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Sample records for fluorescent dye staining

  1. Chlorination effect on the fluorescence of nucleic acid staining dyes.

    PubMed

    Phe, M H; Dossot, M; Block, J C

    2004-10-01

    An alternative to culture methods for the control of drinking water disinfection would use fluorescent dyes that could evidence the nucleic acid damages provoked by sodium hypochlorite treatment. The two dyes selected in this study, SYBR Green II RNA gel stain and TOTO-1 iodide, efficiently stain nucleic acids (DNA and RNA) and quite poorly the other biomolecules considered (Bovine serum albumin, palmitic acid and dextrane). After treatment of nucleic acid solutions with increasing amounts of sodium hypochlorite, a decrease of fluorescence intensity is observed for both DNA and RNA stained with either SYBR-II or TOTO-1. However, the two fluorochromes do not lead to the same results, which shows that the two dyes are not bound to nucleic acids in the same way. Contrary to TOTO-1, SYBR-II reveals to be sufficiently sensitive to indicate both DNA or RNA damages as soon as the latter are in contact with hypochlorite even at concentrations of HClO lower than 10 micromol/L. Moreover, SYBR-II offers the opportunity to make quantitative titration of chlorine treated DNA and therefore seems to be the appropriate candidate to control the efficiency of the hypochlorite disinfection process of drinking water samples. PMID:15350425

  2. Ex Vivo Sentinel Node Mapping in Colon Cancer Combining Blue Dye Staining and Fluorescence Imaging

    PubMed Central

    Schaafsma, Boudewijn E.; Verbeek, Floris P.R.; van der Vorst, Joost R.; Hutteman, Merlijn; Kuppen, Peter J.K.; Frangioni, John V.; van de Velde, Cornelis J.H.; Vahrmeijer, Alexander L.

    2013-01-01

    Background The sentinel lymph node procedure has been proposed to improve nodal staging in colon cancer patients. The aim of this study was to assess the added value of near-infrared fluorescence imaging to conventional blue dye staining for ex vivo sentinel lymph node mapping. Materials and Methods Twenty-two consecutive patients undergoing surgery for colon cancer were included. After tumor resection, a premixed cocktail of the near-infrared lymphatic tracer HSA800 and blue dye was submucosally injected around the tumor for detection of sentinel lymph nodes. The Mini-FLARE imaging system was used for fluorescence imaging. Results In 95% of the patients, at least one sentinel lymph node was identified. Overall, a total of 77 sentinel lymph nodes were identified, of which 77 were fluorescent (100%) and 70 (91%) were blue. Sentinel lymph nodes that were located deeper in the mesenteric fat could easily be located by NIR fluorescence. In 4 out of 5 patients with lymph node metastases, tumor cells were present in at least 1 of the sentinel lymph nodes. Conclusions This study shows the successful use and added value of the near-infrared fluorescence tracer HSA800 to conventional blue dye for the ex vivo sentinel lymph node procedure in colon cancer. PMID:23391167

  3. Development of pathological diagnostics of human kidney cancer by multiple staining using new fluorescent Fluolid dyes.

    PubMed

    Wuxiuer, Dilibaier; Zhu, Yun; Ogaeri, Takunori; Mizuki, Keiji; Kashiwa, Yuki; Nishi, Kentaro; Isobe, Shin-ichiro; Aoyagi, Tei-ichiro; Kiyama, Ryoiti

    2014-01-01

    New fluorescent Fluolid dyes have advantages over others such as stability against heat, dryness, and excess light. Here, we performed simultaneous immunostaining of renal tumors, clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, acquired cystic disease-associated RCC (ACD-RCC), and renal angiomyolipoma (AML), with primary antibodies against Kank1, cytokeratin 7 (CK7), and CD10, which were detected with secondary antibodies labeled with Fluolid-Orange, Fluolid-Green, and Alexa Fluor 647, respectively. Kank1 was stained in normal renal tubules, papillary RCC, and ACD-RCC, and weakly or negatively in all other tumors. CK7 was positive in normal renal tubules, papillary RCC, and ACD-RCC. In contrast, CD10 was expressed in renal tubules and clear cell RCC, papillary RCC, AML, and AC-RCC, and weakly in chromophobe RCC. These results may contribute to differentiating renal tumors and subtypes of RCCs. We also examined the stability of fluorescence and found that fluorescent images of Fluolid dyes were identical between a tissue section and the same section after it was stored for almost three years at room temperature. This indicates that tissue sections can be stored at room temperature for a relatively long time after they are stained with multiple fluorescent markers, which could open a door for pathological diagnostics. PMID:24995295

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

    PubMed Central

    Tashyreva, Daria; Elster, Josef; Billi, Daniela

    2013-01-01

    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

  5. Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes.

    PubMed

    Shigano, Miyuki; Takashima, Rie; Takasawa, Hironao; Hamada, Shuichi

    2016-04-01

    The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly those that require metabolic activation. For this assay, hepatocytes (HEPs) can be isolated by collagenase treatment but without requirement for in situ liver perfusion. Consequently, the liver MN assay can be integrated into a general repeated-dose (RD) toxicity study. The method is also applicable to liver MN assays involving partial hepatectomy or the use of juvenile rats. Here, we propose an improved method for staining HEPs prepared from formalin-fixed liver tissues for MN assays, without collagenase treatment. HEP suspensions are prepared by treating the tissues with concentrated KOH and a fluorescent dye, SYBR(®) Gold (SYGO), is used for staining. Visualization of the MN in SYGO-stained HEPs is clearer than with Wright-Giemsa staining. We compared the induction of MN as measured with our new method versus the conventional method using collagenase dispersion. Our method not only enables the integration of the liver MN assay into a general RD toxicity study but also allows it to be conducted retrospectively. PMID:27085473

  6. Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

    PubMed Central

    2012-01-01

    Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested. PMID:22867517

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

    NASA Astrophysics Data System (ADS)

    Thomas, Marlon Sheldon

    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

  8. Safranine fluorescent staining of wood cell walls.

    PubMed

    Bond, J; Donaldson, L; Hill, S; Hitchcock, K

    2008-06-01

    Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy. PMID:18802812

  9. Chromosome characterization using single fluorescent dye

    DOEpatents

    Crissman, Harry A.; Hirons, Gregory T.

    1995-01-01

    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.

  10. Lipophilic dye staining of Cryptococcus neoformans extracellular vesicles and capsule.

    PubMed

    Nicola, André Moraes; Frases, Susana; Casadevall, Arturo

    2009-09-01

    Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNA-selective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components. PMID:19465562

  11. Vital staining of nerve structures with fluorescent dyes and optical determination of acetylcholine in the somatic muscle of the earthworm Lumbricus terrestris.

    PubMed

    Volkov, M E

    2012-11-01

    Experiments with fluorescent dyes showed that high concentrations of K(+) ions in the medium depolarize the membrane and enhance exo-endocytosis in nerve structures, which is accompanied by an increase in acetylcholine concentration in the somatic muscle of earthworm. In the presence of BAPTA and without Ca(2+) exo-endocytosis is sharply decelerated, the level of acetylcholine in the muscle decreases, but remains relatively high. PMID:23330101

  12. Fiberized fluorescent dye microtubes

    NASA Astrophysics Data System (ADS)

    Vladev, Veselin; Eftimov, Tinko

    2013-03-01

    In the present work we study the effect of the length of fluorescent dye-filled micro-capillaries on the fluorescence spectra. Two types of micro-capillaries have been studied: a 100 μm inner diameter fused silica capillary with a transparent coating and one of the holes of a fiber optic glass ferrule with 125 μm inner diameter. The tubes were filled with solutions of Rhodamine 6G dissolved in ethanol and then in glycerin. Experimental data show that the maximum fluorescence and the largest spectral widths are observed for a sample length of about 0.25 mm for the used concentration. This results show that miniature tunable fiberized dye lasers can be developed using available standard micro-and fibre-optic components.

  13. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  14. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  15. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  16. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  17. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  18. A New Organic Dye-Based Staining for The Detection of Plant DNA in Agarose Gels.

    PubMed

    Sönmezoğlu, Özlem Ateş; Özkay, Kerime

    2015-01-01

    Ethidium bromide (EtBr) is used to stain DNA in agarose gel electrophoresis, but this dye is mutagenic and carcinogenic. We investigated N-719, which is a visible, reliable and organic Ruthenium-based dye, and five fluorescent alternatives for staining plant DNA. For prestaining and poststaining, N-719, GelRed, and SYBR Safe stained both DNA and PCR product bands as clearly as EtBr. SYBR Green I, methylene blue, and crystal violet were effective for poststaining only. The organic dye N-719 stained DNA bands as sensitively and as clearly as EtBr. Consequently, organic dyes can be used as alternatives to EtBr in plant biotechnology studies. PMID:26158569

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

    PubMed

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

    1999-03-15

    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

  20. Stain and dye stability over a 30-year period: a comparison of certified dye powders by the Biological Stain Commission.

    PubMed

    Penney, D P; Frank, M; Fagan, C; Willis, C

    2009-02-01

    The Biological Stain Commission (BSC) Assay Laboratory has received numerous inquiries during the past several years regarding the long-term stability of stain and dye powders, particularly since packaging requirements call for expiration dates on reagents. We have conducted a study to examine the long-term stability of selected dye powders. We used the standard procedures of the BSC for testing biological stains for certification to give an indication of the long-term chemical stability as well as staining performance of the dye powders. An earlier study by Emmel and Stotz examined the stability of various dye powders after a five-year storage period. The present study is a follow-up project covering the same dyes after storage for 30 years. The dye samples chosen for the study are the same samples used in the five-year storage period study and give comparative results for all three time periods. The results of this study affirm the generally held speculation that dye powders are stable for many years and thus have a substantial shelf-life. PMID:19096966

  1. Real-time histological imaging of kidneys stained with food dyes using multiphoton microscopy.

    PubMed

    Nagao, Yasuaki; Kimura, Kazushi; Wang, Shujie; Fujiwara, Takeshi; Mizoguchi, Akira

    2015-10-01

    We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases. PMID:26260138

  2. Sizing of single fluorescently stained DNA fragments by scanning microscopy

    PubMed Central

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

    2003-01-01

    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. PMID:14602931

  3. Image analysis of dye stained patterns in soils

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    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.

  4. Methods for preparing fluorescent and neutral red-stained whole mounts of mouse mammary glands

    PubMed Central

    Landua, John D.; Visbal, Adriana P.; Lewis, Michael T.

    2010-01-01

    Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly-developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained, and some fluorescent preparations, can be used for several common downstream analyses. PMID:19936989

  5. Methods for preparing fluorescent and neutral red-stained whole mounts of mouse mammary glands.

    PubMed

    Landua, John D; Visbal, Adriana P; Lewis, Michael T

    2009-12-01

    Whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionally-localized phenotypes that would be difficult to detect or analyze using other techniques. We present three newly developed methods for preparing whole mounts of mammary glands from genetically-engineered mice expressing fluorescent proteins, as well as using either neutral red or a variety of fluorescent dyes. Unlike traditional hematoxylin- or carmine-stained preparations, neutral red-stained and some fluorescent preparations can be used for several common downstream analyses. PMID:19936989

  6. A new and permanent staining method for starch granules using fluorescence microscopy.

    PubMed

    Revilla, M A; Tolivia, D; Tarrágo, J F

    1986-05-01

    A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time. PMID:2425462

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    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.

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

    DOEpatents

    Glazer, Alexander N.; Benson, Scott C.

    1996-01-01

    Novel fluorescent DNA-staining 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.

  9. Reactive Fluorescent Dyes For Urethane Coatings

    NASA Technical Reports Server (NTRS)

    Willis, Paul B.; Cuddihy, Edward F.

    1991-01-01

    Molecules of fluorescent dyes chemically bound in urethane conformal-coating materials to enable nondestructive detection of flaws in coats through inspection under ultraviolet light, according to proposal. Dye-bonding technique prevents outgassing of dyes, making coating materials suitable for use where flaw-free coats must be assured in instrumentation or other applications in which contamination by outgassing must be minimized.

  10. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    SciTech Connect

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  11. Improved Charge-Transfer Fluorescent Dyes

    NASA Technical Reports Server (NTRS)

    Meador, Michael

    2005-01-01

    Improved charge-transfer fluorescent dyes have been developed for use as molecular probes. These dyes are based on benzofuran nuclei with attached phenyl groups substituted with, variously, electron donors, electron acceptors, or combinations of donors and acceptors. Optionally, these dyes could be incorporated as parts of polymer backbones or as pendant groups or attached to certain surfaces via self-assembly-based methods. These dyes exhibit high fluorescence quantum yields -- ranging from 0.2 to 0.98, depending upon solvents and chemical structures. The wavelengths, quantum yields, intensities, and lifetimes of the fluorescence emitted by these dyes vary with (and, hence, can be used as indicators of) the polarities of solvents in which they are dissolved: In solvents of increasing polarity, fluorescence spectra shift to longer wavelengths, fluorescence quantum yields decrease, and fluorescence lifetimes increase. The wavelengths, quantum yields, intensities, and lifetimes are also expected to be sensitive to viscosities and/or glass-transition temperatures. Some chemical species -- especially amines, amino acids, and metal ions -- quench the fluorescence of these dyes, with consequent reductions in intensities, quantum yields, and lifetimes. As a result, the dyes can be used to detect these species. Another useful characteristic of these dyes is a capability for both two-photon and one-photon absorption. Typically, these dyes absorb single photons in the ultraviolet region of the spectrum (wavelengths < 400 nm) and emit photons in the long-wavelength ultraviolet, visible, and, when dissolved in some solvents, near-infrared regions. In addition, these dyes can be excited by two-photon absorption at near-infrared wavelengths (600 to 800 nm) to produce fluorescence spectra identical to those obtained in response to excitation by single photons at half the corresponding wavelengths (300 to 400 nm). While many prior fluorescent dyes exhibit high quantum yields

  12. Pasteurella pestis detection in fleas by fluorescent antibody staining*

    PubMed Central

    Hudson, Bruce W.; Kartman, Leo; Prince, Frank M.

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study. This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation. The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  13. Contact urticaria caused by a fluorescent dye.

