimes. The major lifetime components are identified with different sites of solubilization in the membrane. The variation\\u000a of the lifetime of the membrane-bound dye was studied

M. M. G. Krishna; N. Periasamy



Fluorescent dye particles as pollen analogues for measuring pollen dispersal in an insect-pollinated forest herb.  


In flowering plants, pollen dispersal is often the major contributing component to gene flow, hence a key parameter in conservation genetics and population biology. A cost-effective method to assess pollen dispersal consists of monitoring the dispersal of fluorescent dyes used as pollen analogues. However, few comparisons between dye dispersal and realized pollen dispersal have been performed to validate the method. We investigated pollen dispersal in two small populations of the insect-pollinated herb Primula elatior from urban forest fragments using direct (paternity analyses based on microsatellite DNA markers) and indirect (fluorescent dyes) methods. We compared these methods using two approaches, testing for the difference between the distance distributions of observed dispersal events and estimating parameters of a dispersal model, and related these results to dye dispersal patterns in three large populations. Dye and realized (based on paternity inference) pollen dispersal showed exponential decay distributions, with 74.2-94.8% of the depositions occurring at <50 m and a few longer distance dispersal events (up to 151 m). No significant difference in curve shape was found between dye and realized pollen dispersal distributions. The best-fitting parameters characterizing the dye dispersal model were consistent with those obtained for realized pollen dispersal. Hence, the fluorescent dye method may be considered as reliable to infer realized pollen dispersal for forest herbs such as P. elatior. However, our simulations reveal that large sample sizes are needed to detect moderate differences between dye and realized pollen dispersal patterns because the estimation of dispersal parameters suffers low precision. PMID:20703887

Van Rossum, Fabienne; Stiers, Iris; Van Geert, Anja; Triest, Ludwig; Hardy, Olivier J



Micelles assembled with carbocyanine dyes for theranostic near-infrared fluorescent cancer imaging and photothermal therapy.  


It is an emerging focus to explore a theranostic nanocarrier for simultaneous cancer imaging and therapy. Herein, we demonstrate a theranostic micelle system for cancer near infrared fluorescent (NIRF) imaging with enhanced signal to noise ratio and superior photothermal therapy. The copolymers consisting of monomethoxy poly(ethylene glycol) and alkylamine-grafted poly(l-aspartic acid) are assembled with carbocyanine dyes into theranostic micelles, which exhibit small size, high loading capacity, good stability, sustained release behavior, and enhanced cellular uptake. The micelles achieve the preferable biodistribution and long-term retention of carbocyanine dyes at tumor, which result in enhanced NIRF imaging by generating stable retention of NIRF signals at both hypervascular and hypovascular tumors during a long-term imaging period of up to 8 day, accompanying with negligible noise at normal tissues. The photostability of carbocyanine dye (Cypate) plays an important role for long-term cancer imaging with enhanced SNR. Moreover, the micelles exhibit severe photothermal damage on cancer cells via the destabilization of subcellular organelles upon photoirradiation, causing superior photothermal tumor regress. The micelles act as a powerful theranostic nanocarrier for simultaneous cancer imaging with high contrast and superior photothermal therapy. PMID:24008037

Yang, Hong; Mao, Huajian; Wan, Zhihui; Zhu, Aijun; Guo, Miao; Li, Yanli; Li, Xinming; Wan, Jiangling; Yang, Xiangliang; Shuai, Xintao; Chen, Huabing



UV laser interaction with a fluorescent dye solution studied using pulsed digital holography.  


A frequency tripled Q-switched Nd-YAG laser (wavelength 355 nm, pulse duration 12 ns) has been used to pump Coumarin 153 dye solved in ethanol. Simultaneously, a frequency doubled pulse (532 nm) from the same laser is used to probe the solvent perpendicularly resulting in a gain through stimulated laser induced fluorescence (LIF) emission. The resulting gain of the probe beam is recorded using digital holography by blending it with a reference beam on the detector. Two digital holograms without and with the pump beam were recorded. Intensity maps were calculated from the recorded digital holograms and used to calculate the gain of the probe beam due to the stimulated LIF. In addition numerical data of the local temperature rise was calculated from the corresponding phase maps using Radon inversion. It was concluded that about 15% of the pump beam energy is transferred to the dye solution as heat while the rest is consumed in the radiative process. The results show that pulsed digital holography is a promising technique for quantitative study of fluorescent species. PMID:24150372

Amer, Eynas; Gren, Per; Sjödahl, Mikael



Solvatochromic behavior on the absorption and fluorescence spectra of Rose Bengal dye in various solvents.  


The absorption and fluorescence spectra of Rose Bengal dye were studied in various solvents. It was found that solvent effects on the absorption wavelength are consistent with the solvatochromic model of Kamlet, Abboud and Taft. The solvent polarizability value pi* was found to have a linear relationship with the absorption wavelength of the dye in various solvents. Additionally, the normalized transition energy value (E(T)(N)) showed some scattering when plotted versus Deltanu(af). Density functional calculations were used to assign the absorption in the region 540-570 nm to a pi-pi* transition between the HOMO and LUMO of the anion. Experimental ground state and excited state dipole moments were calculated by using the solvatochromatic shifts of absorption and fluorescence spectra as a function of the dielectric constant (epsilon) and refractive index (n). The dipole moment for Rose Bengal was found to be 1.72 Debye in the ground state, whereas this value was 2.33 Debye in the excited state. PMID:18986828

Rauf, M A; Graham, John P; Bukallah, Saeed B; Al-Saedi, Mariam A S



Near-Infrared Fluorescent Dye-Doped Semiconducting-Polymer Dots  

PubMed Central

Near-infrared (NIR) fluorescence sensing is desirable for in vivo biological measurements. But the method is currently limited by the availability of NIR fluorescent markers as well as by their poor performance, such as self-aggregation and dim fluorescence, in a physiological environment. To address this issue, this paper describes a NIR fluorescent polymer dot (Pdot) that emits at 777 nm. This Pdot was comparable in size to a water-soluble NIR quantum dot that emits at 800 nm (ITK Qdot800) but was about 4 times brighter and with a narrower emission peak. We formed the NIR Pdot by doping the NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), into the matrix of poly (9,9-dioctylfluorene-co-benzothiadiazole) (PFBT) as the Pdot formed using a nanoscale precipitation technique. Free molecules of NIR775 aggregate in aqueous solution but encapsulating them into the hydrophobic Pdot matrix effectively introduced them into aqueous solution for use in biological studies. Most importantly, the brightness of NIR775 was dramatically enhanced because of the excellent light harvesting ability of PFBT and the very efficient energy transfer from PFBT to NIR775. We anticipate this bright NIR Pdot will be useful in biological measurements and cellular imaging where strong NIR emission is beneficial.

Jin, Yuhui; Ye, Fangmao; Zeigler, Maxwell; Wu, Changfeng; Chiu, Daniel T.



Characterization of core-shell lattices by near-IR fluorescent cyanine dyes  

NASA Astrophysics Data System (ADS)

Cyanine-based near-IR fluorescent probes have been applied as labels to determine aldehyde-groups in polymeric materials. The advantage of such probes is that they can be applied to polymers which are autofluorescent or strongly colored or to strongly scattering polymer emissions. The example which is discussed in this paper is the labelling of a polymeric core-shell latex, comprising a polystyrene core and a glycidylmethacrylate shell, which has been hydrolyzed and subsequently oxidized to form aldehyde functional groups, by application of a Cyanine with a hydrazide functionality. The Cyanine which has been applied absorbs and fluoresces in the 650 - 700 nm range. In addition to near-IR fluorescence, the Cyanine dyes have strong absorption (with extinction coefficients in the order of 2 X 105 I/ It is shown that by application of reflection absorption measurements also sensitive detection of labelled functional groups can be realized. The combined application of fluorescence and reflection measurements allows for the detection of concentration and distribution of functional groups in polymeric systems down to very low levels.

Hofstraat, Johannes W.; van Houwelingen, Gerrit D.; Nuijens, Marcel J.; Gooijer, Cees; Velthorst, Nel H.



Histometrics: improvement of the dynamic range of fluorescently stained proteins resolved in electrophoretic gels using hyperspectral imaging.  


Most image-based analyses, using absorbance or fluorescence of the spatial distribution of identifiable structures in complex biological systems, use only a very small number of dimensions of possible spectral data for the generation and interpretation of the image. We here extend the concepts of hyperspectral imaging, being developed in remote sensing, into analytical biotechnology. The massive volume of information contained in hyperspectral spectroscopic images requires multivariate analysis in order to extract the chemical and spatial information contained within the data. We here describe the use of multivariate statistical methods to map and quantify common protein staining fluorophores (SYPRO Red, Orange and Tangerine) in electrophoretic gels. Specifically, we find (a) that the 'background' underpinning limits of detection is due more to proteins that have not migrated properly than to impurities or to ineffective destaining, (b) the detailed mechanisms of staining of SYPRO red and orange are apparently not identical, and in particular (c) that these methods can provide two orders of magnitude improvement in the detection limit per pixel, to levels well below the limit observable optically. PMID:11922594

Woodward, A M; Kaderbhai, N; Kaderbhai, M; Shaw, A; Rowland, J; Kell, D B



Utility of intensely fluorescent cyanine dyes (CY3) for assay of gap junctional communication by dye-transfer  

Microsoft Academic Search

Utilization of a class of florescent cyanine dyes (Cy3™) for the assay of gap junctional communication by the dye transfer method was examined. When comp ed with Lucifer Yellow (LY), a commonly used tracer, microinjected Cy3 dye was found to yield similar degrees of cell coupling. Blockade of the transfer of both tracers by 12-O-tetradecanoylphorbol-13 acetate (TPA), which is known

M. Z Hossain; L. A Ernst; J. I Nagy



Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.  


Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 ?g of material. The approach is robust for both soluble and detergent-solubilized membrane proteins. PMID:24091556

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H



Dye fluorescence enhancement and quenching by gold nanoparticles: Direct near-field microscopic observation of shape dependence  

NASA Astrophysics Data System (ADS)

We investigated the enhancement and quenching of dye fluorescence by single gold nanoparticles using an aperture-type scanning near-field optical microscope to evaluate the shape dependence. Enhancement or reduction of fluorescence was found to be strongly dependent on the shape of the particle. Gold nanoplates showed significant fluorescence enhancement, as did aggregated nanoparticles for the most part. On the other hand, gold nanorods with high aspect ratios showed quenching effects. The results suggest that the electromagnetic near-field in the vicinity of the surface of nanoparticles, not just near the apex or the tip but the whole surface, is important for understanding fluorescence enhancement and quenching.

Horimoto, Noriko Nishizawa; Imura, Kohei; Okamoto, Hiromi



Resist Pattern Inspection Using Fluorescent Dye-Doped Polystyrene Thin Films in Reactive-Monolayer-Assisted Thermal Nanoimprint Lithography  

NASA Astrophysics Data System (ADS)

Fluorescent dye-doped polystyrene (PS) thin films were studied for defect inspection of PS resist patterns by fluorescence microscopy in reactive-monolayer-assisted thermal nanoimprint lithography using a photoreactive monolayer. A fluorescent dye of N,N\\aku '-bis(2,6-dimethylphenyl-perylene-3,4,9,10-tetracarboxylic diimide doped in a PS resist thin film maintained an almost identical fluorescence intensity after annealing at a temperature necessary for thermal nanoimprinting. To avoid degradation of a dye doped in a resist film owing to exposure to ultraviolet light for preparing a PS graft layer on the photoreactive monolayer, a double coating method for preparing a dye-doped PS resist layer on the PS graft layer was adopted. It was demonstrated by the fluorescent microscopic defect inspection that resist pattern defects due to unleveled residual layers after thermal nanoimprinting were significantly decreased by adding low-molecular-weight PS (5,100 g mol-1) to high-molecular-weight PS (360,000 g mol-1). The rheological study revealed that the low-molecular-weight PS obviously functioned as a plasticizer, which flattened residual layers and decreased their thickness.

Kubo, Shoichi; Sato, Yuko; Hirai, Yoshihiko; Nakagawa, Masaru



CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye.  


Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100-500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100-500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100-200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR. PMID:23417332

Valdés, Alberto; García-Cañas, Virginia; Cifuentes, Alejandro



The evaluation of a novel method comparing quantitative light-induced fluorescence (QLF) with spectrophotometry to assess staining and bleaching of teeth  

Microsoft Academic Search

This study reports the development and evaluation of a novel method using quantitative light-induced fluorescence (QLF), which\\u000a enables its use for quantifying and assessing whole tooth surface staining and tooth whitening. The method was compared with\\u000a a spectrophotometer to assess reliability. Two experimental phases, intrinsic stain formation and tooth whitening, were conducted\\u000a in vitro on 16 extracted bovine teeth. Intrinsic

A. A. Adeyemi; F. D. Jarad; E. de Josselin de Jong; N. Pender; S. M. Higham



Silica cross-linked nanoparticles encapsulating fluorescent conjugated dyes for energy transfer-based white light emission and porphyrin sensing.  


This work demonstrated that water-soluble fluorescent hybrid materials can be successfully synthesized by use of silica cross-linked micellar nanoparticles (SCMNPs) as scaffolds to encapsulate fluorescent conjugated dyes for pH sensing, porphyrin sensing and tunable colour emission. Three dyes were separately encapsulated inside SCMNPs (short to dye-SCMNPs). Each of the dye-SCMNPs indicated longer lifetime in water than that of free dye dissolved in organic solvent. The 7-(hexadecyloxy) coumarin-3-ethylformate (HCE) encapsulated inside SCMNPs (HCE-SCMNPs) exhibited fluorescence quenching by pH change in aqueous media. Furthermore, it was confirmed that the radiative and nonradiative energy transfer processes both occurred between HCE-SCMNPs and tetraphenyl-porphyrin (TPP), which were used to synthesize the water-soluble TPP sensor. Significantly, HCE-SCMNPs doped with 5,12-dicotyl-quinacridone (8CQA) and TPP showed water-soluble white light emission (CIE (0.29, 0.34)) upon singlet excitation of 376 nm due to colour adjustment of 8CQA and energy transfer from HCE (donor) to TPP (acceptor). PMID:22930394

Gai, Fangyuan; Zhou, Tianlei; Zhang, Ligong; Li, Xiang; Hou, Weijia; Yang, Xinchun; Li, Yantao; Zhao, Xiaogang; Xu, Da; Liu, Yunling; Huo, Qisheng



Synthesis and characterization of monodisperse, mesoporous, and magnetic sub-micron particles doped with a near-infrared fluorescent dye  

SciTech Connect

Recently, multifunctional silica nanoparticles have been investigated extensively for their potential use in biomedical applications. We have prepared sub-micron monodisperse and stable multifunctional mesoporous silica particles with a high level of magnetization and fluorescence in the near infrared region using an one-pot synthesis technique. Commercial magnetite nanocrystals and a conjugated-NIR-dye were incorporated inside the particles during the silica condensation reaction. The particles were then coated with polyethyleneglycol to stop aggregation. X-ray diffraction, N{sub 2} adsorption analysis, TEM, fluorescence and absorbance measurements were used to structurally characterize the particles. These mesoporous silica spheres have a large surface area (1978 m{sup 2}/g) with 3.40 nm pore diameter and a high fluorescence in the near infrared region at {lambda}=700 nm. To explore the potential of these particles for drug delivery applications, the pore accessibility to hydrophobic drugs was simulated by successfully trapping a hydrophobic ruthenium dye complex inside the particle with an estimated concentration of 3 wt%. Fluorescence imaging confirmed the presence of both NIR dye and the post-grafted ruthenium dye complex inside the particles. These particles moved at approximately 150 {mu}m/s under the influence of a magnetic field, hence demonstrating the multifunctionality and potential for biomedical applications in targeting and imaging. - Graphical Abstract: Hydrophobic fluorescent Ruthenium complex has been loaded into the mesopores as a surrogate drug to simulate drug delivery and to enhance the multifunctionality of the magnetic NIR emitting particles. Highlights: > Monodisperse magnetic mesoporous silica particles emitting in the near infrared region are obtained in one-pot synthesis. > We prove the capacity of such particles to uptake hydrophobic dye to mimic drug loading. > Loaded fluorescent particles can be moved under a magnetic field in a microfluidic device.