    PubMed

    Sugiura, Keiji; Sugiura, Mariko; Sasaki, Kazumi; Mori, Ayumi

    2010-08-01

    A 28-year-old man developed urticaria while he was working in the garage. We suspected contact urticaria, which can be caused by some products used in his field, and we carried out a prick test using his work gloves and shoes. His gloves were orange and black in colour, his shoes were black and white in colour, and the materials they were made of were unknown. The results of the prick test using the gloves and shoes were positive. Gas chromatography/mass spectrometry was applied, and a fluorescent dye was found to be present in his gloves and shoes. The results of a prick test using the fluorescent dye were positive. His urticaria improved after he stopped using these gloves and shoes. This was a rare case of contact urticaria caused by a fluorescent dye in clothing. PMID:20695863

  14. Use of Fluorescent Dyes for Readily Recognizing Sperm Damage

    PubMed Central

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

    2013-01-01

    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. PMID:24163795

  15. Facial nerve identification with fluorescent dye in rats.

    PubMed

    de Melo, Giulianno Molina; Cervantes, Onivaldo; Covolan, Luciene; Baptista, Heloisa Allegro; Ferreira, Elenn Soares; Abrahao, Marcio

    2016-02-01

    PURPOSE The parotidectomy technique still has an elevated paresis and paralysis index, lowering patient life's quality. The correct identification of the facial nerve can prevent nerve damage. Fluorescent dye identifies nerves in experimental studies but only few articles focused its use on facial nerve study in parotidectomies. We aimed to stain the rat facial nerve with fluorescent dye to facilitate visualization and dissection in order to prevent injuries. METHODS Forty adult male Wistar rats were submitted to facial injection of saline solution (Gsf-control group, 10) or fluorescent dye solution (Gdye group, 30) followed by parotidectomy preserving the facial nerve, measuring the time for localization and facility of localization (LocTime and LFN). Nerve function was assessed using the Vibrissae Movements (PMV) and Eyelid Closure Motion (PFP) scores. RESULTS Nerve localization was faster in Gdye group, with 83% Easy LFN rate. The Gdye group presented with low nerve injury degree and better PMV and PFP scores, with high sensitivity and accuracy. CONCLUSIONS This experimental method of facial nerve fluorescence was effective for intraoperative nerve visualization, identification and preservation. The technique may be used in future facial nerve studies, translated to humans, contributing to the optimization of parotid surgery in the near future. PMID:26959618

  16. Staining Fission Yeast Filamentous Actin with Fluorescent Phalloidin Conjugates.

    PubMed

    Hagan, Iain M

    2016-01-01

    The Schizosaccharomyces pombe filamentous (F)-actin cytoskeleton drives cell growth, morphogenesis, endocytosis, and cytokinesis. The protocol described here reveals the distribution of F-actin in fixed cells through the use of fluorescently conjugated phalloidin. Simultaneous staining of cell wall landmarks (with calcofluor) and chromatin (with 4',6-diamidino-2-phenylindole, or DAPI) makes this rapid staining procedure highly effective for staging cell cycle progression, monitoring morphogenetic abnormalities, and assessing the impact of environmental and genetic changes on the integrity of the F-actin cytoskeleton. PMID:27250943

  17. Fluorescent indicator dyes for calcium ions

    NASA Technical Reports Server (NTRS)

    Tsien, Roger Y. (Inventor); Grynkiewicz, Grzegorz (Inventor)

    1986-01-01

    The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, quin2, the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues. The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.

  18. Shock wave diagnostics using fluorescent dye probes

    NASA Astrophysics Data System (ADS)

    Banishev, Alexandr; Christensen, James; Dlott, Dana

    2015-06-01

    Fluorescent probes are highly developed, and have found increasing use in a wide variety of applications. We have studied shock compression of various materials with embedded dye probes used as high speed probes of pressure and temperature. Under the right conditions, dye emission can be used to make a map of the pressure distribution in shocked microstructured materials with high time (1 ns) and space (1 micrometer) resolution. In order to accomplish this goal, we started by studying shock compression of PMMA polymer with rhodamine 6G dye (R6G), as a function of shock pressure and shock duration. We observed the shock-induced spectral redshift and the shock-induced intensity loss. We investigated the fundamental mechanisms of R6G response to pressure. We showed that the time response of a dye probe is limited by its photophysical behavior under shock. We developed superemissive ultrafast dye probes by embedding R6G in a silica nanoparticle. More recently, we have searched for dye probes that have better responses. For instance, we have found that the dye Nile Red embedded in the right polymer matrix has 1.7 times larger pressure-induced redshift than R6G.

  19. Pasteurella pestis detection in Fleas by fluorescent antibody staining.

    PubMed

    Hudson, B W; Kartman, L; Prince, F M

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study.This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation.The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  20. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

    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.

  1. Spiculogenesis in the siliceous sponge Lubomirskia baicalensis studied with fluorescent staining.

    PubMed

    Annenkov, Vadim V; Danilovtseva, Elena N

    2016-04-01

    Siliceous sponges are the most primitive multicellular animals whose skeleton consists of spicules - needle-like constructions from silicon dioxide surrounding organic axial filaments. Mechanisms of spicule formation have been intensively studied due to the high ecological importance of sponges and their interest to materials science. Light and electron microscopy are not appropriate enough to display the process from silicon-enriched cells to mature spicules because of composite structure of the sponge tissues. In this article, spiculogenesis in the siliceous sponge has been studied for the first time with the use of fluorescent microscopy. Fluorescent vital dye NBD-N2 was applied to stain growing siliceous structures in the sponge and primmorph cell system. The main stages of spicule growth in the fresh-water sponge Lubomirskia baicalensis (Pallas, 1773) were visualized: silicon accumulation in sclerocytes; formation of an organic filament protruding from the cell; further elongation of the filament and growth of the spicule in a spindle-like form with enlargement in the center; merger with new sclerocytes and formation of the mature spicule. Fluorescent microscopy combined with SEM allows us to overcome the virtual differentiation between intra- and extracellular mechanisms of spicule growth. The growing spicule can capture silicic acid from the extracellular space and merge with new silicon-enriched cells. Visualization of the growing spicules with the fluorescent dye allows us to monitor sponge viability in ecological or toxicological experiments and to apply genomic, proteomic and biochemical techniques. PMID:26821342

  2. DAPI staining and fluorescence microscopy techniques for phytoplasmas.

    PubMed

    Andrade, Nancy M; Arismendi, Nolberto L

    2013-01-01

    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

  3. Highly Fluorescent dye-nanoclay Hybrid Materials Made from Different Dye Classes.

    PubMed

    Grabolle, Markus; Starke, Marian; Resch-Genger, Ute

    2016-04-12

    Nanoclays like laponites, which are commercially avaible in large quantities for a very moderate price, provide a facile solubilization strategy for hydrophobic dyes without the need for chemical functionalization and can act as a carrier for a high number of dye molecules. This does not require reactive dyes, amplifies fluorescence signals from individual emitters due to the high number of dyes molecules per laponite disk, and renders hydrophobic emitters applicable in aqueous environments. Aiming at the rational design of bright dye-loaded nanoclays as a new class of fluorescent reporters for bioanalysis and material sciences and the identification of dye structure-property relationships, we screened a series of commercial fluorescent dyes, differing in dye class, charge, and character of the optical transitions involved, and studied the changes of their optical properties caused by clay adsorption at different dye loading concentrations. Upon the basis of our dye loading density-dependent absorption and fluorescence measurements with S2105 and Lumogen F Yellow 083, we could identify two promising dye-nanoclay hybrid materials that reveal high fluorescence quantum yields of the nanoclay-adsorbed dyes of at least 0.20 and low dye self-quenching even at high dye-loading densities of up to 50 dye molecules per laponite platelet. PMID:27007448

  4. Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia☆

    PubMed Central

    Zou, Tiejun; Liu, Xiang; Ding, Shangshu; Xing, Junping

    2010-01-01

    Objective The recent advent of flow cytometry (FCM), coupled with fluorescent dyes, has been successfully applied to assess mitochondrial function. The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium (Rh123/PI) dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia. Methods Twenty-five fertile men (with normal sperm parameters) and 230 infertile patients were examined. Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups: asthenospermia (n = 30) and oligoasthenozoospermia (n = 25). Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function. Results Significant differences were found between the normal and abnormal semen samples (P < 0.05) when Rh123+/PI−, Rh123−/PI+ and Rh123−/PI− sperm were examined by FCM, but there was no significant difference between the asthenospermia (P = 0.469) and oligoasthenozoospermia group (P = 0.950) when Rh123+/PI− and Rh123−/PI+ sperm were then examined; however, a significant difference was found between the 2 groups (P = 0.003) when Rh123−/PI− sperm were examined. There was no correlation between Rh123−/PI− sperm and semen parameters in the normal group, but there was a significant negative correlation between the sperm concentration and Rh123−/PI− sperm in asthenospermia and oligoasthenozoospermia patients (r = -0.509, -0.660; P = 0.018, 0.038). Conclusion Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia. PMID:23554656

  5. Brazilwood, sappanwood, brazilin and the red dye brazilein: from textile dyeing and folk medicine to biological staining and musical instruments.

    PubMed

    Dapson, R W; Bain, C L

    2015-01-01

    Brazilin is a nearly colorless dye precursor obtained from the heartwood of several species of trees including brazilwood from Brazil, sappanwood from Asia and the Pacific islands, and to a minor extent from two other species in Central America, northern South America and the Caribbean islands. Its use as a dyeing agent and medicinal in Asia was recorded in the 2(nd) century BC, but was little known in Europe until the 12(th) century AD. Asian supplies were replaced in the 16(th) century AD after the Portuguese discovered vast quantities of trees in what is now Brazil. Overexploitation decimated the brazilwood population to the extent that it never fully recovered. Extensive environmental efforts currently are underway to re-create a viable, sustainable population. Brazilin is structurally similar to the better known hematoxylin, thus is readily oxidized to a colored dye, brazilein, which behaves like hematein. Attachment of the dye to fabric is by hydrogen bonding or in conjunction with certain metallic mordants by coordinative bonding. For histology, most staining procedures involve aluminum (brazalum) for staining nuclei. In addition to textile dyeing and histological staining, brazilin and brazilein have been and still are used extensively in Asian folk medicine to treat a wide variety of disorders. Recent pharmacological studies for the most part have established a scientific basis for these uses and in many cases have elucidated the biochemical pathways involved. The principal use of brazilwood today is for the manufacture of bows for violins and other stringed musical instruments. The dye and other physical properties of the wood combine to produce bows of unsurpassed tonal quality. PMID:25893688

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

    SciTech Connect

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

    2000-05-01

    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.

  7. Fluorescent thin gel films using organic dyes and pigments

    NASA Astrophysics Data System (ADS)

    Nakazumi, Hiroyuki; Takashi, Tarao; Taniguchi, Shin-ichi; Nanto, Hidehito

    1997-10-01

    New organic-inorganic fluorescent thin gel films included with laser dyes or fluorescent organic pigments have been prepared for display application. The florescent dyes (benzoxazolium, pyrromethene, and rhodamine dyes) and super-fine particles of fluorescent pigments (coumarin and perylene) were successfully incorporated into thin silicate gel films prepared from tetraethoxysilane (TEOS), methyltriethoxysilane (MTES), and methoxysilane oligomer (MTSO) under acid catalyzed hydrolysis. The blue, green, and red luminescence were observed from these thin films (thickness: 100 - 400 nm), respectively. Fluorescence spectra, fluorescent quantum yield and lifetime of thin gel films are examined. Fluorescent peaks for most of dyes and pigments used in gel films were similar to those in solution, and fluorescent lifetime for dyes and pigments used in gel films were 2.9 - 4.5 ns. Photostability of fluorescent gel films is dependent on fluorescent organic dyes and pigments used and/or silicate gel matrixes. Coumarin and perylene pigments have higher fluorescent quantum yield in gel film prepared from MTSO. The large Stokes shift was observed in fluorescent gel film using coumarin and benzoxazolium dyes. The coumarin and perylene pigments are significantly photo- stable in gel film prepared from MTSO, and photodegradation of perylene red after irradiation of 500 W Xi-lamp for 30 min is below 20%.

  8. Synthesize dye-bioconjugates to visualize cancer cells using fluorescence microscopy

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    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.

  9. Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye

    SciTech Connect

    Wu Weibing; Liu Chang; Wang Mingliang; Huang Wei; Zhou Shengrui; Jiang Wei; Sun Yueming; Cui Yiping; Xu Chunxinag

    2009-04-15

    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.

  10. How to use Nile Red, a selective fluorescent stain for microalgal neutral lipids.

    PubMed

    Alemán-Nava, Gibrán S; Cuellar-Bermudez, Sara P; Cuaresma, María; Bosma, Rouke; Muylaert, Koenraad; Ritmann, Bruce E; Parra, Roberto

    2016-09-01

    The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125mg/mL), or EDTA (3.0 to 3.8mg/mL). Temperatures between 30 and 40°C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25μg/mL and 2.0μg/mL, and the cell concentration >5×10(4)cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain. PMID:27432343

  11. Deep-red to near-infrared fluorescent dyes: Synthesis, photophysical properties, and application in cell imaging.

    PubMed

    Li, Qi; Liu, Weimin; Wu, Jiasheng; Zhou, Bingjiang; Niu, Guangle; Zhang, Hongyan; Ge, Jiechao; Wang, Pengfei

    2016-07-01

    More and more attention has been paid to the design of new fluorescent imaging agents with good photostability and water solubility, especially those with emissions in the deep-red and near-infrared regions. In this work, we designed and synthesized four novel fluorescent dyes with deep-red or NIR fluorescence by hybridizing coumarin and pyronin moieties based on our previous work. Introduction of carboxylic acid in the dyes not only imparted the dyes with water solubility but also provided a versatile sensing platform for designing the fluorescent probes and sensors of biomolecules. The photophysical properties of these new dyes were investigated through absorption and fluorescence spectroscopy. Cell imaging experiments showed that esterification products could selectively stain lysosomes with good photostability, thereby indicating that they could be useful in the development of fluorescent probes for bioimaging. PMID:27060414

  12. Deep-red to near-infrared fluorescent dyes: Synthesis, photophysical properties, and application in cell imaging

    NASA Astrophysics Data System (ADS)

    Li, Qi; Liu, Weimin; Wu, Jiasheng; Zhou, Bingjiang; Niu, Guangle; Zhang, Hongyan; Ge, Jiechao; Wang, Pengfei

    2016-07-01

    More and more attention has been paid to the design of new fluorescent imaging agents with good photostability and water solubility, especially those with emissions in the deep-red and near-infrared regions. In this work, we designed and synthesized four novel fluorescent dyes with deep-red or NIR fluorescence by hybridizing coumarin and pyronin moieties based on our previous work. Introduction of carboxylic acid in the dyes not only imparted the dyes with water solubility but also provided a versatile sensing platform for designing the fluorescent probes and sensors of biomolecules. The photophysical properties of these new dyes were investigated through absorption and fluorescence spectroscopy. Cell imaging experiments showed that esterification products could selectively stain lysosomes with good photostability, thereby indicating that they could be useful in the development of fluorescent probes for bioimaging.

  13. Staining of Platyhelminthes by herbal dyes: An eco-friendly technique for the taxonomist

    PubMed Central

    Kumar, Niranjan; Mehul, Jadav; Das, Bhupamani; Solanki, J. B.