Le Guevel, Xavier, E-mail: [Biomedical Diagnostics Institute, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9 (Ireland); Nooney, Robert; McDonagh, Colette; MacCraith, Brian D. [Biomedical Diagnostics Institute, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9 (Ireland)



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

Microsoft Academic Search

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 in- teractions in vitro between a class of spinal cord neu- rons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes,

Marcia G. Honig; Richard I. Hume



PHYSICAL PROCESSES IN LASER MEDIA: Saturation of the fluorescence of dye solutions exposed to pulsed laser excitation  

Microsoft Academic Search

The fluorescence saturation curves of dye molecules were calculated for optically thin layers of solutions exposed to pulsed laser excitation. Intercombination conversion of molecules from an excited singlet state to a triplet state was taken into account for various distributions of the photon flux density of the exciting radiation as a function of time and in the beam cross section.

S. Ya Dzhasim; N. Ya Serov; V. V. Fadeev; A. M. Chekalyuk



Single-lane single-fluor sequencing using dideoxy-labeled, heavy-atom-modified near-IR fluorescent dyes  

NASA Astrophysics Data System (ADS)

Using a near-IR (NIR) fluorescence detection system and labels synthesized in our laboratories, electropherograms of oligonucleotides separated by capillary gel electrophoresis and detected using NIR fluorescence will be presented. The sequence of nucleotide bases was determined using a single-lane, single-dye technique. The molar concentrations of the ddNTP's used during extension reactions were varied in order to achieve a ratio of 4:2:1:0 (A:C:G:T) which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the template, M13mp18, using standard Sanger dideoxy chain termination techniques, the modified T7 DNA polymerase, and a NIR-labeled M13 primer. The data indicated reliable sequence determination up to 300 bases with a base-calling accuracy of 90%. In order to eliminate the need for dye-labeled primers and the T7 DNA polymerase enzyme, we have developed a sequencing strategy which utilizes dye-labeled dideoxy nucleotides in a single-lane, single-fluor approach. Base-calling is accomplished by measuring the fluorescence lifetime of intramolecular heavy-atom modified dyes.

Williams, Daryl C.; Flanagan, James H.; Legendre, Benjamin L.; Hammer, Robert P.; Soper, Steven A.



Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: A comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies  

SciTech Connect

Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.

Khan, Faaizah; Gnudi, Luigi [Metabolic Unit, King's College London School of Medicine, Guy's Hospital, London SE1 9RT (United Kingdom); Pickup, John C. [Metabolic Unit, King's College London School of Medicine, Guy's Hospital, London SE1 9RT (United Kingdom)], E-mail:



Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes. Presence of positive and false positive beads.  


To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr. PMID:19072229

Marani, Mariela M; Martínez Ceron, María C; Giudicessi, Silvana L; de Oliveira, Eliandre; Côté, Simon; Erra-Balsells, Rosa; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A


Apparatus for eliminating background interference in fluorescence measurements  


The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser which excites a different stained component of the same biological particle.

Martin, J.C.; Jett, J.H.



Apparatus for eliminating background interference in fluorescence measurements  


The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle.

Martin, John C. (Los Alamos, NM); Jett, James H. (Los Alamos, NM)



Apparatus for eliminating background interference in fluorescence measurements  


The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle. 8 figs.

Martin, J.C.; Jett, J.H.



Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red.  


The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting A?1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of A?1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures. PMID:21279219

Mishra, Rajesh; Sjölander, Daniel; Hammarström, Per



Comparison of Quantitative Fluorescent Staining Methods for Flow-System Analysis of DNA, Protein, and Nuclear-to-Cytoplasmic Size in Cultured Cells, Tumor Cells, and Human Gynecological Specimens.  

National Technical Information Service (NTIS)

Three groups of reagents having different mechanisms of action for DNA binding were used for quantitative fluorescent staining and analysis of cellular DNA. The reagents used included the fluorescent antitumor antibiotics mithramycin, chromomycin A sub 3 ...

H. A. Crissman J. A. Steinkamp



Internal labelling of the eggs of the aphidophagous hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) with a fluorescent dye.  


A method was developed to label the egg load of Episyrphus balteatus (De Geer) (Diptera: Syrphidae) by feeding adult females with the fluorescent dye Rhodamine B. In a preliminary experiment aimed at selecting an appropriate dye concentration, gravid females reared in insectary conditions were provided for two successive days with a 50% honey-water solution enriched with Rhodamine B at concentrations of 0.1%, 0.01% and 0.001%. Eggs deposited on aphid-infested broad bean plants by syrphid females fed with Rhodamine B 0.01% showed the most intense marking. The higher dye concentration induced irregular marking among the eggs, presumably due to its repellent taste and consequently lower intake by the flies. A second series of trials focused on the persistence of the marker, prior and after oviposition, at the selected (optimal) dilution. A reduction in the marking degree of eggs was observed when the first egg deposition was delayed for one day after removing the dye from the cage. When marked females were allowed to deposit further batches of eggs the subsequent days, fewer eggs were laid and their fluorescence properties was reduced. Exposure to direct sunlight during 10 hours had no detectable effect on the fluorescence level of marked eggs deposited on a bean plant. The egg-marking technique examined in this study could be useful to determine the crop area accommodating eggs deposited by marked syrphid females migrating from a release point into cultivated fields. PMID:16628909

Bribosia, E; Bylemans, D; Migon, M; van Impe, G



A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.  


Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques. PMID:20600953

Moreno, Alfredo; SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Alvarez, Javier



Structure elucidation of laser dye coumarin-540A by joint application of X-ray diffraction, X-ray fluorescence, prompt fluorescence, UV and visible spectroscopy  

Microsoft Academic Search

X-ray, ultraviolet, and visible light induced photophysical changes of coumarin-540A in ethanol have been studied by the joint applications of X-ray, ultraviolet, and visible spectroscopy. Some impurities were found by X-ray fluorescence measurements. During the high power optical pumping, coumarin showed photochemical changes. Photoproduct emission spectra characteristics showed that photoproduct molecules can also be used as a laser dye at

Sinan S. Keskin; Necdet Aslan; Fuat Bayrakçeken



Structure elucidation of laser-dye coumarin-540A by joint application of X-ray diffraction, X-ray fluorescence, prompt fluorescence, UV and visible spectroscopy  

Microsoft Academic Search

X-ray, ultraviolet, and visible light induced photophysical changes of coumarin-540A in ethanol has been studied by the joint applications of X-ray, ultraviolet, and visible spectroscopy. Some impurities were found by X-ray fluorescence measurements. During the high power optical pumping, coumarin showed photochemical changes. Photoproduct emission spectra characteristics showed that photoproduct molecules could also be used as a laser-dye at a

Sinan S. Keskin; Necdet Aslan; Fuat Bayrakçeken



Identification of hybridomas derived from mouse CD5+ B lymphocytes by fluorescent staining for cytoplasmic CD5 expression.  

PubMed Central

The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immunoglobulin gene repertoire and antibody specificity of this B-lymphocyte subpopulation. However, hybridomas derived from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignment of the generated hybridomas to the CD5+ B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be detected in the cytoplasm of some mouse hybridomas, indicating that although surface expression has been lost, they may have been generated from CD5+ B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expression of the CD5 gene occurred as a direct consequence of the hybridization process. To confirm that cytoplasmic CD5 expression in the hybridomas is in fact directly related to the B-lymphocyte origin we have generated hybridomas from FACS-sorted CD5+ and CD5- murine splenic B lymphocytes, and determined their surface and cytoplasmic CD5 expression by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5+ B lymphocytes (nine hybridomas) were negative for surface but positive for cytoplasmic CD5 expression, whereas all hybridomas derived from CD5- B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This finding shows that staining for cytoplasmic CD5 expression provides an accurate method of determining the cellular origin of murine B-lymphocyte hybridomas.

Sadigh, S; Scott, B B; Mageed, R A; Malcolm, A; Andrew, E M; Maini, R N



Theoretical investigation on saturated Förster-Resonant-Energy-Transfer Microscopy using FRET dye pairs as fluorescent probes  

NASA Astrophysics Data System (ADS)

Saturated Förster-Resonant-Energy-Transfer (FRET) microscopy (SFM) employing tiny silica nanoparticles densely co-doped with Cy3 and Cy5 molecules as fluorescent labels has been proposed and analyzed with rigorous rate equations previously, showing that optical resolutions beyond the diffraction-limit in both lateral and axial directions can be realized by exploiting the saturation effect in FRET process. Here, using a set of similar rate equations, we show that SFM can also be realized with single FRET dye pairs as fluorescent labels. A similar nonlinear optical response to excitation beam is revealed for Cy3-Cy5 dye pairs, in comparison to the silica nanoparticles co-doped with Cy3 and Cy5 molecules.

Chen, Jianfang; Cheng, Ya



A highly fluorescent pH sensing membrane for the alkaline pH range incorporating a BODIPY dye.  


A robust and re-usable dipstick-type fluorescent pH sensor for the alkaline pH range was developed by embedding a brightly fluorescent boron-dipyrromethene (BODIPY) dye bearing an acidic phenol moiety into a polyurethane matrix immobilized on a 3D epoxy-functionalized polymer support. The sensor strip has a dynamic working range of pH 10.0-13.1, i.e., operates in strongly basic media where pH glass electrodes can suffer from alkaline errors, and tolerates a high electrolyte background such as simulated seawater and sewage. This work describes the preparation of the sensing material and provides insight into the features that a hydrogel sensing membrane can bestow on an embedded pH-responsive dye by means of optical spectroscopic investigations. PMID:23091817

Hecht, Mandy; Kraus, Werner; Rurack, Knut



Bioconjugatable azo-based dark-quencher dyes: synthesis and application to protease-activatable far-red fluorescent probes.  


We describe the efficient synthesis and one-step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher-3 (BHQ-3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, ?-iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water-soluble (sulfonated derivative) and aldehyde-modified BHQ-3 dyes, the direct preparation of which failed by means of conventional azo-coupling reactions. The aldehyde- and ?-iodoacetyl-containing fluorescence quenchers were readily conjugated to aminooxy- and cysteine-containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red- or far-red-emitting rhodamine or cyanine dyes through sequential and/or one-pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis. PMID:23255474

Chevalier, Arnaud; Massif, Cédrik; Renard, Pierre-Yves; Romieu, Anthony



Resonance Rayleigh Scattering for the Determination of Berberine in Tablet Form with some Acidic Xanthene Fluorescent Dyes  

Microsoft Academic Search

.  ?A simple determination method of berberine with a limit of determination at the nanogram level is proposed under the use\\u000a of a common spectrofluorometer to detect the intensity of resonance Rayleigh scattering (RRS). In aqueous solution at pH 4–5,\\u000a berberine reacts with acidic xanthene fluorescent dyes such as eosine Y, erythrosine, ethyl eosin, phloxin and Rose Bengal\\u000a to form an

Shaopu Liu; Ping Feng



Absorption and fluorescence studies on interaction between cationic dyes and Klebsiella K7 capsular polysaccharide.  


Interaction of cationic dyes, pinacyanol chloride, acridine orange and phenosafranin, with Klebsiella K7 capsular polysaccharide has been investigated by spectrophotometric and spectrofluorometric measurements. The acidic polysaccharide induce a metachromatic blue shift of the absorption band of pinacyanol chloride from 600 nm to 495 nm, indicating strong metachromasy. Stoichiometry of polyanion and dye cation (1:1.5) in the polymer-dye compound formed by the interaction between pinacyanol chloride dye and K7 polymer indicate that both glucuronic acid and pyruvic acid act as the potential anionic sites for interaction. Both spectrophotometric titration of pinacyanol chloride and spectrofluorometric titration of acridine orange and phenosafranin dyes by the polymer gave quite comparable equivalent weights for the polymer. Dye-polymer interaction studies indicated induction of metachromasy in the cationic dye by the anionic biopolymer, establishing its chromotropic character. PMID:1512016

Mitra, A; Chakraborty, A K



Charge-Recombination Fluorescence from Push-Pull Electronic Systems Constructed around Amino-Substituted Styryl-BODIPY Dyes.  


A small series of donor-acceptor molecular dyads has been synthesized and fully characterized. In each case, the acceptor is a dicyanovinyl unit and the donor is a boron dipyrromethene (BODIPY) dye equipped with a single styryl arm bearing a terminal amino group. In the absence of the acceptor, the BODIPY-based dyes are strongly fluorescent in the far-red region and the relaxed excited-singlet states possess significant charge-transfer character. As such, the emission maxima depend on both the solvent polarity and temperature. With the corresponding push-pull molecules, there is a low-energy charge-transfer state that can be observed by both absorption and emission spectroscopy. Here, charge-recombination fluorescence is weak and decays over a few hundred picoseconds or so to recover the ground state. Overall, these results permit evaluation of the factors affecting the probability of charge-recombination fluorescence in push-pull dyes. The photophysical studies are supported by cyclic voltammetry and DFT calculations. PMID:24038505

Nano, Adela; Ziessel, Raymond; Stachelek, Patrycya; Harriman, Anthony



Tricolour fluorescence detection of sequence-specific DNA with a new molecular beacon and a nucleic acid dye TOTO-3.  


We have developed a tricolor fluorescence quantitative method for sequence-specific DNA detection using a new molecular beacon (MB) and a nucleic acid dye TOTO-3. This new MB is designed with two fluorophores of FAM and TAMRA instead of one fluorophore and one quencher of traditional MB, and a nucleotide with guanine base is attached directly to FAM as a quencher. In the absence of target DNA, MBs are in the stem-loop state. The fluorescence of FAM is absorbed by TAMRA, and the fluorescence of TAMRA is quenched by guanine base. Meanwhile, the interaction between TOTO-3 and MBs is very weak. In the presence of target DNA, MBs hybridize with target DNA to form a double-stranded structure. TAMRA is separated from FAM and guanine base, and the fluorescence of FAM and TAMRA recovers simultaneously. At the same time TOTO-3 binds to double-stranded DNA, the fluorescence of TOTO-3 significantly enhances. In this strategy, the false-positive signals of MBs caused by non-specific interactions can be distinguished by the change of the ratio of the total fluorescence intensities of FAM and TAMRA to that of TOTO-3 at different concentrations of target DNA. In the simple sample, the detection of target DNA can be achieved with the total fluorescence intensity of three dyes, which results in a significant improvement of the detection sensitivity. In the complex sample, the detection of target DNA can be achieved with the fluorescence intensity of TOTO-3 which can overcome the false-positive signals of MBs and improve the detection accuracy. PMID:23113317

Xiang, Dongshan; Zhang, Cuiling; Chen, Lu; Ji, Xinghu; He, Zhike



Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles  

NASA Astrophysics Data System (ADS)

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems. Electronic supplementary information (ESI) available: Cell culture preparation for dose/response imaging experiments. See DOI: 10.1039/c3nr02639f

Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.