    2015-01-01

    Aim: An environment compatible technique to stain Platyhelminthes, Fasciola gigantica, Gastrothylax crumenifer, Taenia solium, and Moniezia expansa using aqueous and alcoholic extract of sugar beet (Beta vulgaris), China rose (Hibiscus rosa-sinensis), and red rose (Rosa hybrida) were described to minimized the deleterious effects of the synthetic dyes. Materials and Methods: Aqueous/ethanolic extracts of roses were extracted from the flowers while red beet was extracted from the roots. Results: Stained helminthes acquired a comparable level of pigmentation with the distinction of their internal structure in these natural dyes. The flukes (liver and rumen) internal structure, oral and ventral/posterior sucker, cirrus sac, gravid uterus, testes, ovary, and vitallaria were appeared pink color in aqueous and alcoholic extract of either China or red rose and yellow to brown color in sugar beet stain. The interior of the proglottid of T. solium and M. expansa took yellow to brown color with good contrast in sugar beet stain and of pink to pink-red in China and red rose stain. Conclusion: The extract of roses (red rose followed by China rose) followed by red beet possess the potential to replace the conventional stains in the taxonomic study of Platyhelminthes parasites. PMID:27047037

  14. Detection of Acid Fast Bacilli in Saliva using Papanicolaou Stain Induced Fluorescence Method Versus Fluorochrome Staining: An Evaluative Study

    PubMed Central

    (Munot), Priya P Lunawat; Mhapuskar, Amit A; Ganvir, S M; Hazarey, Vinay K; Mhapuskar, Madhavi A; Kulkarni, Dinraj

    2015-01-01

    Background: Fifty years after effective chemotherapy, tuberculosis (TB) still remains leading infectious cause of adult mortality. The aim of present study was to evaluate diagnostic utility of papanicolaou (Pap) stain induced fluorescence microscopic examination of salivary smears in the diagnosis of pulmonary TB. Materials and Methods: Cross-sectional study of 100 individuals clinically suspected of suffering from active pulmonary TB. Control group – 50 individuals are suffering from any pulmonary disease other than TB such as pneumonia or bronchiogenic carcinoma. Fluorescence microscopic examination of two salivary smears stained by Pap stain and auramine-rhodamine (A-R) stain respectively for each patient. Ziehl–Neelsen stained sputum smear examined under the light microscope for each patient. Culture was done in all the patients for microbiological confirmation. McNemar's Chi-square analysis, Kappa test, and Z-test. Results: The sensitivities of the three staining methods using culture as a reference method were 93.02%, 88.37% and 87.20% for Pap, A-R and Ziehl–Neelson respectively. Conclusion: Pap-induced fluorescence of salivary smears is a safe, reliable and rapid method, which can prove as a valuable diagnostic tool for diagnosis of TB. PMID:26229384

  15. Indian ink vs tissue marking dye: a quantitative comparison of two widely used macroscopical staining tool.

    PubMed

    Kosemehmetoglu, Kemal; Guner, Gunes; Ates, Deniz

    2010-07-01

    The evaluation of the surgical margins is a major concern in surgical pathology, and marking of surgical margins with substances such as alcian blue, Tipp-ex, artist's pigments, colored gelatin, starch, erythrocyte layers, etc. was recommended for this purpose; Indian ink and tissue marking dyes are widely used. As there is no systematic study comparing tissue marking dyes and Indian ink as the most common substances used for the purpose, this study was conducted to compare the two. Penetration into the tissue, brightness under the microscope, the spreading area of one drop of dye on tissue paper, the intensity of colors, and unit price were compared for each of the five colors of Rotring's Indian ink and Thermo-Shandon's tissue marking dyes, applied on reduction mammoplasty specimens. Rotring's Indian ink is proved to be just as effective as Thermo-Shandon's tissue marking dye and bares the majority of the characteristics of a perfect staining substance, which are easily applied, quickly fixed, durable and cheap, contain no potential contaminants, be work safe, would not smudge/stain surrounding tissues, and look bright under the microscope without obscuring the view. PMID:20559655

  16. Facile and eco-friendly synthesis of green fluorescent carbon nanodots for applications in bioimaging, patterning and staining

    NASA Astrophysics Data System (ADS)

    Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Wen, Xiangping; Zhang, Guomei; Yang, Jun; Dong, Chuan; Shuang, Shaomin

    2015-04-01

    We report a facile and eco-friendly strategy for the fabrication of green fluorescent carbon nanodots (CDs), and demonstrate their applications for bio-imaging, patterning, and staining. A one-pot hydrothermal method using various plant petals yields bright green-emitting CDs, providing an easy way for the production of green fluorescent CDs without the need for a tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. The as-prepared CDs show small size distribution and excellent dispersibility. Their strong green fluorescence is observed when the excitation wavelength is between 430 nm and 490 nm. Moreover, they exhibit high tolerance to various external conditions, such as pH values, external cations, and continuous excitation. Due to minimum toxicity as well as good photoluminescence properties, these CDs can be applied to in vitro and in vivo imaging, patterning, and staining. According to confocal fluorescence imaging of human uterine cervical squamous cell carcinoma cells, CDs penetrate into the cell and enter the cytoplasm and the nucleus. More strikingly, carp is directly fed with CDs for in vivo imaging and shows bright green fluorescence at an excitation wavelength of 470 nm. In addition, the obtained CDs are used as fluorescent inks for drawing luminescence patterns. Finally, we also apply the CDs as a fluorescent dye. Interestingly, the absorbent filter paper with staining emits dramatic fluorescence under 470 nm excitation.We report a facile and eco-friendly strategy for the fabrication of green fluorescent carbon nanodots (CDs), and demonstrate their applications for bio-imaging, patterning, and staining. A one-pot hydrothermal method using various plant petals yields bright green-emitting CDs, providing an easy way for the production of green fluorescent CDs without the need for a tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. The as-prepared CDs show small size

  17. A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo.

    PubMed

    Press, Adrian T; Ungelenk, Luisa; Rinkenauer, Alexandra C; Gröger, Marko; Lehmann, Frank; Mosig, Alexander; Schubert, Ulrich S; Clemens, Mark G; Bauer, Michael

    2016-09-01

    Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability. PMID:26563981

  18. Extrinsic Fluorescent Dyes as Tools for Protein Characterization

    PubMed Central

    Hawe, Andrea; Sutter, Marc

    2008-01-01

    Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization. PMID:18172579

  19. Multianalyte Microphysiometry Reveals Changes in Cellular Bioenergetics Upon Exposure to Fluorescent Dyes

    PubMed Central

    Shinawi, Tesniem F.; Kimmel, Danielle W.; Cliffel, David E.

    2014-01-01

    Fluorescent dyes have been designed for internal cellular component specificity, and have been used extensively in the scientific community as a means to monitor cell growth, location, morphology, and viability. However, it is possible that the introduction of these dyes influences the basal function of the cell and, in turn, the results of these studies. Electrochemistry provides a non-invasive method for probing the unintended cellular affects of these dyes. The multianalyte microphysiometer (MAMP) is capable of simultaneous electrochemical measurement of extracellular metabolites in real-time. In this study, analytes central to cellular metabolism, glucose, lactate, oxygen, as well as extracellular acidification were monitored to determine the immediate metabolic effects of nuclear stains, including SYTO®, DAPI dilactate, Hoechst 33342, and FITC dyes upon the pheochromocytoma PC-12 cells and RAW 264.7 macrophages. The experimental results revealed that SYTO dye 13 significantly decreased glucose and oxygen consumption and increased extracellular acidification and lactate production in both cell lines, indicating a shift to anaerobic respiration. No other dyes caused significantly definitive changes in cellular metabolism upon exposure. This study shows that fluorescent dyes can have unintended effects on cellular metabolism and care should be taken when using these probes to investigate cellular function and morphology. PMID:24228839

  20. Near-Infrared Fluorescence of the NBT/BCIP Chromogenic Stain

    NASA Astrophysics Data System (ADS)

    McCutchen, M. D.; Bumm, L. A.; McCauley, D. W.; Trinh, L. A.; Bonner-Fraser, M.; Fraser, S. E.

    2007-03-01

    We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). Although the product is a solid with strong optical absorption, its fluorescence enables high cellular resolution imaging of gene expression. We use spectrofluorometry to identify NBT diformazan as the component of the stain that is the fluorophore exhibiting the strong fluorescence signal. The fluorescence shows an intense emission signal (780-910 nm) that is well separated from excitation (645-685 nm). The NBT diformazan fluorescence is also photostable. Because NBT/BCIP is a widely used chromogenic stain, existing staining protocols can also be applied to fluorescence imaging techniques to increase the resolution of gene expression patterns.

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

    PubMed

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

    2000-05-01

    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. PMID:10788401

  2. NIR fluorescent dyes: versatile vehicles for marker and probe applications

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Chapman, Gala; Beckford, Garfield; Henary, Maged

    2013-02-01

    The use of the NIR spectral region (650-900 nm) is advantageous due to the inherently lower background interference and the high molar absorptivities of NIR chromophores. Near-Infrared (NIR) dyes are increasingly used in the biological and medical field. The binding characteristics of NIR dyes to biomolecules are possibly controlled by several factors, including hydrophobicity, size and charge just to mention a few parameters. Binding characteristics of symmetric carbocyanines and found that the hydrophobic nature of the NIR dye is only partially responsible for the binding strength. Upon binding to biomolecules significant fluorescence enhancement can be observed for symmetrical carbocyanines. This fluorescence amplification facilitates the detection of the NIR dye and enhances its utility as NIR reporter. This manuscript discusses some probe and marker applications of such NIR fluorescent dyes. One application discussed here is the use of NIR dyes as markers. For labeling applications the fluorescence intensity of the NIR fluorescent label can significantly be increased by enclosing several dye molecules in nanoparticles. To decrease self quenching dyes that have relatively large Stokes' shift needs to be used. This is achieved by substituting meso position halogens with amino moiety. This substitution can also serve as a linker to covalently attach the dye molecule to the nanoparticle backbone. We report here on the preparation of NIR fluorescent silica nanoparticles. Silica nanoparticles that are modified with aminoreactive moieties can be used as bright fluorescent labels in bioanalytical applications. A new bioanalytical technique to detect and monitor the catalytic activity of the sulfur assimilating enzyme using NIR dyes is reported as well. In this spectroscopic bioanalytical assay a family of Fischer based n-butyl sulfonate substituted dyes that exhibit distinct variation in absorbance and fluorescence properties and strong binding to serum albumin as its

  3. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

    PubMed Central

    Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

    2015-01-01

    Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

  4. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    PubMed

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

    2015-11-01

    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

  5. Bioconjugation Methods for Coupling Targeting Ligands with Fluorescent Dyes.

    PubMed

    Ling, Xiaoxi

    2016-01-01

    Targeted molecular imaging probes are essential tools for visualization of specific molecular processes in cells and living systems. Among these, targeted fluorescent probes are widely used due to the high sensitivity and resolution of fluorescence imaging. The conventional strategy for developing targeted fluorescent probes is to couple targeting ligands with fluorescent dyes by covalent bond via bioconjugation. Here, we describe several commonly used bioconjugation methods, from traditional amide and thiol coupling, to metal-catalyzed coupling reaction and catalyst free cycloaddition. PMID:27283413

  6. Fluorescent aggregates Cyan 40 and Thiazole Orange dyes in solution

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    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.

  7. The use of epifluorescence microscopy and fluorescent dyes for visualization of Oxyuris equi eggs.

    PubMed

    Barros, Hélio L; Marques, Sandra M T; Stefani, Valter

    2016-08-15

    This study presents a new method for visualization of Oxyuris equi eggs collected by means of a clear adhesive tape applied to the perianal region of horses. The obtained results indicate that this protocol permits a quick, easy, clear and selective visualization of Oxyuris equi eggs even in the presence of feces and other material. Another advantage of this method is that it can be used with fluorescent dyes solubilized in water, which will stain biologic material without dissolving or altering the adhesive tape and is also environmentally safe. Other dyes currently used for staining biologic materials use organic solvents, which may be combined with acids or bases in their formulation, preventing their use with the tape method. PMID:27514902

  8. Maximizing dye fluorescence via incorporation of metallic nanoparticles in solution

    NASA Astrophysics Data System (ADS)

    Xu, Yang; Lei, Guangyin; Booker, Annette C.; Linares, Katherine A.; Fleming, Dara L.; Meehan, Kathleen; Lu, Guo-Quan; Love, Nancy G.; Love, Brian J.

    2004-12-01

    Gram-negative bacteria initiate a stress response in which the cells efflux potassium when electrophilic toxins are introduced into their environment. Hence, measurement of K+ concentration in the surrounding water using a fluorescence-based potassium-selective optode has been proposed for environmental and homeland security applications. Unfortunately, the fluorophore commonly used in such an optode is inefficient. Surface enhanced fluorescence (SEF) can be used to increase its fluorescence efficiency, which will improve the sensor's performance. To understand this phenomenon before applying it to the optode system, Rose Bengal (RB), an inexpensive and well characterized dye, in solution with gold and silver nanoparticles was studied. As expected, fluorescence from RB-gold solutions was low since alignment of gold's surface plasmon resonance (SPR) peak and absorption and fluorescence energies in RB favored energy transfer from RB to the gold nanoparticles. The alignment of the silver's SPR peak and the RB transitions favored transfer from silver to RB. SEF was observed in solutions with large dye-to-silver separation. However, little fluorescence was observed when the solution was pumped at the silver's SPR peak. Fluorescence from the dye decreased as dye-to-silver separation decreased. An explanation for these observations is presented; additional research is needed to develop a complete understanding.

  9. Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence.

    PubMed

    Kang, Chulhun; Kim, Hyun Jung; Kang, Donghoon; Jung, Duk Young; Suh, Myungkoo

    2003-10-01

    Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. PMID:14595675

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

    PubMed Central

    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

    1990-01-01

    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 PMID:1693631

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

    DOEpatents

    Glazer, Alexander N.; Benson, Scott C.

    1998-01-01

    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.

  12. Effects of purification and fluorescent staining on viability of Mycobacterium leprae.

    PubMed

    Lahiri, Ramanuj; Randhawa, Baljit; Krahenbuhl, James L

    2005-09-01

    Over the years, researchers have carried out experiments with Mycobacterium leprae obtained from either human multibacillary lesions, or infected armadillo tissues, or infected footpad tissues of conventional mice as well as athymic nu/nu mice. In general, these sources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies, but less than satisfactory for studies in cell biology and immunology where contaminating host tissues pose a serious problem. We examined the utility of a procedure for eliminating mouse footpad tissue from M. leprae suspension using sodium hydroxide solution and its subsequent effect on the viability of the organism by determining the rate of palmitic acid oxidation, bacterial membrane integrity, and growth in the mouse footpad. We found that treating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1N NaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely affecting the viability of the organism. This is a simple and rapid method to get suspensions of nu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a research reagent for studying the host-pathogen relationship in leprosy. We also report here a method for labeling M. leprae with the fluorescent dye PKH26, without compromising on the viability of the organism. This method may be useful in intracellular trafficking studies of M. leprae or in other cell biology studies that require tracking of the bacteria using fluorescent tag. We observed the staining to be stable in vitro over considerable lengths of time and did not affect the viability of the bacteria. PMID:16830641

  13. Estimation of Fluorescent Dye Amount in Tracer Dye Test

    NASA Astrophysics Data System (ADS)

    Pekkan, Emrah; Balkan, Erman; Balkan, Emir

    2015-04-01

    Karstic groundwater is more influenced by human than the groundwater that disperse in pores. On the other hand karstic groundwater resources, in addition to providing agricultural needs, livestock breeding, drinking and domestic water in most of the months of the year, they also supply drinking water to the wild life at high altitudes. Therefore sustainability and hydrogeological investigation of karstic resources is critical. Tracing techniques are widely used in hydrologic and hydrogeologic studies to determine water storage, flow rate, direction and protection area of groundwater resources. Karanfil Mountain (2800 m), located in Adana, Turkey, is one of the karstic recharge areas of the natural springs spread around its periphery. During explorations of the caves of Karanfil mountain, a 600 m deep cave was found by the Turkish and Polish cavers. At the bottom of the cave there is an underground river with a flow rate of approximately 0.5 m3/s during August 2014. The main spring is located 8 km far from the cave's entrance and its mean flow rate changes between 3.4 m3/s and 0.21 m3/s in March and September respectively according to a flowrate observation station of Directorate of Water Works of Turkey. As such frequent storms, snowmelt and normal seasonal variations in rainfall have a significant and rapid effect on the volume of this main spring resource. The objective of our research is to determine and estimate dye amount before its application on the field inspired from the previously literature on the subject. This estimation is intended to provide a preliminary application of a tracer test of a karstic system. In this study dye injection, inlet point will be an underground river located inside the cave and the observation station will be the spring that is approximately 8 km far from the cave entrance. On the other hand there is 600 meter elevation difference between cave entrance and outlet spring. In this test Rodamin-WT will be used as tracer and the

  14. Several hemicyanine dyes as fluorescence chemosensors for cyanide anions

    NASA Astrophysics Data System (ADS)

    Liang, Muhan; Wang, Kangnan; Guan, Ruifang; Liu, Zhiqiang; Cao, Duxia; Wu, Qianqian; Shan, Yanyan; Xu, Yongxiao

    2016-05-01

    Four hemicyanine dyes as chemosensors for cyanide anions were synthesized easily. Their photophysical properties and recognition properties for cyanide anions were investigated. The results indicate that all the dyes can recognize cyanide anions with obvious color, absorption and fluorescence change. The recognition mechanism analysis basing on in situ 1H NMR and Job plot data indicates that to the compounds with hydroxyl group, the recognition mechanism is intramolecular hydrogen bonding interaction. However, to the compounds without hydroxyl group, cyanide anion is bonded to carbon-carbon double bond in conjugated bridge and induces N+ CH3 to neutral NCH3. Fluorescence of the compounds is almost quenched upon the addition of cyanide anions.

  15. Update on flashlamp pumped pulsed dye laser treatment for port wine stains (capillary malformation) patients

    PubMed Central

    Hsiao, Yen-Chang; Chang, Cheng-Jen

    2011-01-01

    Background and Aims: Currently, the method of choice for the treatment of port-wine stains is laser photocoagulation. Because of evolving treatment options, it is no longer enough for port-wine stains merely to be lightened through laser treatment. The best course of management consists of the most appropriate laser that will produce the most complete clearing of a lesion in the fewest treatment sessions with the least morbidity. The goal is generally accomplished with the use of yellow-light lasers. Materials (Subjects) and Methods: Absorption of laser energy by melanin causes localized heating in the epidermis, which may, if not controlled, produce permanent complications such as hypertrophic scarring or dyspigmentation. Refinements of the results can be achieved by using the flashlamp-pumped pulsed dye laser (FLPDL) in conjunction with the cryogen spray cooling (CSC) system. In our related studies, the infrared thermal image instrument is used for doctors in determining the optimum laser light dosage and preventing the side effects caused by FLPDL. Topic application of angiogenesis inhibitor (Imiquimod) in conjunction with pulsed dye laser treatment for the PWS patients has been assessed for improvement of FLPDL treatment. Results: We present the clinical effect of FLPDL, and the efficacy and safety of cooled laser treatment of PWS birthmarks. Our clinical outcome in the laser treatment of patients with PWS has been achieved to maximize thermal impact on targeted vessels, while minimizing adverse complications. Conclusions: CSC in conjunction with FLPDL can improve the treatment of PWS. The infrared image instrument is helpful for doctors in determining the optimum laser light dosage. Topic application of angiogenesis inhibitor (Imiquimod) in conjunction with laser treatment for the PWS patients is promising in the near future. PMID:24155536

  16. Development of Highly Fluorescent Materials Based on Thiophenylimidazole Dyes

    NASA Technical Reports Server (NTRS)

    Santos, Javier; Bu, Xiu R.; Mintz, Eric A.; Meador, Michael A. (Technical Monitor)

    2000-01-01

    Organic fluorescent materials are expected to find many potential applications in optical devices and photo-functionalized materials. Although many investigations have been focused on heterocyclic compounds such as coumarins, bipyridines, rhodamines, and pyrrole derivatives, little is known for fluorescent imidazole materials. We discovered that one particular class of imidazole derivatives is highly fluorescent. A series of monomeric and polymeric based fluorescent dyes were prepared containing a thiophene unit at the second position of the imidazole ring. Dependence of fluorescence efficiency on parameters such as solvent polarity and substituent groups has been investigated. It was found that a formyl group at the 2-position of the thiophene ring dramatically enhance fluorescence properties. Ion recognition probes indicated their potential as sensor materials. These fluorophores have flexibility for introduction of versatile substituent groups that could improve the fluorescence efficiency and sensor properties.

  17. Electrowetting actuation of a dye-doped fluorescent droplet

    NASA Astrophysics Data System (ADS)

    Guerrero, Raphael A.; Mero, Rea Divina C.

    2014-11-01

    We report tunable color output from a fluorescent dye-doped droplet actuated by electrowetting. The system design, based on a planar electrowetting set-up, is compact and straightforward, with minimal voltage requirements for effective actuation. Fluorescent droplets are sourced from a 1 mM solution of rhodamine 6G in distilled water. Initial contact angle for a dye-doped droplet is 72.1°. At a maximum applied voltage of 20 V, the contact angle decreases to 56.5°. Emission spectra are collected as the droplet fluoresces under UV illumination. Over an electrowetting voltage range of 0 to 20 V, the peak fluorescence wavelength shifts from 568 to 546 nm.

  18. Optimization strategies for a fluorescent dye with bimodal excitation spectra: application to semiautomated proteomics

    NASA Astrophysics Data System (ADS)

    Patton, Wayne F.; Berggren, Kiera N.; Lopez, Mary F.

    2001-04-01

    Facilities engaged in proteome analysis differ significantly in the degree that they implement automated systems for high-throughput protein characterization. Though automated workstation environments are becoming more routine in the biotechnology and pharmaceutical sectors of industry, university-based laboratories often perform these tasks manually, submitting protein spots excised from polyacrylamide gels to institutional core facilities for identification. For broad compatibility with imaging platforms, an optimized fluorescent dye developed for proteomics applications should be designed taking into account that laser scanners use visible light excitation and that charge-coupled device camera systems and gas discharge transilluminators rely upon UV excitation. The luminescent ruthenium metal complex, SYPRO Ruby protein gel stain, is compatible with a variety of excitation sources since it displays intense UV (280 nm) and visible (470 nm) absorption maxima. Localization is achieved by noncovalent, electrostatic and hydrophobic binding of dye to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream microchemical characterization techniques such as matrix-assisted laser desorption/ionization-mass spectrometry is assured. Protocols have been devised for optimizing fluorophore intensity. SYPRO Ruby dye outperforms alternatives such as silver staining in terms of quantitative capabilities, compatibility with mass spectrometry and ease of integration into automated work environments.

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

    NASA Astrophysics Data System (ADS)

    Bhattacharjee, Shubhadeep; Jose, Iven

    2011-02-01

    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-β 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.

  20. Measurement of cation movement in primary cultures using fluorescent dyes.

    PubMed

    Reynolds, I J

    2001-05-01

    Ca(2+), Na(+), K(+), and Mg(2+) have a central role in neuronal excitability. The concentration of these cations in the cytoplasm of neurons (generically termed [ion(+)]i) provides a marker of the excitation state of the neurons, and may also illuminate the activity of specific signaling mechanisms that involve Ca(2+)- or Mg(2+)-activated enzymes. The measurement of [ion(+)]i in cultured neurons is achieved with the use of an ion-sensitive fluorescent dye in combination with equipment designed to quantitatively measure fluorescence. Specificity is obtained by choosing dyes with the appropriate selectivity for the ion of interest. Measurements of steady state ion concentrations can be made, as well as measurements of the net difference between ion movement into the cytoplasm (in response to a stimulus) and the physiological buffering of that ion. The procedures in this unit for loading and recording from dyes are broadly similar for each ion when ratiometric dyes are used as described, and can readily be modified for use with single-wavelength dyes. Support protocols are provided for calibration of individual dyes, which can be more problematic. PMID:18428522

  1. Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

    NASA Astrophysics Data System (ADS)

    Yamada, Kengo; Yoshida, Katsuyuki; Sonoda, Ryoichi

    Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

  2. Bright and photostable cyanine-styryl chromophores with green and red fluorescence colour for DNA staining

    NASA Astrophysics Data System (ADS)

    Bohländer, Peggy R.; Wagenknecht, Hans-Achim

    2015-12-01

    The synthesis and optical characterisation of a series of green- and red-emitting cyanine and cyanine-styryl dyes is presented that were developed based on the cyanine-indole-quinolinium and based on the thiazole red type structure. For the green emitting fluorophores the quinolinium part was replaced by a pyridinium group. The bridge to the indole group was attached either to the 2-position or to the 4-position of the pyridinium moiety. For the red-emitting dyes the connection to the indole moiety is at the 4-position of the quinolinium part. In each set of dyes a methyl group at the indole-NH and/or a phenyl group at the 2-position of the indole part were introduced to tune the optical properties and photostability. Additionally, two dyes were modified with a cyano group to tune the photophysical properties and to enhance the photostabilities. The developed dyes show good photostabilities and bright green or red fluorescence intensities in the presence of DNA. Thus, these dyes represent important and promising candidates for fluorescent molecular imaging of nucleic acids inside living cells.

  3. 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...

  4. Superior optical nonlinearity of an exceptional fluorescent stilbene dye

    SciTech Connect

    He, Tingchao; Sreejith, Sivaramapanicker; Zhao, Yanli; Gao, Yang; Grimsdale, Andrew C.; Lin, Xiaodong E-mail: hdsun@ntu.edu.sg; Sun, Handong E-mail: hdsun@ntu.edu.sg

    2015-03-16

    Strong multiphoton absorption and harmonic generation in organic fluorescent chromophores are, respectively, significant in many fields of research. However, most of fluorescent chromophores fall short of the full potential due to the absence of the combination of such different nonlinear upconversion behaviors. Here, we demonstrate that an exceptional fluorescent stilbene dye could exhibit efficient two- and three-photon absorption under the excitation of femtosecond pulses in solution phase. Benefiting from its biocompatibility and strong excited state absorption behavior, in vitro two-photon bioimaging and superior optical limiting have been exploited, respectively. Simultaneously, the chromophore could generate efficient three-photon excited fluorescence and third-harmonic generation (THG) when dispersed into PMMA film, circumventing the limitations of classical fluorescent chromophores. Such chromophore may find application in the production of coherent light sources of higher photon energy. Moreover, the combination of three-photon excited fluorescence and THG can be used in tandem to provide complementary information in biomedical studies.

  5. Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs

    PubMed Central

    Qu, Haiyong; Xing, Wenxi; Wu, Fenfen; Wang, Yongzhang

    2016-01-01

    The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca2+ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca2+ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs. PMID:27055240

  6. Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs.

    PubMed

    Qu, Haiyong; Xing, Wenxi; Wu, Fenfen; Wang, Yongzhang

    2016-01-01

    The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca2+ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca2+ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs. PMID:27055240

  7. Neoplasm diagnostics based on fluorescence of polymethine dyes

    NASA Astrophysics Data System (ADS)

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

    2002-05-01

    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.

  8. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    NASA Astrophysics Data System (ADS)

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-01

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  9. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    SciTech Connect

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-10

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  10. Hematoxylin shortages: their causes and duration, and other dyes that can replace hemalum in routine hematoxylin and eosin staining.

    PubMed

    Dapson, R; Horobin, R W; Kiernan, J

    2010-02-01

    The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa. PMID:19562570

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

    PubMed

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

    2007-01-01

    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

  12. Designer metal-nanoantennae/dye complexes for maximum fluorescence enhancement

    NASA Astrophysics Data System (ADS)

    Meng, Xiang; Yang, Hao; Grote, Richard R.; Dadap, Jerry I.; Panoiu, Nicolae C.; Osgood, Richard M.

    2015-09-01

    We theoretically investigate the fluorescence enhancement of a representative set of dye-molecules excited by three classes of nanoantennae, using a fully vectorial three-dimensional finite-difference time-domain (3D FDTD) method. Through these 3D FDTD calculations, in conjunction with analytic guidance using temporal coupled-mode (TCM) theory, we develop a design procedure for antennae assemblies that allow achieving fluorescence enhancements of 200-900 over the emission intensity in the bare dye molecule. The enhancement from these commercially available fluorochrome conjugates, namely, CFTM568, CFTM660R and CFTM790 are fully investigated using spherical-dimer, elliptical-dimer, and bowtie nanoantennae. These results demonstrate a method for rationally designing arbitrary metallic nanoparticle/emitter assemblies prior to their synthesis and assembly to achieve optimum fluorescence enhancement.

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

    PubMed

    Fröhlich, J; König, H

    1999-03-01

    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

  14. Fluorescence properties of dye doped mesoporous silica

    NASA Astrophysics Data System (ADS)

    Carbonaro, Carlo M.; Corpino, Riccardo; Ricci, Pier Carlo; Chiriu, Daniele; Cannas, Carla

    2014-10-01

    In this paper we present a review of the main results we obtained studying the emission properties of organic-inorganic hybrids obtained combining mesoporous silica and Xantene dyes, in particular the standard referenc Rhodamine 6G. The purpose of the review is to show the possibility to efficiently "dope" the transparent inorganic porous matrix to obtain promising systems for photonic and biomedical applications. The strategies to solve the concentration effect and the leaching phenomenon are discussed within the framework of the single exciton theory.