Wood stains  


Wood stains are products used for wood finishing. Wood stain poisoning occurs when someone swallows these substances. This is ... Various wood stains Note: This list does not include all sources of wood stain.


Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles.  


A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or "free" surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems. PMID:24056530

Wang, Wei; Nallathamby, Prakash D; Foster, Carmen M; Morrell-Falvey, Jennifer L; Mortensen, Ninell P; Doktycz, Mitchel J; Gu, Baohua; Retterer, Scott T



Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.  


Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110?nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5?nM). PMID:23180379

Astakhova, I Kira; Wengel, Jesper



Ex-vivo imaging of excised tissue using vital dyes and confocal microscopy  

PubMed Central

Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex-vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture based studies.

Johnson, Simon; Rabinovitch, Peter



Ex vivo imaging of excised tissue using vital dyes and confocal microscopy.  


Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture-based studies. PMID:22752953

Johnson, Simon; Rabinovitch, Peter



Inhibition of fluorescent dyes for the design of efficient activatable probes dedicated to non-invasive small animal imaging  

NASA Astrophysics Data System (ADS)

The framework of fluorescent targeting probes for optical imaging is similar to that of contrast agents for other modalities. They generally include a biological ligand, specific of the biological process to image, and a label, which confers the probe its optical properties. Moreover, more sophisticated labeling functions, termed "activatable" can be designed. An "activatable probe" will be initially non fluorescent. Only a specific molecular process, such as an enzymatic reaction or cell internalization, is able to activate the probe fluorescence. Such probes are particularly easy to design for the optical imaging modality, because of the easily triggered and well known fluorescence inhibition processes. The optical properties of different commercially available organic dyes and their fluorescence inhibition are therefore examined. Three classes of activatable probes have been listed: (i) activatable probes which rely on the selfquenching of the label; (ii) activatable probes which use RET (Resonance Energy Transfer) between the label acting as a donor, and an organic non-emissive acceptor; (iii) activatable probes which use an inorganic nanostructure as the inhibitor, such as a gold nano-particle. Whereas activatable probes of class (i) can lead to higher fluorescence levels after activation, their initial fluorescence inhibition can be hindered by structural constraints. Probes of class (ii) can therefore be more interesting according to the probe design. The efficiency of probes of class (iii) using nanometer gold particles is reduced because of their plasmon band lying in the visible and not near-infrared domain.

Texier, Isabelle; Heinrich, Emilie



Comparison of the effects of ophthalmic solutions on human corneal epithelial cells using fluorescent dyes.  


Abstract Purpose: To investigate the effect of differently preserved ophthalmic solutions on the viability and barrier function of human corneal epithelial cells (HCEC) using fluorescent dyes. Methods: HCEC monolayers were exposed to the ophthalmic solutions containing benzalkonium chloride (BAK), edetate disodium, polyquad, stabilized oxychloro complex (Purite), sodium perborate, or sorbic acid for 5?min, 15?min, and 1?h. At 24?h after exposure, the cultures were assessed for metabolic activity using alamarBlue. The enzyme activity, membrane integrity, and apoptosis were evaluated using confocal microscopy. Barrier function was assessed using sodium fluorescein. Results: The metabolic assay showed that the BAK-preserved ophthalmic solutions significantly reduced cell viability after a 5-min exposure compared to the phosphate buffered saline treated control (P<0.05). Using confocal microscopy, the micrographs showed that BAK caused a reduction in the enzyme activity, increased membrane permeability, and decreased the number of viable cells. Ophthalmic solutions with new preservatives had varying time-dependent adverse effects on cell viability, and the preservative-free solution had the least effect on HCEC. Sodium fluorescein permeability showed that HCEC monolayers treated with BAK-preserved solutions were more permeable to sodium fluorescein than those treated by the other ophthalmic solutions (P<0.05). Conclusions: BAK-preserved solutions had greater adverse effects on metabolic activity, enzyme activity, membrane integrity, cell viability, and barrier function than the solutions that were not preserved with BAK. Our study suggests that BAK-free especially, preservative-free ophthalmic solutions are safer alternatives to BAK-preserved ones. PMID:23905770

Xu, Manlong; Sivak, Jacob G; McCanna, David J



In vivo fluorescence imaging of lysosomes: a potential technique to follow dye accumulation in the context of PDT?  

NASA Astrophysics Data System (ADS)

Lysosomes and intracellular acidic compartments seem to play an important role in the context of PDT. Some photosensitizers are localized in the lysosomes of tumor-associated macrophages. Liposomes, which are lysosomotropic drug carriers, are used to deliver photosensitizers in tumors. Liposomes are taken up by the liver cells after intravenous injection. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cell separation, and observation by electronic microscopy. Little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH-sensitive probe. We have already demonstrated the ability of fluorescence spectroscopy and imaging using a pH-dependent probe to monitor pH in living tissues. As pH of lysosome is very low, the kinetic of liposome uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Liposomes-encapsulated carboxyfluorescein are prepared by the sonication procedure. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the peinil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a rapid fluorescence increase followed by a slow phase of fluorescence decrease. pH decreases from physiological value to 6.0. After sacrifice and flush with cold saline solution, pH of liver ex vivo is found to be 5.0 - 5.5. These data show a rapid clearance of released dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of pH.

Devoisselle, Jean-Marie; Mordon, Serge R.; Soulie, Sylvie



In vivo fluorescence imaging of lysosomes: a potential technique to follow dye accumulation in the context of PDT?  

NASA Astrophysics Data System (ADS)

Lysosomes and intracellular acidic compartments seem to play an important role in the context of PDT. Some photosensitizers are localized in the lysosomes of tumor-associated macrophages. Liposomes, which are lysosomotropic drug carriers, are used to deliver photosensitizers in tumors. Liposomes are taken up by the liver cells after intravenous injection. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cell separation, and observation by electronic microscopy. Little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH-sensitive probe. We have already demonstrated the ability of fluorescence spectroscopy and imaging using a pH-dependent probe to monitor pH in living tissues. As pH of lysosome is very low, the kinetic of liposome uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Liposomes-encapsulated carboxyfluorescein are prepared by the sonication procedure. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the peinil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a rapid fluorescence increase followed by a slow phase of fluorescence decrease. pH decreases from physiological value to 6.0. After sacrifice and flush with cold saline solution, pH of liver ex vivo is found to be 5.0 - 5.5. These data show a rapid clearance of released dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of pH.

Devoisselle, Jean-Marie; Mordon, Serge R.; Soulie-Begu, Sylvie



Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe  

Microsoft Academic Search

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence

Yongnian Ni; Daiqin Lin; Serge Kokot



Lasing Spectral Blue Shifts of Fluorescent Saturable Absorbing Dye in Microdroplets  

Microsoft Academic Search

We report blue shifts of emission spectra of microdroplet lasers containing fluores- cent saturable absorbing dye as compared with those of conventional dye lasers. The results cannot be simply explained by matching the lasing peaks to the minima of threshold condition without taking into account cavity enhancement. We find that the microdroplet lasers have quality factors of lo4 with a

I.-Ju Yu; Wen-Feng Hsieh



Kinetics of White Blood Cell Staining by Intravascular Administration of Rhodamine 6G  

Microsoft Academic Search

Rhodamine 6G is a vital dye accumulating in the mitochondria of cells. It is used in intravital fluorescence microscopy for contrast enhancement of white blood cells (WBC), enabling visualization of WBC in the microvasculature even at high center flow velocity. The aim of this study was to examine the kinetics of WBC staining after intravascular administration of rhodamine 6G in

H. Baatz; M. Steinbauer; A. G. Harris; F. Krombach



Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red  

PubMed Central

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein ?-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of ?-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with ?-galactosidase aggregates led to a shift of the emission maximum (?max) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated ?-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native ?-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with ?-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Oliveira, Sabrina; Sanders, Niek N.; Lucas, Bart; van Hoek, Arie; Hink, Mark A.; Visser, Antonie J. W. G.; De Smedt, Stefaan C.; Hennink, Wim E.; Jiskoot, Wim



A novel, Golgi-Cox-based fluorescent staining method for visualizing full-length processes in primary rat neurons.  


The Golgi method, a well-known method used for staining whole dendrites and axonal trees of neurons, has been used widely for studying dendritic growth in vivo. Although detailed structural examination of neurons and their processes stained by the Golgi method has elucidated the complicated neuronal circuit, application of the method in cultured neurons has been unsuccessful to date. Here, we report the development of a stable, highly sensitive Golgi-Cox method that allows visualization of full-length processes, including the dendritic spines and the growth cones, of cultured rat neurons. This modified staining method requires: (1) rat cultured neurons fixed with a mixture containing 4% paraformaldehyde and 12.5% glutaraldehyde before impregnation with mercury; (2) rapid freezing of the fixed neurons using dry ice; and (3) immersion of the fixed neurons in Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin-G antibody (Molecular Probes, Eugene, OR) for visualization after impregnation. PMID:23619398

Koyama, Yoshihisa; Tohyama, Masaya



Self-assembly of highly fluorescent semiconductor nanorods into large scale smectic liquid crystal structures by coffee stain evaporation dynamics  

NASA Astrophysics Data System (ADS)

We deposit droplets of nanorods dispersed in solvents on substrate surfaces and let the solvent evaporate. We find that strong contact line pinning leads to dense nanorod deposition inside coffee stain fringes, where we observe large scale lateral ordering of the nanorods with the long axis of the rods oriented parallel to the contact line. We observe birefringence of these coffee stain fringes by polarized microscopy and we find the direction of the extraordinary refractive index parallel to the long axis of the nanorods.

Nobile, Concetta; Carbone, Luigi; Fiore, Angela; Cingolani, Roberto; Manna, Liberato; Krahne, Roman



Design of fluorescence dye-conjugated peptides for the receptor-targeted optical imaging of tumors using solid-phase peptide libraries  

NASA Astrophysics Data System (ADS)

We describe the design and properties of cyanine dye-peptide conjugates synthesized on solid supports for screening assays. With this approach, we demonstrate the feasibility of including a diagnostic molecule into a spot synthesis of membrane-bound peptide libraries in order to permit screening of complete dye-peptide conjugates for their potential as fluorescent contrast agents in biomedical optical imaging. A cellulose support, which is modified with a linker permitting cleavage of the dye-peptide conjugates from the support, was prepared. The attachment of dyes to the peptides is exemplified with carboxy-substituted indotricarbocyanines, which can be covalently linked to the N-terminal amino group or a lysin in the course of the synthesis. Several dye-peptide conjugates were obtained by automated peptide synthesis on resins. Furthermore, model sequences consisting of up to 11 amino acids were synthesized on cellulose in sufficient amounts and purity, thus permitting direct testing of these compounds in cell- based assays and fluorescence microscopy. The dyes show only negligible alterations in their absorption and fluorescence properties when attached to low molecular weight peptides. In conclusion, membrane-bound peptide libraries provide a powerful tool to generate large diversities of dye-labeled peptides and to make these compounds available for biological screening assays.

Licha, Kai; Bhargava, Sarah; Becker, Andreas; Hessenius, Carsten; Schneider-Mergener, Jens; Volkmer-Engert, Rudolph



The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange.  


Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells. PMID:3508914

Steven, F S; Suresh, U; Wong, T L; Griffin, M M



Nanoscopic heterogeneities in adsorption and electron transfer processes of perylene diimide dye on TiO 2 nanoparticles studied by single-molecule fluorescence spectroscopy  

Microsoft Academic Search

The interfacial electron transfer processes between a fluorescent water-soluble perylene diimide dye (WS-PDI) and TiO2 nanoparticles were investigated using single-molecule fluorescence spectroscopy. Based on the single-molecule fluorescence spectral measurements, it was suggested that the local environment and\\/or the structural conformation of single WS-PDI molecules play important roles in the efficiency of the electron injection from WS-PDI in the singlet excited

Takashi Tachikawa; Shi-Cong Cui; Sachiko Tojo; Mamoru Fujitsuka; Tetsuro Majima



Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues  

Microsoft Academic Search

Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures

Ana María Díez-Navajas; Charles Greif; Anne Poutaraud; Didier Merdinoglu



Non-fluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.  


In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for whole mount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy. PMID:24048924

Jussen, David; Urbach, Rolf



A universal and label-free aptasensor for fluorescent detection of ATP and thrombin based on SYBR Green I dye.  


A facile and universal aptamer-based label-free approach for selective and sensitive fluorescence detection of proteins and small biomolecules by using the SYBR Green I (SGI) dye is developed. This robust versatile biosensing strategy relies on fluorescence turn-off changes of SGI, resulting from target-induced structure switching of aptamers. Upon binding with the targets, the aptamers dissociate from the respective cDNA/aptamer duplexes, leading to the release of the dsDNA-intercalated SGI into solution and the quenching of the corresponding fluorescence intensities. Such target-induced conformational changes and release of aptamers from the DNA duplexes essentially lead to the change in the fluorescence signal of the SGI and thus constitute the mechanism of our aptamer-based label-free fluorescence biosensor for specific target analyses. Under optimized conditions, our method exhibits high sensitivity and selectivity for the quantification of ATP and thrombin with low detection limits (23.4 nM and 1.1 nM, respectively). Compared with previous reported methods for aptamer-based detection of ATP and thrombin, this label-free approach is selective, simple, convenient and cost-efficient without any chemical labeling of the probe or the target. Therefore, the present strategy could be easily applicable to biosensors that target a wide range of biomolecules. PMID:23202351

Kong, Ling; Xu, Jin; Xu, Yunying; Xiang, Yun; Yuan, Ruo; Chai, Yaqin



Near Infrared Voltage Sensitive Fluorescent Dyes Optimized for Optical Mapping in Blood-Perfused Myocardium  

PubMed Central

Background Styryl voltage-sensitive dyes (e.g. di-4-ANEPPS) have been successfully used for optical mapping in cardiac cells and tissues. However, their utility for probing electrical activity deep inside the myocardial wall and in blood-perfused myocardium has been limited because of light scattering and high absorption by endogenous chromophores and hemoglobin at blue-green excitation wavelengths. Objectives The purpose of this study was to characterize two new styryl dyes Di-4-ANBDQPQ (JPW-6003) and Di-4-ANBDQBS (JPW-6033) optimized for blood-perfused tissue and intramural optical mapping. Methods Voltage-dependent spectra were recorded in a model lipid bilayer. Optical mapping experiments were conducted in 4 species (mouse, rat, guinea pig, and pig). Hearts were Langendorff-perfused using Tyrode’s solution and blood (pig). Dyes were loaded via bolus injection into perfusate. Transillumination experiments were conducted in isolated coronary-perfused pig right ventricular wall preparations. Results The optimal excitation wavelength in cardiac tissues (650nm) was >70nm beyond the absorption maximum of hemoglobin. Voltage-sensitivity of both dyes was ~10–20%. Signal decay half-life due to dye internalization was 80–210 minutes, which is 5–7 times slower than for di-4-ANEPPS. In transillumination mode, ?F/F was as high as 20%. In blood-perfused tissues, ?F/F reached 5.5% (1.8 times higher than for di-4-ANEPPS). Conclusion We have synthesized and characterized two new NIR dyes with excitation/emission wavelengths shifted >100nm to the red. They provide both high voltage-sensitivity, and 5–7 times slower internalization rate compared to conventional dyes. The dyes are optimized for deeper tissue probing and optical mapping of blood-perfused tissue, and can also be used for conventional applications.