  15. Fluorescence properties of dye doped mesoporous silica

    SciTech Connect

    Carbonaro, Carlo M. Corpino, Riccardo Ricci, Pier Carlo Chiriu, Daniele; Cannas, Carla

    2014-10-21

    In this paper we present a review of the main results we obtained studying the emission properties of organic-inorganic hybrids obtained combining mesoporous silica and Xantene dyes, in particular the standard reference Rhodamine 6G. The purpose of the review is to show the possibility to efficiently 'dope' the transparent inorganic porous matrix to obtain promising systems for photonic and biomedical applications. The strategies to solve the concentration effect and the leaching phenomenon are discussed within the framework of the single exciton theory.

  16. Tailoring Fluorescent Dyes To Optimize a Hybrid RGD-Tracer.

    PubMed

    Bunschoten, Anton; van Willigen, Danny M; Buckle, Tessa; van den Berg, Nynke S; Welling, Mick M; Spa, Silvia J; Wester, Hans-Jürgen; van Leeuwen, Fijs W B

    2016-05-18

    Quantitative assessment of affinity and kinetics is a critical component in the development of (receptor-targeted) radiotracers. For fluorescent tracers, such an assessment is currently not yet applied, while (small) changes in chemical composition of the fluorescent component might have substantial influence on the overall properties of a fluorescent tracer. Hybrid imaging labels that contain both a radiolabel and a fluorescent dye can be used to evaluate both the affinity (fluorescent label) and the in vivo distribution (radiolabel) of a targeted tracer. We present a hybrid label oriented and matrix-based scoring approach that enabled quantitative assessment of the influence of (overall) charge and lipophilicity of the fluorescent label on the (in vivo) characteristics of αvβ3-integrin targeted tracers. Systematic chemical alterations in the fluorescent dye were shown to result in a clear difference in the in vivo distribution of the different hybrid tracers. The applied evaluation technique resulted in an optimized targeted tracer for αvβ3-integrin, which combined the highest T/M ratio with the lowest uptake in other organs. Obviously this selection concept would also be applicable during the development of other (receptor-targeted) imaging tracers. PMID:27074375

  17. Fluorescence of Alexa fluor dye tracks protein folding.

    PubMed

    Lindhoud, Simon; Westphal, Adrie H; Visser, Antonie J W G; Borst, Jan Willem; van Mierlo, Carlo P M

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding. PMID:23056480

  18. DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

    PubMed Central

    Beierlein, Frank R.; Paradas Palomo, Miguel; Sharapa, Dmitry I.; Zozulia, Oleksii; Mokhir, Andriy; Clark, Timothy

    2016-01-01

    Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries. PMID:27467071

  19. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia

    PubMed Central

    Klinger-Strobel, Mareike; Ernst, Julia; Lautenschläger, Christian; Pletz, Mathias W; Fischer, Dagmar; Makarewicz, Oliwia

    2016-01-01

    Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(d,l-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. PMID:26917959

  20. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia.

    PubMed

    Klinger-Strobel, Mareike; Ernst, Julia; Lautenschläger, Christian; Pletz, Mathias W; Fischer, Dagmar; Makarewicz, Oliwia

    2016-01-01

    Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO(®) 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. PMID:26917959

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

    PubMed Central

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

    2011-01-01

    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. PMID:21339175

  2. Following the dyeing of wool in real time using fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Bradley, Grant; Collins, Stephen; Davidson, R. Stephen

    1996-06-01

    The novel design of a heated stage (miniature dye bath) for use with an inverted fluorescence microscope is described. The heated stage allows investigation of wool dyeings to be followed in situ in real time. The photometer attachment of the microscope allows for quantitative analysis. The progress of the dyeing of wool with a fluorescent whitening agent and a fluorescent dye have been monitored in real time.

  3. Tissue diagnosis of intestinal microsporidiosis using a fluorescent stain with Uvitex 2B.

    PubMed Central

    Franzen, C; Müller, A; Salzberger, B; Fätkenheuer, G; Eidt, S; Mahrle, G; Diehl, V; Schrappe, M

    1995-01-01

    AIMS--To detect intestinal microsporidiosis in paraffin wax embedded biopsy specimens using a fluorescence technique incorporating optical brighteners. METHODS--Eight HIV infected patients with confirmed intestinal microsporidiosis (six with Enterocytozoon bieneusi, one with Encephalitozoon intestinalis and one with Encephalitozoon cuniculi infection) and 10 without infection were studied. Tissue sections of paraffin wax embedded duodenal biopsy specimens were stained with 1% Uvitex 2B, coded and analysed independently by two investigators. RESULTS--In all eight cases with confirmed intestinal microsporidian infection, spores could be detected easily in tissue sections using the fluorescence technique. Spores or other elements consistent with microsporidiosis were not found in the 10 patients without infection. CONCLUSION--Staining of tissue sections from paraffin wax embedded intestinal biopsy specimens with stains incorporating Uvitex 2B is a rapid and easy technique for the diagnosis of intestinal microsporidiosis. Images PMID:8543621

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

    SciTech Connect

    Yasokawa, Toshiki; Ishimaru, Ichirou; Kuriyama, Shigeki; Masaki, Tsutomu; Takegawa, Kaoru; Tanaka, Naotaka

    2007-09-24

    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.

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

    NASA Astrophysics Data System (ADS)

    Renfro, Lisa; Geronemus, Roy G.

    1992-06-01

    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.

  6. Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions.

    PubMed

    Bhadauria, V; Miraz, P; Kennedy, R; Banniza, S; Wei, Y

    2010-04-01

    Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil-C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems. PMID:19669979

  7. Influence of selected fluorescent dyes on small aquatic organisms

    NASA Astrophysics Data System (ADS)

    Rowiński, Paweł; Chrzanowski, Marcin

    2011-02-01

    Rhodamine B and Rhodamine WT are fluorescent dyes commonly used as tracers in hydrological investigations. Since introducing intensely red substances into rivers raises understandable doubts of ecological nature, the authors aimed at examining the influence of these dyes on small water fauna using bioindication methods. Quantitative results, calculated with the use of Bliss-Weber probit statistical method, were achieved by means of standardized ecotoxicological tests containing ready-to-hatch resting forms of fairy shrimp (Thamnocephalus platyurus). Qualitative studies included observation of water flea crustacean (Daphnia magna) and horned planorbis snail (Planorbis corneus), both typically present in rivers and representative for temperate climate, as well as guppy fish (Poecilla reticulata), paramecium protozoan (Paramaecium caudatum) and the above-mentioned fairy shrimp. The investigation revealed that both dyes in concentrations used for hydrological purposes are low enough to exert almost no toxic impact on water fauna considered.

  8. Fluorescent labeling of dendritic spines in cell cultures with the carbocyanine dye “DiI”

    PubMed Central

    Cheng, Connie; Trzcinski, Olivia; Doering, Laurie C.

    2014-01-01

    Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system. PMID:24847216

  9. Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

    PubMed Central

    Goodwin, P M; Johnson, M E; Martin, J C; Ambrose, W P; Marrone, B L; Jett, J H; Keller, R A

    1993-01-01

    Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis. Images PMID:8451182

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

    PubMed Central

    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.

    2000-01-01

    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. PMID:11010903

  11. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    PubMed

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. PMID:26629954

  12. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    NASA Astrophysics Data System (ADS)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

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

    USGS Publications Warehouse

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

    2006-01-01

    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

  14. The relationship between the color of carious dentin stained with a caries detector dye and bacterial infection.

    PubMed

    Iwami, Yukiteru; Shimizu, Ayako; Narimatsu, Masahiro; Kinomoto, Yoshifumi; Ebisu, Shigeyuki

    2005-01-01

    This in vitro study aimed to design a method for the objective evaluation of carious dentin using numerical values. This study also investigated the relationship between the color of carious dentin stained with a caries detector dye using this objective method and the rate of bacterial detection as detected by a polymerase chain reaction (PCR). In 15 molars with occlusal dentin caries and three extracted sound molars, dentin was removed in multiple steps with 300 microm removed each step. Before and after every removal, images of a color-matching sticker and carious surfaces stained with a caries detector dye were acquired simultaneously using a CCD camera and dentinal tissue samples were removed with a round bur. Next, corrected L*, a* and b* values of the carious surfaces (CIE 1976 L*a*b* color system) were calculated from the color changes of the stickers in the images. In addition, bacterial DNA in the dentinal tissue was detected by PCR. From evaluations of the receiver operating characteristic curves for the L*, a* and b* values, the L* value was determined to be a more useful parameter than a* or b* for detecting bacterial infection using the caries detector dye. The bacterial detection rates of carious dentin decreased as the L* values of carious dentin stained with the dye increased. When the L* values were more than 60, the dentin had no bacterial infection. This study clarified the relationship between the colors of lesions stained with a caries detector dye and the rates of bacterial detection. PMID:15765962

  15. Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

    PubMed Central

    Hayashi-Takanaka, Yoko; Stasevich, Timothy J.; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2014-01-01

    To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye∶protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red). PMID:25184362

  16. Particle Image Velocimetry Applications Using Fluorescent Dye-Doped Particles

    NASA Technical Reports Server (NTRS)

    Petrosky, Brian J.; Maisto, Pietro; Lowe, K. Todd; Andre, Matthieu A.; Bardet, Philippe M.; Tiemsin, Patsy I.; Wohl, Christopher J.; Danehy, Paul M.

    2015-01-01

    Polystyrene latex sphere particles are widely used to seed flows for velocimetry techniques such as Particle Image Velocimetry (PIV) and Laser Doppler Velocimetry (LDV). These particles may be doped with fluorescent dyes such that signals spectrally shifted from the incident laser wavelength may be detected via Laser Induced Fluorescence (LIF). An attractive application of the LIF signal is achieving velocimetry in the presence of strong interference from laser scatter, opening up new research possibilities very near solid surfaces or at liquid/gas interfaces. Additionally, LIF signals can be used to tag different fluid streams to study mixing. While fluorescence-based PIV has been performed by many researchers for particles dispersed in water flows, the current work is among the first in applying the technique to micron-scale particles dispersed in a gas. A key requirement for such an application is addressing potential health hazards from fluorescent dyes; successful doping of Kiton Red 620 (KR620) has enabled the use of this relatively safe dye for fluorescence PIV for the first time. In this paper, basic applications proving the concept of PIV using the LIF signal from KR620-doped particles are exhibited for a free jet and a twophase flow apparatus. Results indicate that while the fluorescence PIV techniques are roughly 2 orders of magnitude weaker than Mie scattering, they provide a viable method for obtaining data in flow regions previously inaccessible via standard PIV. These techniques have the potential to also complement Mie scattering signals, for example in multi-stream and/or multi-phase experiments.

  17. Several hemicyanine dyes as fluorescence chemosensors for cyanide anions.

    PubMed

    Liang, Muhan; Wang, Kangnan; Guan, Ruifang; Liu, Zhiqiang; Cao, Duxia; Wu, Qianqian; Shan, Yanyan; Xu, Yongxiao

    2016-05-01

    Four hemicyanine dyes as chemosensors for cyanide anions were synthesized easily. Their photophysical properties and recognition properties for cyanide anions were investigated. The results indicate that all the dyes can recognize cyanide anions with obvious color, absorption and fluorescence change. The recognition mechanism analysis basing on in situ (1)H NMR and Job plot data indicates that to the compounds with hydroxyl group, the recognition mechanism is intramolecular hydrogen bonding interaction. However, to the compounds without hydroxyl group, cyanide anion is bonded to carbon-carbon double bond in conjugated bridge and induces N(+)CH3 to neutral NCH3. Fluorescence of the compounds is almost quenched upon the addition of cyanide anions. PMID:26921604

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

    PubMed

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

    2008-02-14

    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

  19. Synthesis, spectroscopic characteristic of novel fluorescent dyes of pyrazoline compounds

    NASA Astrophysics Data System (ADS)

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

    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.

  20. Fluorescence dye tagging scheme for mercury quantification and speciation

    DOEpatents

    Jiao, Hong; Catterall, Hannah

    2015-09-22

    A fluorescent dye or fluorophore capable of forming complexes with mercury comprises 6,8-difluoro-7-hydroxy-2-oxo-2H-chromene-3-carboxylate amide, wherein the amide is formed by reacting the succinimidyl ester (Pacific Blue.TM.) with an amino acid containing a thiol group, such as cysteine or glutathione. Mercury complexes of the fluorophore fluoresce when excited by a UV or violet laser diode, and the detected intensity can be calibrated to quantify the concentration of mercury in a sample reacted with the fluorophore.

  1. Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Otani, Mieko; Taniguchi, Taizo; Sakai, Akiko; Seta, Jouji; Kadoyama, Keiichi; Nakamura-Hirota, Tooru; Matsuyama, Shogo; Sano, Keiji; Takano, Masaoki

    2011-07-01

    We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4-5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS. PMID:21384102

  2. Near-infrared fluorescent dyes for fiber optic sensing

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Kim, Jun Seok; Medou-Ovono, Martial; Strekowski, Lucjan

    2005-05-01

    Fiber optic sensing requires the use of molecular probes such as fluorescent dyes or indicators that can be induced during analysis to produce a detectable spectral change. Spectroscopic techniques have long been applied to the determination of analytical and bioanalytical measurements using fiber optic sensors; however, relatively few studies have been reported utilizing near-infrared (NIR) absorbing chromophores. This longer wavelength region of the electromagnetic spectrum is more advantageous because of the inherently lower background interference and the high molar absorptivities of NIR absorbing chromophores. Low background interference is especially important in samples containing a complex matrix. The design and operation of an NIR probe are similar to that of conventional UV-visible probes. In principle optical fiber or other optical sensors can be made selective to a particular analyte. The selectivity will be determined primarily by the selectivity of the sensor dye and by the nature of the matrix entrapping the dye if the probe is non-covalently attached. This presentation discusses the development of different NIR dyes for fiber optic sensor applications. Examples are given for determining basic analytical properties, e.g., pH, metal ion concentration, and solvent hydrophobicity. Similarly, NIR dyes are very useful for bioanalytical probes (immunochemistry, etc.) as well.

  3. Fluorescence lifetime imaging with near-infrared dyes

    NASA Astrophysics Data System (ADS)

    Becker, Wolfgang; Shcheslavskiy, Vladislav

    2013-02-01

    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.

  4. Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms.

    PubMed

    Davey, H M; Kell, D B

    1997-08-01

    Flow cytometry is a rapid method for measuring the optical properties of individual cells. The technique has found great utility in the study of mammalian cells, but microbiological applications have been more limited. We here show that UV-excited fluorescent whitening agents, in particular Tinopal CBS-X, are effective stains for both vegetative microbial cells and for spores of Gram-positive bacteria. Pretreatment of samples with ethanol speeds the staining process. Under favourable conditions, Tinopal CBS-X may be used to discriminate among organisms, a fact that may be useful when screening for a target microorganism against a high biological background. PMID:9266751

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

    PubMed

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

    1976-07-01

    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. PMID:61736

  6. Polyacrylamide films as a tool for investigating qualitative and quanitative aspects of the staining of glycosaminoglycans with basic dyes.

    PubMed

    Tas, J

    1977-05-01

    With the introduction of model films of polyacrylamide gel into which purified glycosaminogly cans (GAGs) have been 'incorporated', the direct recording of metachromatic spectra with virtually no interference of the corresponding orthochromatic peaks has become possible. Because this model system yields situations comparable to those of stained sections under the microscope, it is well suited for investigating qualitative and quantitative aspects of histochemical staining procedures. Previous model experiments have shown that under aqueous conditions only minor differences can be observed between the metachromatic peaks of different GAGs complexed with a suitable dye (e.g. Toluidine Blue O, Thionin, Safranin O, Cresyl Violet, Cystal Violet). In non-aqueous media, such as glycerol and ethylene glycol, the complexes with Toluidine Blue O revealed a special pattern for heparin, having a metachromatic peak (517 nm) about 30 nm lower than that of all other GAGs. This observation has formed the basis of a method for the qualitative microspectrophotometric detection of heparin in situ which was worked out by combining model film experiments with microspectrophotometric data obtained from rat mast cells. Since only a limited number of cells in necessary for obtaining reliable data with this method, the presence of heparin in the cytoplasmic granules of normal human mast cells and basophilic granulocytes could thus be proved directly. Alcian Blue 8GX, another basic dye frequently use in GAG histochemistry, has also been investigated with polyacrylamide films. In contrast to the metachromatic dyes, the rate of staining with Alcian Blue depends to a large extent on the rate of penetration of the dye into the model films. The rate of penetration is also a phenomenon of great importance for dye binding in situ, where complex basic protein molecules may form a barrier for the Alcian Blue molecules. The model film studies performed so far have yielded conditions that provide

  7. Treatment of Port-Wine Stains with Flash Lamp Pumped Pulsed Dye Laser on Indian Skin: A Six Year Study

    PubMed Central

    Thajudheen, Chandroth Ponnambath; Jyothy, Kannangath; Priyadarshini, Arul

    2014-01-01

    Context: Port-wine stain (PWS) is one of the commonly encountered congenital cutaneous vascular lesions, with an equal sex distribution. Pulsed dye lasers (PDL) have revolutionized the treatment of both congential and acquired cutaneous vascular lesions. The pulsed dye lasers owing to its superior efficacy and safety profile have become the gold standard for the management of port-wine stains. Aims: To evaluate the efficacy and side effects of pulsed dye laser for the management of Port-wine stain on Indian skin. Materials and Methods: Seventy five patients of Fitzpatrick skin types IV&V with PWS underwent multiple treatments with PDL (V beam-Candela) over a period of six years at monthly intervals. Laser parameters were wavelength 595nm, spot sizes 7-10mm, fluence 6-12 j/cm2, pulse duration 0.45-10ms, along with cryogen cooling. Serial photographs were taken before and after every session. Clinical improvement scores of comparable photographs using a quartile grading (o=<20%, 1=21-40%, 2=41-60%, 3=61-80%, 4=>80%) were judged independently by two dermatologists after the series of treatment. Minimum number of treatments was 6 and maximum 17. They were followed up at six monthly intervals to observe re darkening of PWS. Results: No patient showed total clearance.Grade3 improvement was observed in 70 % of children and 50% of adults after 8-10 sessions. Children showed better and faster response than adults. Thirty percent of patients developed post inflammatory hyper pigmentation which resolved over a period of six to eight weeks. Two patients had superficial scarring due to stacking of pulses. None of the patients showed re darkening of PWS till now. Conclusion: Pulsed dye laser is an effective and safe treatment for port-wine stain in Indian skin. PMID:24761097

  8. Deterministically Polarized Fluorescence from Single Dye Molecules Aligned in Liquid Crystal Host

    SciTech Connect

    Lukishova, S.G.; Schmid, A.W.; Knox, R.; Freivald, P.; Boyd, R. W.; Stroud, Jr., C. R.; Marshall, K.L.

    2005-09-30

    We demonstrated for the first time to our konwledge deterministically polarized fluorescence from single dye molecules. Planar aligned nematic liquid crystal hosts provide deterministic alignment of single dye molecules in a preferred direction.

  9. Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry.

    PubMed

    Freer, Giulia

    2014-01-01

    The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining. PMID:24908309

  10. Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection*

    PubMed Central

    Butt, R. Hussain; Coorssen, Jens R.

    2013-01-01

    Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost. PMID:24043422

  11. Emission Lifetimes of a Fluorescent Dye under Shock Compression.

    PubMed

    Liu, Wei-long; Bassett, Will P; Christensen, James M; Dlott, Dana D

    2015-11-01

    The emission lifetimes of rhodamine 6G (R6G) were measured under shock compression to 9.1 GPa, with the dual intents of better understanding molecular photophysics in extreme environments and assessing the usefulness of fluorescence lifetime microscopy to measure spatially dependent pressure distributions in shocked microstructured media. R6G was studied as free dye dissolved in poly(methyl methacrylate) (PMMA), or dye encapsulated in silica microparticles suspended in PMMA. Thin layers of these materials in impedance-matched geometries were subjected to planar single-stage shocks created by laser-driven flyer plates. A synchronized femtosecond laser excited the dye at selected times relative to flyer plate arrival and the emission lifetimes were measured with a streak camera. Lifetimes decreased when shocks arrived. The lifetime decrease was attributed to a shock-induced enhancement of R6G nonradiative relaxation. At least part of the relaxation involved shock-enhanced intersystem crossing. For free dye in PMMA, the lifetime decrease during the shock was shown to be a linear function of shock pressure from 0 to 9 GPa, with a slope of -0.22 ns·GPa(-1). The linear relationship makes it simple to convert lifetimes into pressures. Lifetime measurements in shocked microenvironments may be better than emission intensity measurements, because lifetimes are sensitive to the surrounding environment, but insensitive to intensity variations associated with the motion and optical properties of a dynamically changing structure. PMID:26469397

  12. Laser velocimetry with fluorescent dye-doped polystyrene microspheres.

    PubMed

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

    2013-04-15

    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 μm 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

  13. Microarray Analysis of Port Wine Stains Before and After Pulsed Dye Laser Treatment

    PubMed Central

    Laquer, Vivian T.; Hevezi, Peter A.; Albrecht, Huguette; Chen, Tina S.; Zlotnik, Albert; Kelly, Kristen M.

    2014-01-01

    Background and Objectives Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Materials and Methods Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. Results There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. Conclusion In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were

  14. High frequency application of nanosecond pulsed electric fields alters cellular membrane disruption and fluorescent dye uptake

    NASA Astrophysics Data System (ADS)

    Steelman, Zachary A.; Tolstykh, Gleb P.; Beier, Hope T.; Ibey, Bennett L.

    2016-03-01

    Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a "threshold" point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

  15. DiOlistics: Delivery of Fluorescent Dyes into Cells

    PubMed Central

    Sherazee, Nyssa; Alvarez, Veronica A.

    2013-01-01

    DiOlistic labeling utilizes a particle-mediated delivery system to incorporate dye into cells. Because of its random nature, this technique generates sparse fluorescent labeling which is well suited for the study of neuronal dendritic branching and dendritic spine morphology. DiOlistics is a quick, reliable and nontoxic method that can be used in combination with other techniques such as immunostaining, biolistic DNA transfection, and retrograde tracing. In this article, we describe the methods for diOlistic labeling of neurons from rodent brain slices using DiI and the imaging of neuronal and synaptic morphology using confocal microscopy. PMID:23104356

  16. A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

    PubMed

    Rabilloud, T; Strub, J M; Luche, S; van Dorsselaer, A; Lunardi, J

    2001-05-01

    A comparison between two fluorescent metal chelates for staining proteins separated by electrophoresis has been carried out. One of these chelates is ruthenium II tris (bathophenanthroline disulfonate) and the other is commercial Sypro Ruby. Both can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. The sensitivity and homogeneity of the stains and the interference with mass spectrometry analysis have been investigated. It appears that both stains perform similarly for protein detection, while ruthenium II tris (bathophenanthroline disulfonate) performs better for mass spectrometry analyses and as cost-effectiveness ratio. However, Sypro Ruby is easier to use as a stain. PMID:11678039

  17. Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser.

    PubMed

    Trägårdh, J; Robb, G; Amor, R; Amos, W B; Dempster, J; McConnell, G

    2015-09-01

    We have studied the wavelength dependence of the two-photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin- and cellulose-staining dye Calcofluor White and Alexa Fluor 350, in the visible and near-infrared wavelength range (540-800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two-photon microscopy. We also find that although the rate of photo-bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution-based measurements. PMID:25946127

  18. Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

    NASA Astrophysics Data System (ADS)

    Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

    2006-02-01

    Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

  19. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    PubMed Central

    2011-01-01

    A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting. PMID:22011343

  20. Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining.

    PubMed

    Verma, Mayank; Murkonda, Bhavani Sr; Asakura, Yoko; Asakura, Atsushi

    2016-01-01

    Skeletal muscle is a highly ordered yet complex tissue containing several cell types that interact with each other in order to maintain structure and homeostasis. It is also a highly regenerative tissue that responds to damage in a highly intricate but stereotypic manner, with distinct spatial and temporal kinetics. Proper examination of this process requires one to look at the three-dimensional orientation of the cellular and subcellular components, which can be accomplished through tissue clearing. While there has been a recent surge of protocols to study biology in whole tissue, it has primarily focused on the nervous system. This chapter describes the workflow for whole mount analysis of murine skeletal muscle for LacZ reporters, fluorescent reporters and immunofluorescence staining. Using this technique, we are able to visualize LacZ reporters more effectively in deep tissue samples, and to perform fluorescent imaging with a depth greater than 1700 μm. PMID:27492170

  1. Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria.

    PubMed

    Zeder, M; Pernthaler, J

    2009-09-01

    Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrated various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in bright-field and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multispot focus measurements, and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions. PMID:19658173

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

    PubMed Central

    Kucki, Melanie; Fuhrmann-Lieker, Thomas

    2012-01-01

    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. PMID:21865248

  3. Clinical approved fluorescent dyes coupled to endomicroscopy for in vivo diagnostic of peritoneal carcinomatosis

    NASA Astrophysics Data System (ADS)

    Abbaci, Muriel; Dartigues, Peggy; Soufan, Ranya; De Leeuw, Frederic; Fabre, Monique; Laplace-Builhé, Corinne

    2015-03-01

    Peritoneal carcinomatosis is metastatic stage aggravating digestive, gynecological or bladder cancer dissemination and the preoperative evaluation of lesions remains difficult. There is therefore a need for minimal invasive innovative techniques to establish a precise preoperative assessment of cancer peritoneal cavity. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of the microarchitecture of tissues during an endoscopy. The PERSEE project proposes new developments in robotics and pCLE for the exploration of the peritoneal cavity during laparoscopy. Two fluorescent dyes, Patent blue V and Indocyanine green have been evaluated on human ex vivo samples to improve the contrast of pCLE images. For a future implementation in clinical study, two topically staining protocols operable in vivo have been validated on 70 specimens from 25 patients with a peritoneal carcinomatosis. The specimens were then imaged by pCLE with an optical probe designed for the application. A histo-morphological correlative study was performed on 350 pCLE images and 70 standard histological preparations. All images were interpreted in a random way by two pathologists. Differential histological diagnostics such as normal peritoneum or pseudomyxoma could be recognized on fluorescence images. The statistical analysis of the correlative study is underway. These dyes already approved for human use are interesting for pCLE imaging because some micromorphological criteria look like to conventional histology and are readable by pathologist. Thus pCLE images using both dyes do not require a specific semiology unlike to what is described in the literature, for pCLE associated with fluorescein for the in vivo imaging of pancreatic cysts.

  4. A comparison of direct fluorescent antibody and Giemsa staining for the post-mortem diagnosis of anaplasmosis.

    PubMed

    Johnston, L A; Trueman, K F; Leatch, G; Wilson, A J

    1980-03-01

    Direct fluorescent antibody (DFA) and Giemsa staining of Anaplasma marginale were compared in smears collected serially at post-mortem (PM) from 11 experimentally infected calves. Once smears had been prepared and air-dried they could be held for at least 5 days before staining with either technique with no noticeable change in staining quality. DFA staining was more sensitive in detecting anaplasms in smears than Giemsa staining. Anaplasma spp could be differentiated from Babesia bovis and B. bigemina by DFA staining but there were cross reactions between A. marginale and A. centrale. Blood smears prepared from subcutaneous vessels in the legs provided better diagnostic material than kidney, heart and lung smears. Brain smears were not suitable for PM diagnosis using either staining technique. PMID:7002139

  5. Evaluation of fluorescent dye degradation indirectly induced by x-ray ionizing radiation.

    PubMed

    Benevides, Clayton Augusto; Duarte de Menezes, Frederico; de Araujo, Renato E

    2015-08-01

    This work evaluated the fluorescent dye degradation indirectly induced by ionizing radiation with high energy photons (50 keV). Aqueous gels of agarose with low concentrations of Rhodamine 6G and Fluorescein were submitted to doses of x-ray radiation up to 200 Gy. The dye degradation was analyzed by fluorescence spectroscopy, using an excitation light-emitting diode with a peak wavelength of 462 nm. A rate equation model of fluorophores and radicals' species populations was developed to describe the degradation time behavior of the fluorescent solutions. The model suggests fluorescent dyes should be used in dosimetry. PMID:26368112

  6. Dimer formation effect on the red-shift in fluorescent spectra of dye solutions

    NASA Astrophysics Data System (ADS)

    Sukprasong, Saksit; Manjit, Yongyut; Limpichaipanit, Apichart; Ngamjarurojana, Athipong

    2015-07-01

    The red-shift on fluorescent dyes spectra at high concentration was investigated by laser induce fluorescence technique. In this research, the fluorescent dyes (Rhodamine 6G, Rhodamine B, Fluorescein and Bromofluorescein) were used. The sample solutions were prepared with methanol solvent in the concentration range of 10-5 to 10-3 Molar and the temperature of sample solution was controlled at 25 °C by temperature control chamber. Then, the sample solution was illuminated by violet laser (405 nm) excitation source and the fluorescence spectra were recorded by CCD spectrometer. The result showed that the fluorescence spectra of all fluorescent dye solutions were dependent on concentration of fluorescent dyes. The position of fluorescence maximum intensity was shifted to a higher wavelength (red-shift) when the concentration increased because the dimer formation rate increases with increasing concentration, but the shifting of wavelength for each fluorescent dye solutions was different, which suggests the different rate of formation of dimer molecules in each fluorescent dye solutions.