Matiukas, Arvydas; Mitrea, Bogdan G.; Qin, Maochun; Pertsov, Arkady M.; Shvedko, Alexander G.; Warren, Mark D.; Zaitsev, Alexey V.; Wuskell, Joseph P.; Wei, Mei-de; Watras, James; Loew, Leslie M.



Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles.  


This paper describes a biosensing concept for exotoxins secreted by Staphylococcus aureus and Pseudomonas aeruginosa based on the toxin mediated breakdown and subsequent fluorescent dye release from phospholipid vesicles (liposomes). The sensitivity of vesicles to toxins was tuned by altering the lipid and fatty acid composition of the membranes such that vesicles could be engineered to respond to toxins/enzymes from S. aureus only; P. aeruginosa only; and both S. aureus and P. aeruginosa. Nineteen types of vesicle were made with varying compositions of phosphocholine (PC), phosphoethanolamine (PE), cholesterol and the photo-polymerizable ampiphile 10,12-tricosadiynoic acid (TCDA). The selectivity of the vesicles was measured via a simple fluorescence "switch on" assay. Sensitivity of the vesicles to 40 clinically derived strains of S. aureus and P. aeruginosa was also demonstrated. This work suggests that this technology could be utilised in a diagnostic tool to discriminate between the species of S. aureus and P. aeruginosa in wound dressings. PMID:23063348

Thet, N T; Hong, S H; Marshall, S; Laabei, M; Toby, A; Jenkins, A



Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe  

NASA Astrophysics Data System (ADS)

The interaction between L-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH = 4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that L-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of L-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

Lin, Jing; Liu, Rutao; Gao, Canzhu



Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.  

PubMed Central

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.

Roth, B L; Poot, M; Yue, S T; Millard, P J



[Determination of four Sudan dyes in chili oil by high performance liquid chromatography with on-line photochemical derivatization and fluorescence detection].  


A method for the measurement of Sudan I, Sudan II, Sudan III and Sudan B in chili oil using high performance liquid chromatography (HPLC) with on-line photochemical derivatization and fluorescence detection has been developed. The Sudan dyes were separated on an SB-C18 column in a single run by the mixed mobile phase of acetonitrile-water with a gradient program. A laboratory-built time/energy programmed photochemical reactor (PCR) with an ultraviolet mercury lamp was installed between a photodiode array detector (PDA) and a fluorescence detector (FLD), and it was applied to convert the non-or weakly fluorescent Sudan dyes into fluorescence emission components. The photochemical derivatization conditions and fluorescence detection parameters have been investigated and optimized. The recoveries of the standards spiked in real chili oil samples for all the dyes were 81.3% - 100.4%. The relative standard deviations (RSDs, n = 6) of the fluorescence signal intensity at the spiked level of 0.8 mg/kg were 2.6% - 3.8%. The limits of detection (LODs) were in the range of 0.009 - 0.054 mg/kg and the limits of quantification (LOQs) were 0.030 - 0.181 mg/kg, which were better than those of the commonly used HPLC coupled with PDA. The developed method which is simple, sensitive, and selective can be applied to the routine analysis of Sudan dyes. PMID:23016298

Liu, Jun; Gong, Zhenbin



DNA complexes with dyes designed for energy transfer as fluorescent markers  


Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figs.

Glazer, A.M.; Benson, S.C.



DNA complexes with dyes designed for energy transfer as fluorescent markers  


Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figs.

Glazer, A.N.; Benson, S.C.



DNA complexes with dyes designed for energy transfer as fluorescent markers  


Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated. 4 figures.

Glazer, A.N.; Benson, S.C.



Spectroscopic characterization of coumarin-stained beads: quantification of the number of fluorophores per particle with solid-state 19F-NMR and measurement of absolute fluorescence quantum yields.  


The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative ((19)F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF(3) group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods. PMID:22404690

Huber, Alexandra; Behnke, Thomas; Würth, Christian; Jaeger, Christian; Resch-Genger, Ute



Indocyanine dyes approach free rotation at the 3' terminus of A-RNA: a comparison with the 5' terminus and consequences for fluorescence resonance energy transfer.  


Cyanine dyes are widely used to study the folding and structural transformations of nucleic acids using fluorescence resonance energy transfer (FRET). The extent to which FRET can be used to extract inter- and intramolecular distances has been the subject of considerable debate in the literature; the contribution of dye and linker dynamics to the observed FRET signal is particularly troublesome. We used molecular dynamics (MD) simulations to study the dynamics of the indocarbocyanine dyes Cy3 and Cy5 attached variously to the 3' or 5' terminal bases of a 16-base-pair RNA duplex. We then used Monte Carlo modeling of dye photophysics to predict the results of single-molecule-sensitive FRET measurements of these same molecules. Our results show that the average value of FRET depends on both the terminal base and the linker position. In particular, 3' attached dyes typically explore a wide region of configuration space, and the relative orientation factor, ?(2), has a distribution that approaches that of free-rotators. This is in contrast to 5' attached dyes, which spend a significant fraction of their time in one or more configurations that are effectively stacked on the ends of the RNA duplex. The presence of distinct dye configurations for 5' attached dyes is consistent with observations, made by others, of multiple fluorescence lifetimes of Cy3 on nucleic acids. Although FRET is frequently used as a molecular "ruler" to measure intramolecular distances, the unambiguous measurement of distances typically relies on the assumption that the rotational degrees of freedom of the dyes can be averaged out and that the donor lifetime in the absence of the acceptor is a constant. We demonstrate that even for the relatively free 3' attached dyes, the correlation time of ?(2) is still too long to justify the use of a free-rotation approximation. We further explore the consequences of multiple donor lifetimes on the predicted value of FRET. PMID:23799279

Milas, Peker; Gamari, Ben D; Parrot, Louis; Krueger, Brent P; Rahmanseresht, Sheema; Moore, James; Goldner, Lori S



Properties of two-photon fluorescence and superradiance of a new organic dye C46H51N2B  

NASA Astrophysics Data System (ADS)

Linear and nonlinear optical properties of a new organic dye, trans-4-[ p-(N-n-butyl-N-n-butylamino)-styryl] -N-methyl-pyridinium tetraphenylborate solution in dimethyl formamide (DMF) have been studied systematically. When excited with mode-locked picosecond 1 064 nm laser beam, intense upconversion fluorescence and superradiance can be obtained. The temporal behaviors of one-photon absorption and two-photon absorption (TPA) fluorescence and superradiance have been studied. The highest upconversion efficiency was found to be 4.1% at a pump energy of 4 mJ. By using an optical parameter amplifier (OPA) as the pump laser, the nonlinear transmittance and upconversion efficiencies of the dye solution at different wavelengths were measured. The strongest linear absorption was found at a wavelength of 930 nm whereas the highest upconversion efficiency was at 1 030 nm. The 100 nm red-shift for the highest upconversion efficiency wavelength compared with the strongest nonlinear absorption are caused by excited state absorption.

Zhou, G.; Wang, D.; Yang, S.; Ren, Y.; Xu, X.; Shao, Z.; Zhao, X.; Jiang, M.; Tian, Y.; Hao, F.; Li, S.; Shi, P.



Highly Photostable Near-Infrared Fluorescent pH Indicators and Sensors Based on BF2-Chelated Tetraarylazadipyrromethene Dyes  

PubMed Central

In this study, a series of new BF2-chelated tetraarylazadipyrromethane dyes are synthesized and are shown to be suitable for the preparation of on/off photoinduced electron transfer modulated fluorescent sensors. The new indicators are noncovalently entrapped in polyurethane hydrogel D4 and feature absorption maxima in the range 660–710 nm and fluorescence emission maxima at 680–740 nm. Indicators have high molar absorption coefficients of ?80?000 M–1 cm–1, good quantum yields (up to 20%), excellent photostability and low cross-sensitivity to the ionic strength. pKa values of indicators are determined from absorbance and fluorescence measurements and range from 7 to 11, depending on the substitution pattern of electron-donating and -withdrawing functionalities. Therefore, the new indicators are suitable for exploitation and adaptation in a diverse range of analytical applications. Apparent pKa values in sensor films derived from fluorescence data show 0.5–1 pH units lower values in comparison with those derived from the absorption data due to Förster resonance energy transfer from protonated to deprotonated form. A dual-lifetime referenced sensor is prepared, and application for monitoring of pH in corals is demonstrated.



Fluorescent Dye Encapsulated ZnO Particles with Cell-specific Toxicity for Potential use in Biomedical Applications  

SciTech Connect

Fluorescein isothiocyanate (FITC)-encapsulated core-shell particles with a nanoscale ZnO finishing layer have been synthesized for the first time as multifunctional “smart” nanostructures for particle tracking and cell imaging using the visible fluorescence emission of the dye or UV fluorescence emission of ZnO, and anti-cancer/antibacterial treatments using the selective toxicity of the nanoscale ZnO outer surface. The chemical phase composition, morphology, size, and the layered core-shell architecture of the particles were characterized using detailed transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and UV-vis-NIR spectrophotometry. Systematic XPS studies after removing nanometer thick layers confirmed the expected layered structure in the order ZnO-SiO2-APTMS-FITC proceeding from the surface to the core of the ~200 nm sized particles. Detailed investigation of the fluorescence properties of these hydrophilic particles in bio-compatible media using fluorescence spectroscopy, flow cytometry and fluorescence confocal microscopy demonstrated that the silica/ZnO outer layer offers considerable protection to the encapsulated dye molecules from photobleaching and quenching due to reactive species such as oxygen in the solvent. These particles showed promise toward cell imaging, for example when the bacterium Escherichia coli was used as a test system, the green fluorescence of the particles allowed confocal microscopy to image the cells. The FITC encapsulated ZnO (FITC-ZnO) particles demonstrated excellent selectivity in preferentially killing Jurkat cancer cells (18% cell viability) without any significant toxicity to normal primary immune cells (75% cell viability) at 60 ?g/mL concentrations and inhibited the growth of both gram-positive and gram negative bacteria at concentrations ? 250-500 ?g/mL (for Staphylococcus aureus and Escherichia coli, respectively). These results indicate that the novel FITC encapsulated multifunctional particles with nanoscale ZnO surface layer are smart nanostructures for particle tracking, cell imaging, antibacterial treatments and cancer therapy.

Wang, Hua; Wingett, Denise; Engelhard, Mark H.; Feris, Kevin; Reddy, K. M.; Turner, Paul; Layne, Janet; Hanley, Cory; Bell, Jason; Tenne, Dmitri; Wang, Chong M.; Punnoose, Alex



Optical Properties of Fluorescent Mixtures: Comparing Quantum Dots to Organic Dyes  

ERIC Educational Resources Information Center

|The study describes and compares the size-dependent optical properties of organic dyes with those of semiconductor nanocrystals or quantum dots (QDs). The analysis shows that mixtures of QDs contain emission colors that are sum of the individual QD components.|

Hutchins, Benjamin M.; Morgan, Thomas T.; Ucak-Astarlioglu, Mine G.; Wlilliams, Mary Elizabeth



Bis-naphthobipyrrolylmethene derived BODIPY complex: an intense near-infrared fluorescent dye.  


Synthesis of a novel ?-extended BODIPY derived from naphthobipyrrole is presented. This dye molecule displays very intense near-infrared (NIR) absorption (? > 400?000 M(-1) cm(-1)) and emission bands (>700 nm), accompanied by high quantum yield (?f = 0.65) owing to its extended ?-conjugation along with imposed structural rigidification. PMID:24030220

Sarma, Tridib; Panda, Pradeepta K; Setsune, Jun-Ichiro



Visualization of Phospholipid Domains in Escherichia coli by Using the Cardiolipin-Specific Fluorescent Dye 10-N-Nonyl Acridine Orange  

Microsoft Academic Search

Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algo- rithm revealed NAO-binding domains in the plane of




Identification of live hair cells in rat cochlear sections in culture with FM1-43 fluorescent dye.  


Cochlear hair cells are presumed to live in culture for many days, yet they are difficult to identify in cultured tissues. We stained hair cells in cochlear sections with FM1-43 and cultured them in collagen matrix. Three rows of outer hair cells and a single row of inner ones were distinguished by staining with FM1-43. Fixation of the sections with paraformaldehyde caused loss of the FM1-43 fluorescence, indicating that FM1-43 stained only live hair cells. In sections cultured for 48 h, almost all hair cells were still positive with FM1-43. Culture with gentamycin caused loss of FM1-43-positive cells. In serum-free, long-term cultures (15 days) performed without antibiotics or neurotrophins, the row alignment of FM1-43-positive hair cells was still maintained. Membranous labyrinth-like vacuoles enveloping hair cells were formed in the collagen matrix. Accordingly, FM1-43 is an efficient marker for identifying live hair cells in cultured tissues. Moreover, cochlear hair cells are revealed to live for weeks in serum-free culture without exogenous neurotrophins. PMID:14729254

Fukuda, Jun; Ishimine, Hisako; Tokunaga, Motohide



[Registration of stable aberrations in peripheral blood lymphocytes using G-differential chromosome staining and fluorescence in situ hybridization methods].  


G-banding analysis and fluorescence in situ hybridization (FISH) for whole chromosomes 1, 2 and 4 were applied in comparative assay for frequency of stable chromosome aberrations in 37 individuals with previous exposure to radiation (15 clean-up workers/liquidators of Chernobyl nuclear power plant accident and 22 residents of radiocontaminated areas) and in 18 individuals of a reference group. In G-banding analysis of stable aberrations, we used classification of Ohtaki K. et al., 1992, in compliance with ISCN, 1985. FISH-assay for translocations is performed in accordance with classification of Tucker J.D. et al., 1995. Comparison of the results reveals statistical trustworthy correlation between the two assays. The results point out that FISH for translocations in as few as three chromosomes, when combined with screening of numerous metaphases, provides sensitivity comparable with that provided by G-banding which covers the whole genome. PMID:9889772

Obukhova, T N; Domracheva, E V


Development of indirect competitive fluorescence immunoassay for 2,2',4,4'-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels  

Technology Transfer Automated Retrieval System (TEKTRAN)

An indirect competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in aqueous samples. A hapten, 2,4,2'-tribromodiphenyl ether-4’-aldehyde was sy...


A multifunctional magnetic nanocarrier bearing fluorescent dye for targeted drug delivery by enhanced two-photon triggered release.  