  7. A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Wu, Changfeng; Barnhill, Hannah; Liang, Xiaoping; Wang, Qian; Jiang, Huabei

    2005-11-01

    A fluorescent probe based on bionanoparticle cowpea mosaic virus has been developed for near-infrared fluorescence tomography. A unique advantage of this probe is that over 30 dye molecules can be loaded onto each viral nanoparticle with an average diameter of 30 nm, making high local dye concentration (∼1.8 mM) possible without significant fluorescence quenching. This ability of high loading of local dye concentration would increase the signal-to-noise ratio considerably, thus sensitivity for detection. We demonstrate successful tomographic fluorescence imaging of a target containing the virus-dye nanoparticles embedded in a tissue-like phantom. Tomographic fluorescence data were obtained through a multi-channel frequency-domain system and the spatial maps of fluorescence quantum yield were recovered with a finite-element-based reconstruction algorithm.

  8. Non-specific fluorescent whitener stains in the rapid recognition of pulmonary dirofilariasis: a report of 20 cases.

    PubMed Central

    Green, L. K.; Ansari, M. Q.; Schwartz, M. R.; Ro, J. Y.; Alpert, L. C.

    1994-01-01

    BACKGROUND--Solitary lung nodules in humans caused by the dog parasite Dirofilaria immitis are steadily increasing in number. The organisms can be easily missed in haematoxylin and eosin stained tissue when they are degenerated and pale staining. METHODS--The value of Tinopal CBS-X (TCBS-X) and Calcofluor white (CFW), two rapid, inexpensive, simple non-specific fluorescent whitening stains, were assessed in the identification of these worms. Deparaffinised rehydrated tissue slides prepared from the pulmonary nodules were stained for one minute in 1% w/v aqueous solutions of TCBS-X or CFW, counterstained, coverslipped, and viewed with a fluorescent microscope. RESULTS--The stains demonstrated the intact worm and worm fragments in 20 cases of pulmonary dirofilariasis collected from hospitals in Houston. The filariae and fragments of filariae stained bright green while the background tissue stained red, delineating the internal structures of the worm. CONCLUSIONS--Dirofilariasis should be included in the differential diagnosis of subpleural masses, and non-specific fluorescent whitening stains can help in the rapid recognition of the fragmented organism in cytological or surgical material. Images PMID:7517073

  9. Hollow mesoporous silica nanoparticles for intracellular delivery of fluorescent dye

    PubMed Central

    2011-01-01

    In this study, hollow mesoporous silica nanoparticles (HMSNs) were synthesized using the sol-gel/emulsion approach and its potential application in drug delivery was assessed. The HMSNs were characterized, by transmission electron microscopy (TEM), Scanning Electron Microscopy (SEM), nitrogen adsorption/desorption and Brunauer-Emmett-Teller (BET), to have a mesoporous layer on its surface, with an average pore diameter of about 2 nm and a surface area of 880 m2/g. Fluorescein isothiocyanate (FITC) loaded into these HMSNs was used as a model platform to assess its efficacy as a drug delivery tool. Its release kinetic study revealed a sequential release of FITC from the HMSNs for over a period of one week when soaked in inorganic solution, while a burst release kinetic of the dye was observed just within a few hours of soaking in organic solution. These FITC-loaded HMSNs was also found capable to be internalized by live human cervical cancer cells (HeLa), wherein it was quickly released into the cytoplasm within a short period of time after intracellular uptake. We envision that these HMSNs, with large pores and high efficacy to adsorb chemicals such as the fluorescent dye FITC, could serve as a delivery vehicle for controlled release of chemicals administered into live cells, opening potential to a diverse range of applications including drug storage and release as well as metabolic manipulation of cells. PMID:21208421

  10. Modelling of microcracks image treated with fluorescent dye

    NASA Astrophysics Data System (ADS)

    Glebov, Victor; Lashmanov, Oleg U.

    2015-06-01

    The main reasons of catastrophes and accidents are high level of wear of equipment and violation of the production technology. The methods of nondestructive testing are designed to find out defects timely and to prevent break down of aggregates. These methods allow determining compliance of object parameters with technical requirements without destroying it. This work will discuss dye penetrant inspection or liquid penetrant inspection (DPI or LPI) methods and computer model of microcracks image treated with fluorescent dye. Usually cracks on image look like broken extended lines with small width (about 1 to 10 pixels) and ragged edges. The used method of inspection allows to detect microcracks with depth about 10 or more micrometers. During the work the mathematical model of image of randomly located microcracks treated with fluorescent dye was created in MATLAB environment. Background noises and distortions introduced by the optical systems are considered in the model. The factors that have influence on the image are listed below: 1. Background noise. Background noise is caused by the bright light from external sources and it reduces contrast on the objects edges. 2. Noises on the image sensor. Digital noise manifests itself in the form of randomly located points that are differing in their brightness and color. 3. Distortions caused by aberrations of optical system. After passing through the real optical system the homocentricity of the bundle of rays is violated or homocentricity remains but rays intersect at the point that doesn't coincide with the point of the ideal image. The stronger the influence of the above-listed factors, the worse the image quality and therefore the analysis of the image for control of the item finds difficulty. The mathematical model is created using the following algorithm: at the beginning the number of cracks that will be modeled is entered from keyboard. Then the point with random position is choosing on the matrix whose size is

  11. Toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three popular DNA intercalating fluorescent dyes.

    PubMed

    Sayas, Enric; García-López, Federico; Serrano, Ramón

    2015-09-01

    We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells. PMID:26108459

  12. Identification Of Natural Dyes On Archaeological Textile Objects Using Laser Induced Fluorescent Technique

    SciTech Connect

    Abdel-Kareem, O.; Eltokhy, A.; Harith, M. A.

    2011-09-22

    This study aims to evaluate the use of Laser Fluorescent as a non-destructive technique for identification of natural dyes on archaeological textile objects. In this study wool textile samples were dyed with 10 natural dyes such as cochineal, cutch, henna, indigo, Lac, madder, safflower, saffron, sumac and turmeric. These dyes common present on archaeological textile objects to be used as standard dyed textile samples. These selected natural dyes will be used as known references that can be used a guide to identify unknown archaeological dyes. The dyed textile samples were investigated with laser radiation in different wavelengths to detect the best wavelengths for identification each dye. This study confirms that Laser Florescent is very useful and a rapid technique can be used as a non-destructive technique for identification of natural dyes on archaeological textile objects. The results obtained with this study can be a guide for all conservators in identification of natural organic dyes on archaeological textile objects.

  13. Identification Of Natural Dyes On Archaeological Textile Objects Using Laser Induced Fluorescent Technique

    NASA Astrophysics Data System (ADS)

    Abdel-Kareem, O.; Eltokhy, A.; Harith, M. A.

    2011-09-01

    This study aims to evaluate the use of Laser Fluorescent as a non-destructive technique for identification of natural dyes on archaeological textile objects. In this study wool textile samples were dyed with 10 natural dyes such as cochineal, cutch, henna, indigo, Lac, madder, safflower, saffron, sumac and turmeric. These dyes common present on archaeological textile objects to be used as standard dyed textile samples. These selected natural dyes will be used as known references that can be used a guide to identify unknown archaeological dyes. The dyed textile samples were investigated with laser radiation in different wavelengths to detect the best wavelengths for identification each dye. This study confirms that Laser Florescent is very useful and a rapid technique can be used as a non-destructive technique for identification of natural dyes on archaeological textile objects. The results obtained with this study can be a guide for all conservators in identification of natural organic dyes on archaeological textile objects.

  14. Estrogen receptor-targeted optical imaging of breast cancer cells with near-infrared fluorescent dye

    NASA Astrophysics Data System (ADS)

    Jose, Iven; Deodhar, Kodand; Chiplunkar, Shuba V.; Patkar, Meena

    2010-02-01

    Molecular imaging provides the in vivo characterization of cellular molecular events involved in normal and pathologic processes. With the advent of optical molecular imaging, specific molecules, proteins and genes may be tagged with a luminescent reporter and visualized in small animals. This powerful new tool has pushed in vivo optical imaging to the forefront as it allows for direct determination of drug bio-distribution and uptake kinetics as well as an indicator of biochemical activity and drug efficacy. Although optical imaging encompasses diverse techniques and makes use of various wavelengths of light, a great deal of excitement in molecular research lies in the use of tomographic and fluorescence techniques to image living tissues with near-infrared (NIR) light. Nonionizing, noninvasive near-infrared optical imaging has great potential to become promising alternative for breast cancer detection. Fluorescence spectroscopy studies of human tissue suggest that a variety of lesions show distinct fluorescence spectra compared to those of normal tissue. It has also been shown that exogenous dyes exhibit selective uptake in neoplastic lesions and may offer the best contrast for optical imaging. Use of exogenous agents would provide fluorescent markers, which could serve to detect embedded tumors in the breast. In particular, the ability to monitor the fluorescent yield and lifetime may also enable biochemical specificity if the fluorophore is sensitive to a specific metabolite, such as oxygen. As a first step, we have synthesized and characterized one such NIR fluorescent dye conjugate, which could potentially be used to detect estrogen receptors (ER)[2] . The conjugate was synthesized by ester formation between 17-β estradiol and a hydrophilic derivative of indocyanine green (ICG) cyanine dye, bis-1, 1-(4-sulfobutyl) indotricarbocyanine-5- carboxylic acid, sodium salt. The ester formed was found to have an extra binding ability with the receptor cites as

  15. Application of fluorescent dyes for some problems of bioelectromagnetics

    NASA Astrophysics Data System (ADS)

    Babich, Danylo; Kylsky, Alexandr; Pobiedina, Valentina; Yakunov, Andrey

    2016-04-01

    Fluorescent organic dyes solutions are used for non-contact measurement of the millimeter wave absorption in liquids simulating biological tissue. There is still not any certain idea of the physical mechanism describing this process despite the widespread technology of microwave radiation in the food industry, biotechnology and medicine. For creating adequate physical model one requires an accurate command of knowledge concerning to the relation between millimeter waves and irradiated object. There were three H-bonded liquids selected as the samples with different coefficients of absorption in the millimeter range like water (strong absorption), glycerol (medium absorption) and ethylene glycol (light absorption). The measurements showed that the greatest response to the action of microwaves occurs for glycerol solutions: R6G (building-up luminescence) and RC (fading luminescence). For aqueous solutions the signal is lower due to lower quantum efficiency of luminescence, and for ethylene glycol — due to the low absorption of microwaves. In the area of exposure a local increase of temperature was estimated. For aqueous solutions of both dyes the maximum temperature increase is about 7° C caused with millimeter waves absorption, which coincides with the direct radio physical measurements and confirmed by theoretical calculations. However, for glycerol solution R6G temperature equivalent for building-up luminescence is around 9° C, and for the solution of ethylene glycol it's about 15°. It is assumed the possibility of non-thermal effect of microwaves on the different processes and substances. The application of this non-contact temperature sensing is a simple and novel method to detect temperature change in small biological objects.

  16. Thermoresponsive Polymer and Fluorescent Dye Hybrids for Tunable Multicolor Emission.

    PubMed

    Kim, Joo-Ho; Jung, Yongseok; Lee, Dajung; Jang, Woo-Dong

    2016-05-01

    Fully reversible emission color change is achieved by blending a thermoresponsive polymer with dye hybrids. The emission color can be tuned by changing the mixing ratio of each polymer-dye hybrid. PMID:26990858

  17. Method and apparatus for staining immobilized nucleic acids

    DOEpatents

    Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

    2000-01-01

    A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

  18. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

    PubMed Central

    Huang, Chao; Kaza, Aditya K.; Hitchcock, Robert W.; Sachse, Frank B.

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  19. Molecular design and synthesis of a pH independent and cell permeant fluorescent dye and its applications.

    PubMed

    Jiao, Xiaojie; Liu, Chang; Huang, Kun; Zhang, Siwen; He, Song; Zhao, Liancheng; Zeng, Xianshun

    2015-06-21

    Fluorescent dyes have played crucial roles in the field of molecular imaging as fluorescent fluorophores. In this work, a novel water-soluble and pH-independent fluorescent xanthene dye, a hydroxyl regioisomeric 3',4'-benzorhodol, has been designed and synthesized. Compared with those of rhodol dyes, the absorption (ca. 570 nm) and maximum emission (ca. 620 nm) of the dye are largely red-shifted. Due to its ring-opened zwitterion structure in water media, the dye showed good membrane permeability and distributed in the whole cell cytoplasm upon incubation with live cells. Meanwhile, the dye could be easily modified to probes. The hydrazide derivative of the dye exhibited an excellent Hg(2+) selectivity over other relevant metal ions with a detection limit down to 3 nM. Thus, the excellent fluorescence properties and chemical properties of the dye allow it to be designed as a fluorescent chemosensor and biomarker for biological applications. PMID:25990913

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

    PubMed Central

    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.

    2011-01-01

    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. PMID:21755981

  1. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging.

    PubMed

    Reisch, Andreas; Klymchenko, Andrey S

    2016-04-01

    Speed, resolution and sensitivity of today's fluorescence bioimaging can be drastically improved by fluorescent nanoparticles (NPs) that are many-fold brighter than organic dyes and fluorescent proteins. While the field is currently dominated by inorganic NPs, notably quantum dots (QDs), fluorescent polymer NPs encapsulating large quantities of dyes (dye-loaded NPs) have emerged recently as an attractive alternative. These new nanomaterials, inspired from the fields of polymeric drug delivery vehicles and advanced fluorophores, can combine superior brightness with biodegradability and low toxicity. Here, we describe the strategies for synthesis of dye-loaded polymer NPs by emulsion polymerization and assembly of pre-formed polymers. Superior brightness requires strong dye loading without aggregation-caused quenching (ACQ). Only recently several strategies of dye design were proposed to overcome ACQ in polymer NPs: aggregation induced emission (AIE), dye modification with bulky side groups and use of bulky hydrophobic counterions. The resulting NPs now surpass the brightness of QDs by ≈10-fold for a comparable size, and have started reaching the level of the brightest conjugated polymer NPs. Other properties, notably photostability, color, blinking, as well as particle size and surface chemistry are also systematically analyzed. Finally, major and emerging applications of dye-loaded NPs for in vitro and in vivo imaging are reviewed. PMID:26901678

  2. Ultralow detection limits for an organic dye determined by fluorescence spectroscopy with laser diode excitation

    SciTech Connect

    Johnson, P.A.; Barber, T.E.; Smith, B.W.; Winefordner, J.D. )

    1989-04-15

    Fluorescence of IR-140, a laser dye in methanol solution, is excited by a semiconductor laser diode. Analytical figures of merit are compared for three different instrumental configurations, with the dye measured in a cuvette, a liquid jet, and a compact instrument. The best limit of detection, 46,000 molecules, was achieved with a liquid jet. Linear dynamic range was 6 orders of magnitude. The laser diode operates in the near-infrared region, resulting in low background fluorescence.