We report a novel nanoformulation for targeted drug delivery which utilizes nanophotonics through the fusion of nanotechnology with biomedical application. The approach involves an energy-transferring magnetic nanoscopic co-assembly fabricated of rhodamine B (RDB) fluorescent dye grafted gum arabic modified Fe(3)O(4) magnetic nanoparticle and photosensitive linker by which dexamethasone drug is conjugated to the magnetic nano-assembly. The advantage offered by this nanoformulation is the indirect photo-triggered-on-demand drug release by efficient up-converting energy of the near-IR (NIR) light to higher energy and intraparticle energy transfer from the dye grafted magnetic nanoparticle to the linker for drug release by cleavage. The synthesized nanoparticles were found to be of ultra-small size (13.33 nm) and are monodispersed in an aqueous suspension. Dexamethasone (Dexa) drug conjugated to RDB-GAMNP by photosensitive linker showed appreciable release of Dexa by photo-triggered response on exposure to radiation having a wavelength in the NIR region whereas no detectable release was observed in the dark. Photo-triggered response for the nanoformulation not bearing the rhodamine B dye was drastically less as less Dexa was released on exposure to NIR radiation which suggest that the photo-cleavage of linker and release of Dexa mainly originated from the indirect excitation through the uphill energy conversions based on donor-acceptor model FRET. The promising pathway of nanophotonics for the on-demand release of the drug makes this nanocarrier very promising for applications in nanomedicine. PMID:19420604

Banerjee, Shashwat S; Chen, Dong-Hwang



Bacterial population dynamics in a reverse-osmosis water purification system determined by fluorescent staining and PCR-denaturing gradient gel electrophoresis.  


The bacterial population dynamics in an industrial scale reverse-osmosis (RO) water purification system were analyzed by fluorescent staining methods and denaturing gradient gel electrophoresis (DGGE). Bacterial numbers increased with storage in a tank, and bacterial diversity changed during the water purification process. A DNA sequence-based analysis of the major bands on the DGGE gel revealed that Simonsiella sp. (Betaproteobacteria) was abundant in the source water (activated sludge-treated waste effluent), while Bosea sp. and Rhizobium sp. (Alphaproteobacteria), which usually exist in an oligotrophic environment, became abundant during the water purification process. These results suggest the importance of microbiological monitoring by culture-independent methods for quality control in RO water purification systems. These methods could provide an early warning of impending problems and clarify critical steps in controlling specific bacteria contributing to the contamination of RO water systems. PMID:21566369

Baba, Takashi; Matsumoto, Rie; Yamaguchi, Nobuyasu; Nasu, Masao



Fluorescence-on response via CB7 binding to viologen-dye pseudorotaxanes.  


Fluorescence-on sensors typically rely on disrupting photoinduced electron transfer quenching of the excited state through binding the electron donor. To provide a more general fluorescence-on signaling unit, a quencher-fluorophore dyad has been developed in which quenching by electron transfer to a tethered viologen acceptor can be disrupted through complexation of the viologen by cucurbit[7]uril (CB7). Dyads of benzyl viologen-rhodamine B or a BODIPY fluorophore gave upon CB7 complexation 14- and 30-fold fluorescence enhancement, respectively. PMID:22860771

Singh, Anuradha; Yip, Wai-Tak; Halterman, Ronald L



High sensitivity analysis of water-soluble, cyanine dye labeled proteins by high-performance liquid chromatography with fluorescence detection  

Microsoft Academic Search

A water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide ester of 3H-Indolium,1-[(4-carboxyphenyl)methyl]-2-[3-[1-[(4-carboxyphenyl)methyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-(9CI) (sb-cy3-NHS), containing two p-carboxybenzyl groups on nitrogen atoms, previously developed by our laboratory, was for the first time used for protein derivatization, followed by HPLC separation and fluorescence detection. With bovine serum albumin (BSA) as a model protein, effects of various experimental conditions, including denaturant concentration, reaction time and temperature,

Xiaoqiang Qiao; Li Wang; Junfeng Ma; Qiliang Deng; Zhen Liang; Lihua Zhang; Xiaojun Peng; Yukui Zhang



Immunogold silver staining associated with epi-fluorescence for cucumber mosaic virus localisation on semi-thin sections of banana tissues.  


The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone. PMID:17664166

Helliot, B; Panis, B; Busogoro, J P; Sobry, S; Poumay, Y; Raes, M; Swennen, R; Lepoivre, P


Photon Antibunching in the Fluorescence of a Single Dye Molecule Trapped in a Solid.  

National Technical Information Service (NTIS)

The correlation between fluorescence photons emitted by an optically pumped single molecule of pentacene in a p-terphenyl host has been investigated at short times. The correlation function shows photon antibunching, an unique feature of nonclassical radi...

T. Basche W. E. Moerner



Synthesis and characterization of fluorescent acenequinones as dyes for guest-host liquid crystal displays.  


Syntheses and spectroscopic properties of alkoxy-substituted para-acenequinones are reported. These compounds showed excellent alignment in nematic liquid crystals as evidenced by polarized UV-vis absorption and fluorescence measurements. [structure: see text] PMID:17298074

Chen, Zhihua; Swager, Timothy M



Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules.  


Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway. PMID:23482425

Bederska, Magdalena; Borucki, Wojciech; Znojek, Ewa



Covalent labeling of the cytoplasmic or luminal domains of the sarcoplasmic reticulum Ca(2+)-ATPase with fluorescent azido dyes.  


Sarcoplasmic reticulum (SR) vesicles were incubated with azido derivatives of Cascade blue (ACB), Lucifer yellow (ALY), 2,7-naphthalene-disulfonic acid (ANDS), and fluorescein (AF) for 0.1-24 h at 2 degrees C. All four dyes gave intense reaction with the cytoplasmic domain of the Ca(2+)-ATPase on photoactivation after brief incubation. The penetration of the dyes into the luminal space of the SR was determined after centrifugation through Sephadex microcolumns to remove the external dye, followed by photolabeling and gel electrophoresis of the photolabeled proteins. The reaction of ACB and ANDS with the Ca(2+)-ATPase and with calsequestrin increased progressively during incubation up to 24 h indicating their slow accumulation in the luminal space, while ALY and AF did not show significant penetration into the vesicles. The distribution of the covalently attached ACB in the Ca(2+)-ATPase was tested by tryptic proteolysis after labeling exclusively from the outside (OS), from the inside (IS) or from both sides (BS). In all cases intense ACB fluorescence was seen in the A fragment with inhibition of ATPase activity. In the OS preparations the A1, while in IS the A2 fragment was more intensely labeled. There was no significant incorporation of ACB into the region of B fragment identified by FITC fluorescence. The crystallization of the Ca(2+)-ATPase by EGTA + decavanadate was completely inhibited in the BS samples after labeling either in the Ca2E1 or E2V conformation. There was no inhibition of crystallization in the OS preparations. In the IS preparations labeled in the Ca2E1 state the crystallization was impaired, while in the E2V state there was only slight disorganization of the crystals. The total amount of ACB photoincorporated into SR proteins after incubation for 24 h was 1.75 nmol/mg protein; 2/3 of this labeling occurred from the outside and 1/3 from the inside. Similar level of labeling was obtained in media that stabilize the E1 or the E2 conformation of the Ca(2+)-ATPase. PMID:1832561

Molnar, E; Varga, S; Jona, I; Martonosi, A



Flow cytometric detection of micronuclei by combined staining of DNA and membranes  

SciTech Connect

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.

Wessels, J.M.; Nuesse, M. [Institut fuer Biophysikalische Strahlenforschung, Oberschleibheim (Germany)



A femtosecond fluorescence study of vibrational relaxation and cooling dynamics of UV dyes.  


We present a femtosecond broad-band fluorescence up-conversion study of the vibrational relaxation dynamics of two UV chromophores, 2,5-diphenyloxazole (PPO) and para-terphenyl (pTP), pumped with a large excess of vibrational energy (>2000 cm(-1)). The band narrowing of the transient fluorescence spectrum reflects a biphasic cooling process in a few hundreds of fs and a few ps. In the sub-ps regime, our data suggest a structural rearrangement in the excited state, followed by thermalization of the excess energy. These dynamics affect the fluorescence spectra of PPO and pTP in different ways. In PPO, the damping of a low frequency vibrational wavepacket and a significant sub-ps narrowing of the band characterize the vibrational relaxation. In pTP, the latter is faster and appears as a red shift with distortion of the band in <200 fs. PMID:22307066

Braem, Olivier; Penfold, Thomas J; Cannizzo, Andrea; Chergui, Majed



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

PubMed Central

We have stripped small (3 × 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. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

van Kesteren, W. J. P.; van der Schoot, C.; van Bel, A. J. E.



Substrate-selective supramolecular tandem assays: monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles.  


A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO). PMID:19627092

Nau, Werner M; Ghale, Garima; Hennig, Andreas; Bakirci, Hüseyin; Bailey, David M



Diketo-pyrrolo-pyrrole dyes as new colorimetric and fluorescent pH indicators for optical carbon dioxide sensors.  


New indicators for optical CO2 sensors are synthesized in two steps from the commercially available diketo-pyrrolo-pyrrole (DPP) pigments Irgazin Ruby and Irgazin Scarlet. After introduction of bis(2-ethylhexyl) sulfonamide groups via a simple two-step synthesis, the pigments are rendered highly soluble in organic solvents and in polymers and show pH-dependent absorption and emission spectra. The new indicators have molar absorption coefficients in a 20?000-50?000 M(-1) cm(-1) range, possess quantum yields close to unity, and feature good photostability. The indicators along with a quaternary ammonium base are embedded into ethyl cellulose to give optical carbon dioxide sensors. The absorption and emission spectra of the deprotonated form are bathochromically shifted by more than 100 nm compared to the neutral form (?max absorption 496-550 nm; ?max emission 564-587 nm). This enables colorimetric read-out and self-referenced ratiometric fluorescence intensity measurements. Importantly, the dynamic range of the sensors based on the new indicators is significantly different (0-10 kPa and 1-100 kPa CO2) that enables a broad variety of applications. New DPP dyes are conveniently prepared from commercially available pigments and represent a new class of colorimetric and fluorescent pH indicators for optical carbon dioxide sensors. PMID:23421943

Schutting, Susanne; Borisov, Sergey M; Klimant, Ingo



Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.  


We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy. PMID:22763945

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke



Photophysical processes of an intramolecular charge transfer fluorescent dye with carbazole units.  


A carbazole-based compound with intramolecular charge transfer (ICT) characteristics, 3,6-bis-((N-ethylcarbazole-3-)-propene-1-keto)-N-ethylcarbazole (BCzPCz) was synthesized by N-alkylation, acetylation and aldol condensation. BCzPCz was further confirmed by IR and (1)H NMR. The central N-ethylcarbazole was connected with two N-ethylcarbazole units through the propenone group in BCzPCz. N-ethylcarbazole and carbonyl groups were electron donors (D) and acceptors (A), respectively. The UV-vis absorption and fluorescence characteristics of BCzPCz were also investigated in different solvents. Solvatochromism was attributed to ICT complex formation in singlet excited state. Magnitude of the change in the dipole moment was 24.78 D according to Lippert-Mataga equation. Fluorescence of BCzPCz was significantly affected by pH and was quenched in acidic medium. Fluorescence quantum yield of BCzPCz was 0.516 in ethanol. Experimental results showed its potential use as a fluorescence probe and as two-photon absorption material. PMID:22753316

Gao, Shouqin; Hao, Liling; Li, Junfen; Lin, Peihua; Li, Duxin; Shuang, Shaomin; Dong, Chuan



Photon antibunching in the fluorescence of a single dye molecule trapped in a solid  

Microsoft Academic Search

The correlation between fluorescence photons emitted by an optically pumped single molecule of pentacene in a p-terphenyl host has been investigated at short times. The correlation function shows photon antibunching, a unique feature of a nonclassical radiation field, which decreases if two molecules rather than one are pumped at the same time. The peculiarities of the correlation function for a

Th. Basché; W. E. Moerner; M. Orrit; H. Talon



A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds  

Microsoft Academic Search

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive\\u000a staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial\\u000a colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of\\u000a only 0.5 ?g\\/ml, and growth

Patricia Spiekermann; Bernd H. A. Rehm; Rainer Kalscheuer; Dirk Baumeister; Alexander Steinbüchel



Photon antibunching in the fluorescence of a single dye molecule trapped in a solid  

NASA Astrophysics Data System (ADS)

The correlation between fluorescence photons emitted by an optically pumped single molecule of pentacene in a p-terphenyl host has been investigated at short times. The correlation function shows photon antibunching, a unique feature of a nonclassical radiation field, which decreases if two molecules rather than one are pumped at the same time. The peculiarities of the correlation function for a three-level molecule are discussed and the theoretical description outlined.

Basché, Th.; Moerner, W. E.; Orrit, M.; Talon, H.



Photon antibunching in the fluorescence of a single dye molecule trapped in a solid  

NASA Astrophysics Data System (ADS)

The correlation between fluorescence photons emitted by an optically pumped single molecule of pentacene in a p-terphenyl host has been investigated at short times. The correlation function shows photon antibunching, a unique feature of nonclassical radiation field, which decreases if two molecules rather than one are pumped at the same time. The peculiarities of the correlation function for a three-level molecule are discussed and the theoretical description outlined.

Basche, Th.; Moerner, W. E.



Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.  


Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results in destabilization of the latter. The spectral properties of fluorescent intercalating and non-intercalating ligands [acridine orange, pyronin Y(G), DAPI, Hoechst 33258, and Hoechst 33342]-NA complexes were studied in both the relaxed and condensed form. The changes in absorption, excitation, and fluorescence emission spectra and fluorescence yield that followed the condensation were examined. Although some of these effects can be explained by changes in solvation of the fluorophore and its interaction with NA bases and the solvent, the overall effect of condensation on spectral properties of the complex is unpredictable. In particular, no correlation was found between these effects and the ds DNA binding mode of these ligands. Nevertheless, the spectral data associated with polymer condensation can yield information about the composition and structure of NA and can explain some nonspecific interactions of these probes. PMID:1696951

Kapuscinski, J



Spectroscopic and fluorescence studies on Mn(II), Co(II), Ni(II) and Cu(II) complexes with NO donor fluorescence dyes.  


The reactions of the two common dyes [2TMPACT and 4PENI] with Mn(II), Co(II), Ni(II) and Cu(II) ions were done. All the isolated complexes have been characterized by physicochemical and spectroscopic techniques. The IR data reflect the bidentate mode of 2TMPACT towards the mononuclear complex [Mn(II)] even its tetradentate in binuclear complexes [Co(II) and Cu(II)]. However, the bidentate mode is the only behavior of 4PENI ligand towards each metal ion in its mononuclear complexes. The UV-vis spectral analysis beside the magnetic moment measurements are proposed different geometries concerning each metal ions with the two ligands under investigation, as the Mn(II)-2TMPACT complex is an octahedral but Mn(II)-4PENI is a tetrahedral geometry. All the synthesized compounds are thermogravimetrically investigated. The proposed thermal decomposition was discussed for each compound with each step as well as, the kinetic parameters were calculated for all preferrible decomposition steps. The mass spectroscopy tool was used to emphasis on the suitable molecular formula proposed and the fragmentation patterns were displayed. The fluorescence properties of the synthesized ligands and their complexes were studied in DMSO at room temperature. PMID:21763185

Refat, Moamen S; el-Metwaly, Nashwa M



Fluorescence Ratiometric Properties Induced by Nanoparticle Plasmonics and Nanoscale Dye Dynamics  

PubMed Central

Nanoscale transport of merocyanine 540 within/near the plasmon field of gold nanoparticles was recognized as an effective inducer of single-excitation dual-emission ratiometric properties. With a high concentration of the signal transducer (ammonium), a 700% increase in fluorescence was observed at the new red-shifted emission maximum, compared to a nanoparticle free sensor membrane. A previously nonrecognized isosbestic point is demonstrated at 581.4 ± 0.1?nm. The mechanism can be utilized for enhanced and simplified ratiometric optical chemical sensors and potentially for thin film engineering to make solar cells more effective and stable by a broader and more regulated absorption.



Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications  

Microsoft Academic Search

\\u000a \\u000a Abstract  Conjugated polyelectrolytes (CPEs), with highly delocalized electronic backbones and charged ionic side chains, are naturally\\u000a robust light-harvesting antenna for fluorescence resonance energy transfer (FRET) applications. This chapter describes FRET-based\\u000a biosensors using CPEs as energy donors from the viewpoint of sensing mechanism, donor–acceptor selection and detection targets.\\u000a Important information on how to design CPE structures and assay schemes is elucidated, and

Kan-Yi Pu; Bin Liu


Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy  

Microsoft Academic Search

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water- soluble cationic dye, diI-C3-(3), fluoresced only as the




Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification  

NASA Astrophysics Data System (ADS)

A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

Zhu, Xiao; Xing, Da



A simple system for the identification of fluorescent dyes capable of reporting differences in secondary structure and hydrophobicity among amyloidogenic protein oligomers  

NASA Astrophysics Data System (ADS)

Thioflavin T and Congo Red are fluorescent dyes that are commonly used to identify the presence of amyloid structures, ordered protein aggregates. Despite the ubiquity of their use, little is known about their mechanism of interaction with amyloid fibrils, or whether other dyes, whose photophysics indicate that they may be more responsive to differences in macromolecular secondary structure and hydrophobicity, would be better suited to the identification of pathologically relevant oligomeric species in amyloid diseases. In order to systematically address this question, we have designed a strategy that discretely introduces differences in secondary structure and hydrophobicity amidst otherwise identical polyamino acids. This strategy will enable us to quantify and compare the affinities of Thioflavin T, Congo Red, and other, incompletely explored, fluorescent dyes for different secondary structural elements and hydrophobic motifs. With this information, we will identify dyes that give the most robust and quantitative information about structural differences among the complex population of oligomeric species present along an aggregation pathway between soluble monomers and amyloid fibrils, and correlate the resulting structural information with differential oligomeric toxicity.

Yates, Emma



Treatment Using a Long Pulsed Nd:Yag Laser with a Pulsed Dye Laser for Four Cases of Blebbed Port Wine Stains  

PubMed Central

Port wine stains (PWS) are congenital capillary malformations consisting of ectasia of capillaries and venules. At birth,lesions are flat and relatively uniform in color, but evolve with age to become raised, thickened, irregularly surfaced, and deeply colored. Therefore, it is considered optimal to begin treatment of patients at an early age. Conventional treatment modalities, such as electrocautery or excision, require considerable effort and may be cosmetically unsatisfactory. We have performed treatment of blebbed PWS of four patients using a 1,064 nm long pulsed Nd:YAG laser with a contact cooling device. According to their size, most blebs required three or fewer treatment sessions at 8-week intervals. Treatments were well tolerated by all subjects and patients showed moderate to good improvement of blebs. A 1,064 nm long pulsed Nd:YAG laser with contact cooling may be considered as a promising therapeutic option for treatment of blebbed PWS.

Chang, Hee Sun; Kim, Young-Gull



Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions  

Microsoft Academic Search

BACKGROUND: Array-based comparative genomic hybridization (CGH) and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5) are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining

Mubasher Dar; Theresa Giesler; Rob Richardson; Christine Cai; Mike Cooper; Shahin Lavasani; Peter Kille; Thierry Voet; Joris Vermeesch



Lewis Acid-Assisted Isotopic 18F-19F Exchange in BODIPY Dyes: Facile Generation of Positron Emission Tomography/Fluorescence Dual Modality Agents for Tumor Imaging  

PubMed Central

Positron emission tomography (PET) is a powerful technique for imaging biological pathways in vivo, particularly those that are key targets in disease processes. In contrast, fluorescence imaging has demonstrated to be a superior method for image-guided surgery, such as tumor removal. Although the integration of PET and optical imaging could provide an attractive strategy for patient management, there is a significant shortage of established platforms/methods for PET/optical probe construction. In this study, various reaction conditions were explored to develop a simple and fast method allowing for the introduction of [18F]-fluoride into BODIPY dyes. Through a systematic optimization of the reaction conditions, we found that BODIPY dyes, including commercial amine-reactive BODIPY succinimidyl esters, may be converted into their radioactive analogues in the matter of minutes via a 18F-19F isotopic exchange reaction promoted by a Lewis acid such as SnCl4. An integrin-targeting RGD peptide was also conjugated with [18F]BODIPY® R6G , derived from the commercially available BODIPY® R6G fluorescent tag, to provide a [18F]-RGD conjugate in 82% yield. In vivo evaluation of this imaging probe showed a discernible tumor uptake in the U87MG xenograft model. The dual modality imaging properties of the probe was confirmed by ex vivo fluorescence and microPET imaging experiments. In summary, in the matter of minutes, BODIPY dyes were converted into their “hot” radioactive analogues via a 18F-19F isotopic exchange reaction promoted by a Lewis acid. This approach, which can be applied to commercial BODIPY dyes, provides easy access to positron emission tomography/fluorescence dual modality imaging agents.

Liu, Shuanglong; Lin, Tzu-Pin; Li, Dan; Leamer, Lauren; Shan, Hong; Li, Zibo; Gabbai, Francois P.; Conti, Peter S.



Membrane potentials associated with Ca-induced K conductance in human red blood cells: Studies with a fluorescent oxonol dye, WW 781  

Microsoft Academic Search

Summary A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% ?F\\/mV, a detection limit of 10 mV, a response time of less

Jeffrey C. Freedman; Terri S. Novak



Nanoscopic heterogeneities in adsorption and electron transfer processes of perylene diimide dye on TiO 2 nanoparticles studied by single-molecule fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The interfacial electron transfer processes between a fluorescent water-soluble perylene diimide dye (WS-PDI) and TiO 2 nanoparticles were investigated using single-molecule fluorescence spectroscopy. Based on the single-molecule fluorescence spectral measurements, it was suggested that the local environment and/or the structural conformation of single WS-PDI molecules play important roles in the efficiency of the electron injection from WS-PDI in the singlet excited state to TiO 2. The observed single-molecule fluorescence blinking behavior was interpreted in terms of the interfacial electron-transfer dynamics between the single WS-PDI molecules and TiO 2 nanoparticles.

Tachikawa, Takashi; Cui, Shi-Cong; Tojo, Sachiko; Fujitsuka, Mamoru; Majima, Tetsuro



Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia  

NASA Astrophysics Data System (ADS)

A modular video endoscope is developed and tested to allow imaging in different modalities. This system incorporates white light imaging (WLI), cross-polarized imaging (CPI), and vital-dye fluorescence imaging (VFI), using interchangeable filter modules. CPI and VFI are novel endoscopic modalities that probe mucosal features associated with Barrett's neoplasia. CPI enhances vasculature, while VFI enhances glandular architecture. In this pilot study, we demonstrate the integration of these modalities by imaging areas of Barrett's metaplasia and neoplasia in an esophagectomy specimen. We verify that those key image features are also observed during an in vivo surveillance procedure. CPI images demonstrate improved visualization of branching blood vessels associated with neoplasia. VFI images show glandular architecture with increased glandular effacement associated with neoplasia. Results suggests that important pathologic features seen in CPI and VFI are not visible during standard endoscopic white light imaging, and thus the modalities may be useful in future in vivo studies for discriminating neoplasia from Barrett's metaplasia. We further demonstrate that the integrated WLI/CPI/VFI endoscope is compatible with complementary high-resolution endomicroscopy techniques such as the high-resolution microendoscope, potentially enabling two-step ("red-flag" widefield plus confirmatory high-resolution imaging) protocols to be enhanced.

Thekkek, Nadhi; Pierce, Mark C.; Lee, Michelle H.; Polydorides, Alexandros D.; Flores, Raja M.; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca R.



Victoria blue B — a nuclear stain for cytology  

Microsoft Academic Search

The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF4. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye

E. Schulte; D. Wittekind; V. Kretschmer



Flow Cytometric Monitoring of Antibiotic-Induced Injury in Escherichia coli Using Cell-Impermeant Fluorescent Probes  

Microsoft Academic Search

Three fluorescent nucleic acid binding dyes—propidium iodide, TO-PRO-1, and SYTOX green—were eval- uated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells of Escherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining- compromised, as opposed




FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.  

PubMed Central

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude. Images Fig. 3 Fig. 4

Henkel, A W; Lubke, J; Betz, W J



Putting vital stains in context.  


While vital staining remains a cornerstone in the diagnosis of ocular disease and contact lens complications, there are many misconceptions regarding the properties of commonly used dyes by eye-care practitioners and what is and what is not corneal staining after instillation of sodium fluorescein. Similarly, the proper use and diagnostic utility of rose Bengal and lissamine green B, the other two ophthalmic dyes commonly used for assessing ocular complications, have similarly remained unclear. Due to the limitations of vital stains for definitive diagnosis, concomitant signs and symptoms in addition to a complete patient history are required. Over the past decade, there have been many reports of a type of corneal staining--often referred to as solution-induced corneal staining (SICS)--that is observed with the use of multipurpose solutions in combination with soft lenses, more specifically silicone hydrogel lenses. Some authors believe that SICS is a sign of lens/solution incompatibility; however, new research shows that SICS may be neither a measure of lens/solution biocompatibility nor 'true' corneal staining, as that observed in pathological situations. A large component of SICS may be a benign phenomenon, known as preservative-associated transient hyperfluorescence (PATH). There is a lack of correlated signs and/or symptoms with SICS/PATH. Several properties of SICS/PATH, such as appearance and duration, differentiate it from pathological corneal staining. This paper reviews the properties of vital stains, their use and limitations in assessment of the ocular surface, the aetiology of corneal staining, characteristics of SICS/PATH that differentiate it from pathological corneal staining and what the SICS/PATH phenomenon means for contact lens-wearing patients. PMID:23051047

Efron, Nathan



Long-term monitoring of live cell proliferation in presence of PVP-Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE  

PubMed Central

Summary During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone-Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low-power pulsed blue light-emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual-marked cells, during 5-h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.

Penjweini, Rozhin; Loew, Hans G.; Hamblin, Michael R.; Kratky, Karl W.



Purified azure B as a reticulocyte stain  

Microsoft Academic Search

A comparison has been made between reticulocyte preparations stained with purified azure B and with several commerically available batches of brilliant cresyl blue and new methylene blue. Marked variations were observed in the composition and staining performances of the various batches of the two commerically available dyes. Although there were no significant differences in reticulocyte counts obtained with these two

P N Marshall; S A Bentley; S M Lewis



Uniform mesoporous dye-doped silica nanoparticles decorated with multiple magnetite nanocrystals for simultaneous enhanced magnetic resonance imaging, fluorescence imaging, and drug delivery.  


Highly versatile nanocomposite nanoparticles were synthesized by decorating the surface of mesoporous dye-doped silica nanoparticles with multiple magnetite nanocrystals. The superparamagnetic property of the magnetite nanocrystals enabled the nanoparticles to be used as a contrast agent in magnetic resonance (MR) imaging, and the dye molecule in the silica framework imparted optical imaging modality. Integrating a multitude of magnetite nanocrystals on the silica surface resulted in remarkable enhancement of MR signal due to the synergistic magnetism. An anticancer drug, doxorubicin (DOX), could be loaded in the pores and induced efficient cell death. In vivo passive targeting and accumulation of the nanoparticles at the tumor sites was confirmed by both T2 MR and fluorescence imaging. Furthermore, apoptotic morphology was clearly detected in tumor tissues of mice treated with DOX loaded nanocomposite nanoparticles, demonstrating that DOX was successfully delivered to the tumor sites and its anticancer activity was retained. PMID:20017538

Lee, Ji Eun; Lee, Nohyun; Kim, Hyoungsu; Kim, Jaeyun; Choi, Seung Hong; Kim, Jeong Hyun; Kim, Taeho; Song, In Chan; Park, Seung Pyo; Moon, Woo Kyung; Hyeon, Taeghwan



Reverse fluorescent chromosome banding with chromomycin and DAPI  

Microsoft Academic Search

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in

Dieter Schweizer



Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock.  


The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (?5160 nm) and red (?581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p?

Schreiner, Leonhard; Huber-Lang, Markus; Weiss, Manfred E; Hohmann, Harald; Schmolz, Manfred; Schneider, E Marion



Visualization of DNA-containing structures in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR green I dye  

Microsoft Academic Search

We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the\\u000a fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic\\u000a ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time\\u000a PCR), which enabled

M. Vítová; J. Hendrychová; V. Cepák; V. Zachleder



Influence of dehydrated nanotubed titanic acid on charge transport and luminescent properties of polymer light-emitting diodes with fluorescent dye  

NASA Astrophysics Data System (ADS)

In this paper, we discuss the influence of dehydrated nanotubed titanic acid (DNTA) on charge transport and luminescent properties of polymer light-emitting diodes (PLEDs) doped with fluorescent dye. Photoluminescence results confirm the efficient energy transfer from PVK to 4-(dicyanom-ethylene)-2-t-butyl-6-(1,1,7,7-tetramethyljulolidyl-9-enyl)-4H-pyran (DCJTB) and tris-(8-hydroxtquinoline) aluminum (Alq3) in a DNTA-doped device. The device showed lower turn-on voltages and higher charge current by doping with DNTA, which also caused a shift in the exciton’s recombination region.

Qian, Lei; Bera, Debasis; Jin, Zhen-Sheng; Du, Zu-Liang; Xu, Zheng; Teng, Feng; Liu, Wei



Dual Staining of Natural Bacterioplankton with 4',6-Diamidino-2Phenylindole and Fluorescent Oligonucleotide Probes Targeting Kingdom-Level 16S rRNA Sequencest.  

National Technical Information Service (NTIS)

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4', 6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rho...

R. E. Hicks R. I. Amann D. A. Stahl



Fibre-Optic and Nanoparticle-Based Fluorescence Sensing Using Indicator Dyes: Pitfalls, Self Referencing, Application, and Future Trends  

Microsoft Academic Search

Herein is described how reliable measurements of ionic analytes and biomolecules can be performed\\u000a using fibre-optic sensors or sensor nanoparticles. Referenced signal readings are achieved by using ratiometric\\u000a dyes or by combination of indicator dyes with inert luminophores. Thus, it becomes possible to minimize\\u000a effects from light source instability, leaching, photobleaching or sample colouration. Similarly, a combination\\u000a of reference and indicator

Gerhard J. Mohr


Application of non-specific fluorescent dyes for monitoring enantio-selective ligand binding to molecularly imprinted polymers  

Microsoft Academic Search

The displacement of non-specific dyes from molecularly imprinted polymer (MIP) chromatographic stationary phases has been\\u000a used for the detection and quantification of ligand-polymer binding events. A blank polymer and an L-phenylalaninamide-imprinted\\u000a polymer were prepared using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as a crosslinker.\\u000a The MIP is first loaded with dye, and a solution of the

Sergey A. Piletsky; Ewald Terpetschnig; Håkan S. Andersson; Ian A. Nicholls; Otto S. Wolfbeis



A novel, simple and efficient dye laser with low amplified spontaneous emission background for analytical fluorescence and ionization spectroscopy  

SciTech Connect

A new, simple, compact and efficient, grazing- incidence type of dye laser is suggested which has a low level of Amplified Spontaneous Emission. By using a Coumarin dye (LD 5000) pumped with a 20 mJ XeCl excimer laser, and a diffraction grating with 3000 grooves/mm, an efficiency of 11%, a spectral bandwidth of 0.6 cm{sup -1} and a tuning range from 458 to 517 nm have been obtained.

Matveev, Oleg I.; Omenetto, Nicolo' [EC, Joint Research Centre, Environment Institute, 21020 Ispra, Varese (Italy)



New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes  

SciTech Connect

Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.

Del Castillo, P.; Llorente, A.R.; Gomez, A.; Gosalvez, J.; Goyanes, V.J.; Stockert, J.C. (Autonomous Univ., Madrid (Spain))




PubMed Central

Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO2++/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ?. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.

Zobel, C. Richard; Beer, Michael




PubMed Central

A new serologic test for antibodies to Toxoplasma is described, which is based upon inhibition of specific staining with fluorescent antibody. In performing the test, a mixture of the test serum and known fluorescein-labelled antiserum is added to a dried smear of toxoplasms for 1 hour at 37°C. The smear is then rinsed and examined with a fluorescence microscope. Reduction in the brightness of fluorescence, as compared to that of a negative control slide, indicates the presence of antibody in the test serum. A comparison of the results of this test with those of the methylene blue dye test showed a strong parallelism between the two sets of results. On the other hand, the complement-fixation test for toxoplasmosis did not yield nearly as many positives as the inhibition test. The specificity of the new test was studied by comparing it with dye test results and clinical histories in human patients, and by testing a group of animals immunized with a variety of non-Toxoplasma antigens. No evidence of cross-reactions was obtained in the latter series. Some advantages and disadvantages of the inhibition test are discussed.

Goldman, Morris



Bayesian-inference-based fluorescence correlation spectroscopy and single-molecule burst analysis reveal the influence of dye selection on DNA hairpin dynamics.  


Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures. PMID:22279001

Kügel, Wolfgang; Muschielok, Adam; Michaelis, Jens



Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen.  


EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection. PMID:16833086

Sang, Fuming; Ren, Jicun



Monitoring FET flow control and wall adsorption of charged fluorescent dye molecules in nanochannels integrated into a multiple internal reflection infrared waveguide.  


Using Si as the substrate, we have fabricated multiple internal reflection infrared waveguides embedded with a parallel array of nanofluidic channels. The channel width is maintained substantially below the mid-infrared wavelength to minimize infrared scattering from the channel structure and to ensure total internal reflection at the channel bottom. A Pyrex slide is anodically bonded to the top of the waveguide to seal the nanochannels, while simultaneously enabling optical access in the visible range from the top. The Si channel bottom and sidewalls are thermally oxidized to provide an electrically insulating barrier, and the Si substrate surrounding the insulating SiO(2) layer is selectively doped to function as a gate. For fluidic field effect transistor (FET) control, a DC potential is applied to the gate to manipulate the surface charge on SiO(2) channel bottom and sidewalls and therefore their zeta-potential. Depending on the polarity and magnitude, the gate potential can accelerate, decelerate, or reverse the flow. Here, we demonstrate that this nanofluidic infrared waveguide can be used to monitor the FET flow control of charged, fluorescent dye molecules during electroosmosis by multiple internal reflection Fourier transform infrared spectroscopy. Laser scanning confocal fluorescence microscopy is simultaneously used to provide a comparison and verification of the IR analysis. Using the infrared technique, we probe the vibrational modes of dye molecules, as well as those of the solvent. The observed infrared absorbance accounts for the amount of dye molecules advancing or retracting in the nanochannels, as well as adsorbing to and desorbing from the channel bottom and sidewalls. PMID:18231663

Oh, Youn-Jin; Gamble, Thomas C; Leonhardt, Darin; Chung, Chan-Hwa; Brueck, Steven R J; Ivory, Cornelius F; Lopez, Gabriel P; Petsev, Dimiter N; Han, Sang M



Lighting-up of the dye malachite green with mercury(II)-DNA and its application for fluorescence turn-off detection of cysteine and glutathione.  


This work describes a novel strategy for the highly sensitive and selective detection of cysteine (Cys) and glutathione (GSH) based on the Hg(2+)-AGRO100-malachite green (MG) complex system. The dye MG, which has a very low quantum yield in aqueous solution by itself, can bind with the thymine-rich DNA AGRO100 in the presence of Hg(2+) ions to generate a striking fluorescence intensity enhancement of 1000-fold. As sulfur-containing amino acids, Cys and GSH effectively sequester Hg(2+) ions from the Hg(2+)-AGRO100-MG complex structure to switch the 'lit-up' chemosensor to the 'off' state (about a 50-fold fluorescence intensity decrease), thus providing a facile, but effective, method to probe for Cys/GSH. The fluorescence titration, UV absorption, CD, and Raman spectra provide some insight into the structural and chemical basis for the enhancement effect. The formation of the Hg(2+)-AGRO100-MG complex significantly affects the electronic structure and conformation of the MG molecule by leading to an extended ? system, which is the likely origin of the observed striking fluorescence intensity enhancement. Notably, the proposed sensing platform exhibits exquisite selectivity and sensitivity toward Cys/GSH with limits of detection of 5 nM for Cys and 10 nM for GSH, respectively. Furthermore, the straightforward assay design avoids labeling of the probe, uses only commercially available materials, and still displays comparable sensitivity and excellent selectivity. PMID:22944915

Jia, Xiaofang; Li, Jing; Wang, Erkang



Mass spectral compatibility of four proteomics stains.  


With the recent introduction of new fluorescence stains to the proteomics market, there is now more choice available. SYPRO Ruby, LavaPurple, Flamingo, and Krypton total protein stains were compared for ease of use, image quality, and compatibility with protein identification by peptide mass fingerprinting (PMF) (MALDI-TOF). All four stains produced good images but with slightly different staining patterns. SYPRO was found to inhibit identification of cysteine and tryptophan containing peptides, which reduced protein identification. PMID:17929854

Ball, Malcolm S; Karuso, Peter



Genetic targeting of individual cells with a voltage-sensitive dye through enzymatic activation of membrane binding.  


Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of a thousand, as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase-transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining were elucidated by a simple kinetic model to guide further developments of the method. PMID:16440375

Hinner, Marlon J; Hübener, Gerd; Fromherz, Peter



Effectiveness of Dye Setting Treatments on Cotton Fabrics Dyed with Direct, Reactive, and Vat Dyes  

Microsoft Academic Search

Eight treatments were evaluated for their effectiveness in the setting of dyes in new cotton fabrics. Dyes representing three application classes (direct, reactive, and vat) widely used on cotton fabrics were selected. Because of the perceived problems of red dyes bleeding during laundering, five of the eight dyes selected for evaluation were red. Color and staining evaluations were made after

Patricia Cox Crews



Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.  


Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. PMID:9645228

Tatischeff, I; Bomsel, M; de Paillerets, C; Durand, H; Geny, B; Segretain, D; Turpin, E; Alfsen, A



Enhancing the color gamut of white displays using novel deep-blue organic fluorescent dyes to form color-changed thin films with improved efficiency  

NASA Astrophysics Data System (ADS)

This study used novel fluorescence based deep-blue-emitting molecules, namely BPVPDA, an organic fluorescence color thin film using BPVPDA exhibit deep blue fluorine with CIE coordinates of (0.13,0.16). The developed original Organic RGB color thin film technology enables the optimization of the distinctive features of an organic light emitting diode (OLED) and (TFT) LCD display. The color filter structure maintains the same high resolution to obtain a higher level of brightness, in comparison with conventional organic RGB color thin film. The image-processing engine is designed to achieve a sharp text image for a thin-film-transistor (TFT) LCD with organic color thin films. The organic color thin films structure uses organic dye dopent in limpid photo resist. With this technology , the following characteristics can be obtained: (1) high color reproduction of gamut ratio, and (2) improved luminous efficiency with organic color fluorescence thin film. This performance is among the best results ever reported for a color-filter used on TFT-LCD and OLED.

Liu, Wei-ting; Huang, Wen-Yao



Enhancing the color gamut of white displays using novel deep-blue organic fluorescent dyes to form color-changed thin films with improved efficiency  

NASA Astrophysics Data System (ADS)

This study used the novel fluorescence based deep-blue-emitting molecule BPVPDA in an organic fluorescent color thin film to exhibit deep blue color with CIE coordinates of (0.13, 0.16). The developed original organic RGB color thin film technology enables the optimization of the distinctive features of an organic light emitting diode (OLED) and thin-film-transistor (TFT) LCD display. The color filter structure maintains the same high resolution to obtain a higher level of brightness in comparison with conventional organic RGB color thin film. The image-processing engine is designed to achieve a sharp text image for a TFT LCD with organic color thin films. The organic color thin films structure uses an organic dye dopant in a limpid photoresist. With this technology, the following characteristics can be obtained: 1. high color reproduction of gamut ratio, and 2. improved luminous efficiency with organic color fluorescent thin film. This performance is among the best results ever reported for a color-filter used on TFT-LCD or OLED.

Liu, Wei-Ting; Huang, Wen-Yao



A fluorescent codetection system for immunoblotting and proteomics through ECL-Plex and CyDye labeling.  


The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract. PMID:19378088

McManus, Ciara A; Donoghue, Pamela M; Dunn, Michael J



Quantum dot-based, quantitative, and multiplexed assay for tissue staining.  


The excellent optical properties of quantum dots (QDs), such as high brightness, high photostability, continuous absorption, and narrow emission bandwidth, make them ideal as optical labels to develop QD-based immunohistofluorescence (IHF) imaging for multiplexing cancer biomarker detection on formalin-fixed and paraffin-embedded (FFPE) tissues. IHF is very important for the prediction of a patient's response to cancer chemotherapy or radiotherapy. QD-based IHF faces several challenges that differ from those encountered by organic dye based IHF for clinical assays. The current work addresses some of these issues. Initially, the chemical stability of QDs and organic dyes were compared. The results showed that QDs were stable for at least 5 months on FFPE tissue, whereas organic dyes were photobleached shortly after exposure to light. Various staining methods were also studied. QD fluorescence intensity on the tissue stained with primary antibody (Ab, p16, survivin, EF1?) conjugated QDs from our company was comparable to the signal from a commercially available method in which the tissue was stained with a primary p16 Ab and a QD-labeled secondary goat anti mouse Ab respectively. Finally, the effect of the amount of Ab conjugated to QD on tissue imaging was also studied. There was no significant increase in the QD fluorescence signal on tissues when the Ab:QD ratio increased from 5 to 30. In addition, protein G was tested as an adaptor protein to link Ab to QDs for IHF staining. However, the proper blocking of the protein G on QDs was necessary to reduce crosstalk. The biomarker quantification in QD-based IHF was validated by conventional Western blot and immunohistochemistry. The results contained herein demonstrate a promising application of QDs in multiplex detection and quantification of biomarkers. PMID:23551017

Xu, Hong; Xu, Jing; Wang, Xu; Wu, Daqing; Chen, Zhuo Georgia; Wang, Andrew Y



Triggered dye release via photodissociation of nitric oxide from designed ruthenium nitrosyls: turn-ON fluorescence signaling of nitric oxide delivery.  


Two new fluorescein-tethered nitrosyls derived from designed tetradentate ligands with carboxamido-N donors have been synthesized and characterized by spectroscopic techniques. These two diamagnetic {Ru-NO}(6) nitrosyls, namely, [(Me(2)bpb)Ru(NO)(FlEt)] (1-FlEt, Me(2)bpb = 1,2-bis(pyridine-2-carboxamido)5-dimethylbenzene, FlEt = fluorescein ethyl ester) and [((OMe)(2)IQ1)Ru(NO)(FlEt)] (2-FlEt, (OMe)(2)IQ1 = 1,2-bis(isoquinoline-1-carboxamido)-4,5-dimethoxybenzene), display NO stretching frequencies (?(NO)) at 1846 and 1832 cm(-1) in addition to their FlEt carbonyl stretching frequencies (?(CO)) at 1715 and 1712 cm(-1), respectively. Coordination of the dye ligand enhances the absorptivity and NO photolability of these two nitrosyls in the visible region (450-600 nm) of light. Exposure to visible light promotes rapid loss of NO from both {Ru-NO}(6) nitrosyls to generate Ru(III) photoproducts in dry aprotic solvents, such as MeCN and DMF. The FlEt(-) moiety remains bound to the paramagnetic Ru(III) center in such cases, and hence, the photoproducts exhibit very weak fluorescence from the dye unit. In the presence of water, the Ru(III) photoproducts undergo further aquation and loss of the FlEt(-) moiety via protonation. These steps lead to turn-ON fluorescence (from the free FlEt unit) and provide a visual signal of the NO photorelease from 1-FlEt and 2-FlEt in aqueous media. PMID:21815610

Fry, Nicole L; Wei, Julia; Mascharak, Pradip K



Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology  

PubMed Central

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays.

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.



A 'Magnetic' Gram Stain for Bacterial Detection  

PubMed Central

Magnetic stain. Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet (CV), a triarylmethane dye. Here we show, that bioorthogonal modification of crystal violet with transcyclooctene (TCO) can be used to render Gram-positive bacteria magnetic with magneto-nanoparticles-Tetrazine (MNP-Tz). This allows for class specific automated magnetic detection, magnetic separation or other magnetic manipulations.

Budin, Ghyslain; Chung, Hyun Jung; Lee, Hakho



Multiple Labeling of Cellular Constituents by Combining Surface Reflection Interference and Fluorescence Microscopy  

Microsoft Academic Search

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along

Vitauts I. Kalnins



Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches[S  

PubMed Central

The proportions of body fat and fat-free mass are determining factors of adiposity-associated diseases. Work in Caenorhabditis elegans has revealed evolutionarily conserved pathways of fat metabolism. Nevertheless, analysis of body composition and fat distribution in the nematodes has only been partially unraveled because of methodological difficulties. We characterized metabolic C. elegans mutants by using novel and feasible BODIPY 493/503-based fat staining and flow cytometry approaches. Fixative as well as vital BODIPY staining procedures visualize major fat stores, preserve native lipid droplet morphology, and allow quantification of fat content per body volume of individual worms. Colocalization studies using coherent anti-Stokes Raman scattering microscopy, Raman microspectroscopy, and imaging of lysosome-related organelles as well as biochemical measurement confirm our approaches. We found that the fat-to-volume ratio of dietary restriction, TGF-?, and germline mutants are specific for each strain. In contrast, the proportion of fat-free mass is constant between the mutants, although their volumes differ by a factor of 3. Our approaches enable sensitive, accurate, and high-throughput assessment of adiposity in large C. elegans populations at a single-worm level.

Klapper, Maja; Ehmke, Madeleine; Palgunow, Daniela; Bohme, Mike; Matthaus, Christian; Bergner, Gero; Dietzek, Benjamin; Popp, Jurgen; Doring, Frank



Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches.  


The proportions of body fat and fat-free mass are determining factors of adiposity-associated diseases. Work in Caenorhabditis elegans has revealed evolutionarily conserved pathways of fat metabolism. Nevertheless, analysis of body composition and fat distribution in the nematodes has only been partially unraveled because of methodological difficulties. We characterized metabolic C. elegans mutants by using novel and feasible BODIPY 493/503-based fat staining and flow cytometry approaches. Fixative as well as vital BODIPY staining procedures visualize major fat stores, preserve native lipid droplet morphology, and allow quantification of fat content per body volume of individual worms. Colocalization studies using coherent anti-Stokes Raman scattering microscopy, Raman microspectroscopy, and imaging of lysosome-related organelles as well as biochemical measurement confirm our approaches. We found that the fat-to-volume ratio of dietary restriction, TGF-?, and germline mutants are specific for each strain. In contrast, the proportion of fat-free mass is constant between the mutants, although their volumes differ by a factor of 3. Our approaches enable sensitive, accurate, and high-throughput assessment of adiposity in large C. elegans populations at a single-worm level. PMID:21421847

Klapper, Maja; Ehmke, Madeleine; Palgunow, Daniela; Böhme, Mike; Matthäus, Christian; Bergner, Gero; Dietzek, Benjamin; Popp, Jürgen; Döring, Frank



[Effect of deproteinization on the in situ chromatin staining with 7-aminoactinomycin D].  


The possibility of use of 7-amino-actinomycin D (7aAMD)--fluorescent analog of actinomycin D--as a specific dye for DNA staining in the suspended cells was studied by means of laser flow-cytometry. The optimal conditions for staining were obtained: 7aAMD concentration 10(-5) M, pH 7, staining time 20 min, 37 degrees C, ionic strength 0.15 M Na+. In this case the fluorescent signal is proportional to the DNA amount and coefficient of variation is about 0.03. The influence of the stepwise extraction of the proteins from chromatin also was studied. In the course of the salt deproteinization the fluorescence intensity gradually rose thus showing the increase of the binding sides-number. The deproteinization of cells nuclei by 0.1 HCl increased the number of binding sites 2.5 times more. It was shown that the incubation of cells with RNAse at elevated ionic strength (0.3-0.7 M NaCl) leads to an additional increase of the cell fluorescence and produces no effect at low and normal ionic strength. The deproteinizing effect of RNAse and its possible mechanism is discussed. PMID:2460738

Poletaev, A I; Stepanova, N G; Nikitin, S M


Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis  

PubMed Central

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

Bunthof, Christine J.; van Schalkwijk, Saskia; Meijer, Wilco; Abee, Tjakko; Hugenholtz, Jeroen



Hematoxylin-lac-curcuma polychrome stain for mucin.  


A polychrome method for detection of mucin substance in paraffin section is produced by sequential stepwise staining of hematoxylin, crude lac extract (Laccifer lacca), and crude curcuma extract (khamin shan-Curcuma longa). The name LacCur stain is proposed. After a tissue section is deparaffinized and rehydrated, it is stained with Weigert's hematoxylin for 7 minutes. After a quick wash, it is stained for at least 3 hours with lac dye mordanted with aluminum chloride. Washed again and premordanted with ferric chloride for 1 minute, in the last step, it is counterstained with curcuma dye for 5 minutes. With this staining method, the nuclei are stained black, mucin deep red, and organelles and ground substances brownish yellow. The method and outcome colors are comparable to the widely used Mayer's mucicarmine staining method. It costs less than the Mayer's mucicarmine staining method and the procedure is not complicated. PMID:7543924

Sriplung, H; Kietthubthew, S; Boonyaphiphat, P



Basic blue 93. An alternative to the traditional myeloperoxidase stain.  


This report describes the selective staining of lysosomes in cells of neutrophilic granulocytic origin by the azo textile dye basic blue 93. Used as a single agent aqueous stain after fixation, the dye stained lysosomes black. Stained in this way, lysosomes were sharply delimited and easily visualized. Large numbers of black-stained lysosomes occurred in immature granulocytes like promyelocytes and myelocytes. In more mature granulocytes like metamyelocytes, bands, and neutrophils, substantially fewer numbers of lysosomes were detected. In leukemic myeloblasts and promyelocytes stained with basic blue 93, variable numbers of black-colored lysosomes could be detected and usually paralleled the reactions for myeloperoxidase and Sudan black B in the same cells. As an alternative to the traditional stains for myeloperoxidase, basic blue 93 has several advantages, including simplicity of use, ability to stain aged specimens, and stability of the reaction product. PMID:2449070

Kass, L



Using Microcontact Printing as a Novel Method for Patterned Dyeing of Surface-adsorbed DNA  

NASA Astrophysics Data System (ADS)

We use microcontact printing (MCP)^1 to stain individual DNA molecules adsorbed and combed onto a polymer-coated silicon surface. Polydimethylsiloxane (PDMS) stamps with micron-sized features have been used to selectively stain lambda DNA molecules with SyBr Gold dye. DNA was deposited out of dilute solution onto polymethylmethacrylate (PMMA) layers, 70nm thick, spun-coated on Si wafers, producing linearly stretched and aligned molecules. The stamps were soaked in dye solutions for one minute, followed by wiping of excess solution with a swap. The stamp was pressed onto the surface, varying the pressure and time of application (typically 5-10 minutes) to control the staining. The DNA molecules were imaged with a fluorescence microscope equipped with a cooled CCD camera. Single molecules of DNA were successfully dyed and imaged with stamps having a grating pattern either parallel to or perpendicular to the DNA orientation. Supported by NSF-DMR MRSEC program.

Shea, Emily; Budassi, Julia; Zhu, Ke; Sokolov, Jonathan



Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.  


Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches. Several improvements of the original Coomassie protocol(1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution(2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol(3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol(4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins. Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group. PMID:19684561

Dyballa, Nadine; Metzger, Sabine



Laser dye toxicity, hazards and recommended controls  

NASA Astrophysics Data System (ADS)

Laser dyes are complex fluorescent organic compounds which, when in solution with organic solvents, form a lasing medium. The wavelength of a dye laser's output beam can vary with different dyes, concentrations, and solvents, giving it a tunable feature capable of emitting ultraviolet, visible, or infrared radiation. Toxicity information on the approximately 100 commercially available laser dyes is very scarce. Limited animal experimentation has been performed with only a few dyes. This paper summarizes what is known about laser dye toxicity, and offers recommendations for controlling dye hazards. The laser dyes investigated were categorized according to their central chemical structures. Prepared laser dye solutions usually contain very small quantities of dye--typical dye concentrations are 10(+2) to 10(+5) molar. For this reason, the solvent in which the dye is dissolved plays an important role when defining potential hazards. Practically all the solvents used are flammable and toxic by inhalation and skin absorption, and therefore must be controlled properly.

Mosovsky, J. A.


DAPI: a DNA-specific fluorescent probe.  


DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed. PMID:8580206

Kapuscinski, J



Staining efficiency of specific proteins depends on the staining method: wheat gluten proteins.  


To analyze gluten proteins involved in celiac disease (CD) by proteomic analysis, prolamins extracted from hexaploid wheat varieties were analyzed by SDS-PAGE and 2-DE. Differences between staining methods (CBB, silver nitrate, SYPRO Ruby, and CyDye) were analyzed in comparison to immunoblotting. Staining efficiency varied per protein across methods, and complete staining of all gluten proteins could not be achieved by one of these methods. Care should be taken in the selection of staining method especially if one wants to relate the results to data obtained by immunoblotting. PMID:18398878

van den Broeck, Hetty C; America, Antoine H P; Smulders, Marinus J M; Gilissen, Ludovicus J W J; van der Meer, Ingrid M



Sputum stain for mycobacteria  


Acid fast bacilli stain; AFB stain; Tuberculosis smear; TB smear ... Abnormal results show that the stain is positive for: Mycobacterium tuberculosis Mycobacterium avium-intracellular Other mycobacteria or acid-fast bacteria


High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes  

Microsoft Academic Search

We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15,

Yuri B. Yurov; Ilia V. Soloviev; Svetlana G. Vorsanova; Bertrand Marcais; Gerard Roize; Robert Lewis



Intercalating dye as an acceptor in quantum-dot-mediated FRET  

NASA Astrophysics Data System (ADS)

Fluorescence resonance energy transfer (FRET) is a popular tool to study intermolecular distances and characterize structural or conformational changes of biological macromolecules. We investigate a novel inorganic/organic FRET pair with quantum dots (QDs) as donors and DNA intercalating dyes, BOBO-3, as acceptors by using DNA as a linker. Typically, FRET efficiency increases with the number of stained DNA linked to a QD. However, with the use of intercalating dyes, we demonstrate that FRET efficiency at a fixed DNA:QD ratio can be further enhanced by increasing the number of dyes stained to a DNA strand through the use of an increased staining dye/bp ratio. We exploit this flexibility in the staining ratio to maintain a high FRET efficiency of >0.90 despite a sixfold decrease in DNA concentration. Having characterized this new QD-mediated FRET system, we test this system in a cellular environment using nanocomplexes generated by encapsulating DNA with commercial non-viral gene carriers. Using this novel FRET pair, we are able to monitor the configuration changes and fate of the DNA nanocomplexes during intracellular delivery, thereby providing an insight into the mechanistic study of gene delivery.

Lim, Teck Chuan; Bailey, Vasudev J.; Ho, Yi-Ping; Wang, Tza-Huei



Kinetic analysis of DAF-FM activation by NO: toward calibration of a NO-sensitive fluorescent dye.  


Nitric oxide (NO) research in biomedicine has been hampered by the absence of a method that will allow quantitative measurement of NO in biological tissues with high sensitivity and selectivity, and with adequate spatial and temporal resolution. 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) is a NO sensitive fluorescence probe that has been used widely for qualitative assessment of cellular NO production. However, calibration of the fluorescent signal and quantification of NO concentration in cells and tissues using fluorescent probes, have provided significant challenge. In this study we utilize a combination of mathematical modeling and experimentation to elucidate the kinetics of NO/DAF-FM reaction in solution. Modeling and experiments suggest that the slope of fluorescent intensity (FI) can be related to NO concentration according to the equation: ddtFI=2?k(1)NO(2)O(2)DAF-FMkNO+DAF-FM where ? is a proportionality coefficient that relates FI to unit concentration of activated DAF-FM, k(1) is the NO oxidation rate constant, and k was estimated to be 4.3±0.6. The FI slope exhibits saturation kinetics with DAF-FM concentration. Interestingly, the effective half-maximum constant (EC(50)) increases proportionally to NO concentration. This result is not in agreement with the proposition that N(2)O(3) is the NO oxidation byproduct that activates DAF-FM. Kinetic analysis suggests that the reactive intermediate should exhibit NO-dependent consumption and thus NO(2)() is a more likely candidate. The derived rate law can be used for the calibration of DAF-FM fluorescence and the quantification of NO concentration in biological tissues. PMID:23063986

Namin, Shabnam M; Nofallah, Sara; Joshi, Mahesh S; Kavallieratos, Konstantinos; Tsoukias, Nikolaos M



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

PubMed Central

Amoeboid microglial cells (AMC) in the corpus callosum were selectively labelled following a single intraperitoneal (i.p.) injection of the fluorescent dye, rhodamine isothiocyanate (RhIc) into postnatal rats. The frequency of RhIc-labelled cells varied with age, with the largest number occurring in 7-d-old animals. Thereafter, the labelled cells declined drastically in number and fluorescence and were barely detectable in 12-d-old injected rats. Labelled cells were absent in 13-d or older rats given an RhIc injection. When the injected RhIc was followed over a time course sequence, it was first detected in the cerebral blood vessels and their lining endothelia within 5 min after the injection. A variable number of AMC emitting a weaker fluorescence were closely adherent to the outer walls of the blood vessels. With time, the fluorescence in the AMC was progressively enhanced, but that in the blood vessels showed a concomitant reduction. In the rats that received an intravenous (i.v.) injection of RhIc, the labelling pattern of AMC, both in terms of its variation with age and in temporal sequence, paralleled that in rats given i.p. injections. In 12-d-old rats subjected to a stab wound coupled with an i.p. injection of RhIc, a considerable number of AMC not normally labelled at this age were activated. The cells exhibited an intense fluorescence and expressed MHC surface antigen immunoreactivity. It is concluded from this study that when injected i.p. or i.v., RhIc is readily circulated to the cerebral vessels, where it enters brain tissue by transendothelial transport.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figs 1,2 Figs 3,4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 Figs 17,18 Figs 19,20 Figs 21,22

Xu, J; Kaur, C; Ling, E A



Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis and B. cereus: a flow cytometric study.  


For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA. PMID:10192050

Walberg, M; Gaustad, P; Steen, H B



Non-equilibrium partitioning tracer transport in porous media: 2-D physical modelling and imaging using a partitioning fluorescent dye  

Microsoft Academic Search

This paper describes an investigation into non-equilibrium partitioning tracer transport and interaction with non-aqueous-phase liquid (NAPL) contaminated water-saturated porous media using a two-dimensional (2-D) physical modelling methodology. A fluorescent partitioning tracer is employed within a transparent porous model which when imaged by a CCD digital camera can provide full spatial tracer concentrations and tracer breakthrough curves. Quasi one-dimensional (1-D) benchmarking

Edward H. Jones; Colin C. Smith



TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections  

Microsoft Academic Search

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium\\/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems

Karin Bink; Axel Walch; Annette Feuchtinger; Heinrich Eisenmann; Peter Hutzler; Heinz Höfler; Martin Werner



A New Method in Staining Bacteria on Microscope Slides  

Microsoft Academic Search

A NEW method of providing dyes for some of the commoner staining procedures has been developed and tested. A small volume of the required dye solution is prepared according to the appropriate formula. The fluid is placed in a large dish, and rounds of No. 4 Whatman filter paper are soaked in it. After a few seconds the paper is

P. H. H. Gray



Coffee stain on textiles. Mechanisms of staining and stain removal  

Microsoft Academic Search

Coffee stains on textiles are mainly caused by the water-soluble and acidic colored substances in coffee. The acidic nature\\u000a of coffee stain has been shown by ultraviolet and visible spectroscopy of coffee as a function of pH; ion-pair formation with\\u000a a cationic surfactant and titration with Hyamine 1622 and a surfactant-specific electrode; and precipitation of the colored\\u000a components in coffee

Erik Kissa



FISH and Calcofluor staining techniques to detect in situ filamentous fungal biofilms in water.  


Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water. PMID:17196030

Gonçalves, Ana B; Santos, Isabel M; Paterson, R Russell M; Lima, Nelson



Photophysics of Cyanine Dyes Adsorbed onto Surfaces: Sub-Nanosecond Fluorescence Lifetime Measurements of 3,3'-Diethyloxadicarbocyanine Iodide and Photoisomer. TMR Large-Scale Facilities Access Programme.  

National Technical Information Service (NTIS)

3,3'-Diethyloxadicarbocyanine iodide (DODCI) is commonly used as saturable absorber for mode-locking dye lasers and as a laser dye for pulsed operation, tuneable around 660 nm. In recently published work (1) the authors described the behavior of this dye ...

L. F. Vieira Ferreira A. S. Oliveira K. Henbest D. R. Worrall F. Wilkinson C. N. Danson G. Booth



New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe  

Microsoft Academic Search

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single

Marek Romek; Barbara Gajda; Ewa Krzysztofowicz; Mariusz Kepczynski; Zdzis?aw Smorag



Results of a Preliminary Study of the Fluorescent Background Problem.  

National Technical Information Service (NTIS)

The fluorescent background problem arises from a loss in effectiveness of the fluorescent