  3. Phthalocyanine dye as an extremely photostable and highly fluorescent near-infrared labeling reagent

    NASA Astrophysics Data System (ADS)

    Peng, Xinzhan; Draney, Daniel R.; Volcheck, William M.; Bashford, Gregory R.; Lamb, Donald T.; Grone, Daniel L.; Zhang, Yonghong; Johnson, Craig M.

    2006-02-01

    Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations in photostability. This compromises their performance in applications that require a photostable fluorescent reporting group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important for observing and tracking individual molecular events over extended period of time. We report in this paper an extremely photostable and highly fluorescent phthalocyanine dye, IRDye TM 700DX, as a near-infrared fluorescence labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g. Alexa Fluor"R"680, Cy TM 5.5, Cy TM 7 and IRDye TM 800CW dyes; and about 27 times more photostable than tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with IRDye TM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The IRDye TM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in Western blot and cytoblot assays.

  4. 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

    Netzel, T.L.; Nafisi, K.; Zhao, M.; Lenhard, J.R.; Johnson, I.

    1995-12-21

    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.

  5. Fluorescent silica nanoparticles containing covalently bound dyes for reporter, marker, and sensor applications

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Henary, Maged; Chapman, Gala; Emer, Kyle; Crow, Sidney

    2016-03-01

    Silica nanoparticles have proven to be useful in many bioanalytical and medical applications and have been used in numerous applications during the last decade. Combining the properties of silica nanoparticles and fluorescent dyes that may be used as chemical probes or labels can be relatively easy by simply soaking porous silica nanoparticles in a solution of the dye of interest. Under proper conditions the entrapped dye can stay inside the silica nanoparticle for several hours resulting in a useful probe. In spite of the relative durability of these probes, leaching can still occur. A much better approach is to synthesize silica nanoparticles that have the fluorescent dye covalently attached to the backbone structure of the silica nanoparticle. This can be achieved by using appropriately modified tetraethyl orthosilicate (TEOS) analogues during the silica nanoparticle synthesis. The molar ratio of TEOS and modified TEOS will determine the fluorescent dye load in the silica nanoparticle. Dependent on the chemical stability of the reporting dye either reverse micellar (RM) or Stöber method can be used for silica nanoparticle synthesis. If dye stability allows RM procedure is preferred as it results in a much easier control of the silica nanoparticle reaction itself. Also controlling the size and uniformity of the silica nanoparticles are much easier using RM method. Dependent on the functional groups present in the reporting dye used in preparation of the modified TEOS, the silica nanoparticles can be utilized in many applications such as pH sensor, metal ion sensors, labels, etc. In addition surface activated silica nanoparticles with reactive moieties are also excellent reporters or they can be used as bright fluorescent labels. Many different fluorescent dyes can be used to synthesize silica nanoparticles including visible and NIR dyes. Several bioanalytical applications are discussed including studying amoeba phagocytosis.

  6. Spectral Fluorescence Properties of an Anionic Oxacarbocyanine Dye in Complexes with Human Serum Albumin

    NASA Astrophysics Data System (ADS)

    Pronkin, P. G.; Tatikolov, A. S.

    2015-07-01

    The spectral fluorescence properties of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC) were studied in solutions and in complexes with human serum albumin (HSA). Interaction with HSA leads to a significant increase in the fluorescence of the dye. We studied quenching of the fluorescence of OCC in a complex with HSA by ibuprofen and warfarin. Data on quenching of fluorescence by ibuprofen indicate binding of the dye to binding site II of subdomain IIIA in the HSA molecule. Synchronous fluorescence spectra of human serum albumin in the presence of OCC showed that complexation with OCC does not lead to appreciable rearrangement of the protein molecule at the binding site.

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

    SciTech Connect

    Chang, Kaisyang; Force, R.K. )

    1993-01-01

    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.

  8. In Vitro Activity of Fluorescent Dyes against Asexual Blood Stages of Plasmodium falciparum

    PubMed Central

    Joanny, Fanny; Held, Jana

    2012-01-01

    Many successful antimicrobial drugs originate from synthetic dyes. This paper reports the in vitro activity of 14 fluorescent dyes against Plasmodium falciparum. Five of these dyes (Hoechst 33342, MitoRed, DiOC6, SYTO 9, and rhodamine B) show activity at a low nanomolar concentration against two P. falciparum strains in the histidine-rich protein 2 drug sensitivity assay, while toxicity in HeLa cells is low. These dyes may be a starting point for developing new drugs against P. falciparum. PMID:22850520

  9. A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

    1996-01-01

    A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

  10. A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

    PubMed Central

    Ovecka, Miroslav; Bahaji, Abdellatif; Muñoz, Francisco José; Almagro, Goizeder; Ezquer, Ignacio; Baroja-Fernández, Edurne; Li, Jun; Pozueta-Romero, Javier

    2012-01-01

    Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin. PMID:22899048

  11. Comparative analysis of heterochromatin distribution in wild and cultivated Abelmoschus species based on fluorescent staining methods.

    PubMed

    Merita, Keisham; Kattukunnel, Joseph John; Yadav, Shrirang Ramchandra; Bhat, Kangila Venkataramana; Rao, Satyawada Rama

    2015-03-01

    A comparative analysis of fluorochrome-binding pattern in nine taxa of Abelmoschus had shown that the type, amount and distribution pattern of heterochromatin were characteristic for each taxa. The fluorescent chromosome-binding sites obtained by chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) staining in all the nine species showed constitutive heterochromatin CMA(+), DAPI(+) and CMA(+)/DAPI(+). Large amount of heterozygosity was observed with regard to heterochromatin distribution pattern in all the taxa studied. The CMA(+)-binding sites are comparatively less than DAPI(+)-binding sites which is clearly evident as AT-rich regions are more than GC-rich regions in all the nine taxa analysed in Abelmoschus. These CMA(+) and DAPI(+)-binding sites apparently rise with the increased in chromosome numbers of the different species. This pattern of heterochromatin heterogeneity seems to be a general characteristic feature. Therefore, the differential pattern of distribution of GC- and AT-rich sequences might have played an important role in diversification of the genus Abelmoschus. Polyploidy is an important factor in the evolution of Abelmoschus and the sole reason for range in chromosome numbers in this genus. It may be noted that, though often, but not always, the increase of DNA is caused by an increase in the amount of heterochromatin, i.e. increase of non-coding sections indicating restructuring of the heterochromatin. Thus, cumulative small and direct numerical changes might have played a role in the speciation of Abelmoschus. PMID:25300590

  12. Far-Red Emitting Fluorescent Dyes for Optical Nanoscopy: Fluorinated Silicon-Rhodamines (SiRF Dyes) and Phosphorylated Oxazines.

    PubMed

    Kolmakov, Kirill; Hebisch, Elke; Wolfram, Thomas; Nordwig, Lars A; Wurm, Christian A; Ta, Haisen; Westphal, Volker; Belov, Vladimir N; Hell, Stefan W

    2015-09-14

    Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon-rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750-775 nm provides excellent imaging performance and suppresses re-excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far-red emitting dyes (photobleaching, optical resolution, and switch-off behavior) are discussed in detail and compared with those of some well-established fluorophores with similar spectral properties. PMID:26272226

  13. Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

  14. Treatment of resistant port wine stains (PWS) with pulsed dye laser and non-contact vacuum: a pilot study.

    PubMed

    Kautz, Gerd; Kautz, Ingrid; Segal, Jenny; Zehren, Sabrina

    2010-07-01

    The blanching of resistant port wine stains (PWS) with a pulsed dye laser (PDL) requires a large number of treatments, resulting in substantial discomfort to patients, many of them children. Pneumatic skin flattening (PSF - Serenity Pro) is a new technology that generates a vacuum over the skin and reduces pain in laser-based treatments of the skin, while creating contact between the skin and an upper window. The same technology can be utilized to increase skin blood fraction while operated in a non-contact mode. The objective of this study was to test the enhancement in the efficacy of PWS treatment with PDL and Serenity Pro while vacuum is being utilized in the non-contact, blood-enrichment mode. Fifteen patients with resistant PWS underwent 1-4 treatments (interval of 5-20 weeks) under general anesthesia with a 595-nm PDL at 10-14 J/cm(2), 1.5-3 ms pulse duration, and 7-mm spot size. Lesion blanching with DCD chilling and with vacuum were photographed and compared. Better blanching of various degrees was observed on resistant PWS with the blood-enrichment technique in seven out of 11 patients who returned for follow-up. There were no cases of decrease in efficacy. Blood enrichment with the Serenity Pro non-contact vacuum technology has the potential of enhancing the capability of treating resistant port wine stains in over 50% of cases. Further studies will better quantify the number of treatments necessary for better lesion clearance. The vacuum-assisted technique may be of particular importance in view of the fact that achieving complete lesion clearance remains a challenge in PWS treatments. PMID:20013138

  15. Non-invasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-loaded Perfluorocarbon Nanoparticles

    PubMed Central

    Akers, Walter J.; Kim, Chulhong; Berezin, Mikhail; Guo, Kevin; Fuhrhop, Ralph; Lanza, Gregory M.; Fischer, Georg M.; Daltrozzo, Ewald; Zumbusch, Andreas; Cai, Xin; Wang, Lihong V.; Achilefu, Samuel

    2010-01-01

    The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle but significant ways. Design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime (FLT) imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods. PMID:21171567

  16. Cis/trans Fluorescent Recognition by Naphthalimide Dyes ⊂ CB [7] Assembly.

    PubMed

    Li, Junyong; Gu, Xiaomin; Yuan, Xiaosheng; Qiu, Qiqi; Sun, Jie; Wang, Haibo

    2016-07-01

    A novel method to recognize cis/trans isomers was studied here. The naphthalimide dye as guest could bind with host cucurbit [7]uril (CB [7]) and 1:1 naphthalimide dye ⊂ CB [7] assembly was formed. Moreover, this assembly was used as a fluorescent probe to recognized Fumaric acid (FA) and maleic acid (MA) via fluorescence titration. Two carboxyls in MA are in the same side, they could form stable interaction with the assembly and the fluorescence intensity decreased obviously when naphthalimide dye ⊂ CB [7] was titrated by MA (nearly quenched in 1.5 equiv). But two carboxyls in FA are in opposite sides, the interaction between FA and the assembly was weak and not stable, and the fluorescence intensity changed inconspicuously when the assembly was titrated by FA. PMID:27130626

  17. Use of fluorescent NIR dyes in silica nanoparticles and as enzyme substrates in bioanalytical applications

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Chapman, Gala; Beckford, Garfield; Henary, Maged; Ellis, Holly

    2014-03-01

    Near-Infrared (NIR) absorbing carbocyanine dyes have been increasingly used in analytical, biological and medical fields as they can be useful for developing bioanalytical and biomedical methods. The utilization of the NIR spectral region (650-900 nm) is advantageous and is due to the inherently lower background interference and the high molar absorptivities of NIR chromophores. NIR dyes typically have relatively lower fluorescent quantum yield as compared to visible fluorophores, but much higher molar absorptivities which more than compensates for the lower quantum yields regarding detection limits. Fluorescence intensity of NIR dyes significantly increases by enclosing several dye molecules in silica nanoparticles. Self quenching may become a problem for carbocyanines at such high concentrations that may be present in the silica nanoparticles. Dyes that have large Stokes' shift can significantly decrease this problem. Increased Stokes' shift for carbocyanines dyes can be achieved by substituting meso position halogens with a linker containing aliphatic or aromatic amino moiety which also serves as a covalent linker for attaching the dye molecule to the nanoparticle backbone. The primary applications of these particles are for bright fluorescent labels to be used in bioanalytical applications such as immunochemistry, flow cytometry, etc. This work also discusses the use of NIR dyes as enzyme substrates. NIR dyes can be used as enzyme substrates and hence for characterization of enzyme activity. The well characterized alkenesulfonate monooxygenase enzyme was chosen for these studies. Carbocyanines containing alkylsulfonate moieties do not exhibit significant fluorescence change upon binding to biomolecules however otherwise identical NIR dye analogs that contain alkylaldehyde moiety at the same position do exhibit changes which can be utilized for characterization of alkenesulfonate monooxygenase enzyme activity using near infrared dyes as substrates. In this study

  18. Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis.

    PubMed

    Gibbons, Brandon A; Kharel, Prakash; Robinson, Lauren C; Synowicki, Ron A; Model, Michael A

    2016-05-15

    Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl(-)D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport. PMID:26997529

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

    PubMed Central

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

    2013-01-01

    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 (λAbs=782 nm, λFl=810 nm, ε = 261,000 M-1cm-1, ΦFl≈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). PMID:24019854

  20. Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction

    PubMed Central

    Fonin, Alexander V.; Sulatskaya, Anna I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.

    2014-01-01

    Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence

  1. Modulating fluorescence anisotropy of dye-labeled DNA without involving mass amplification.

    PubMed

    Pei, Xiaojing; Huang, Hongduan; Chen, Yang; Li, Chenxi; Liu, Feng; Li, Na

    2016-07-01

    Fluorescence anisotropy, known as a simple, homogeneous and cost-effective analytical technology, is an invaluable technique for studying the micro-environmental changes of the dye associated with the molecular interactions. An in-depth understanding of the variables affecting the fluorescence anisotropy signal can facilitate better experimental designs to effectively improve the analytical performance. This work is a follow-up effort in evaluating the factors that can significantly influence fluorescence anisotropy. We systematically studied fluorescence anisotropy of dsDNA with the changing length based on dye-DNA interactions, with the fluorophores in the end-labeling, the middle-site-labeling, and multiple number of labeling manners. The fluorescence anisotropy value and the base-pair response dynamic range could be expanded by labeling the fluorophores in the middle of dsDNA and increasing the number of labels on dsDNA. The C overhang configuration in the end-labeling manner could enhance the fluorescence anisotropy signal but not expand the base-pair response range. Results from all the labeling fluorophores reinforced the leveling-off effect, i.e., the fluorescence anisotropy signal does not response to the increased length of the DNA duplex when the length is larger than a critical number of base pairs. These findings provide perspectives about choosing appropriate fluorescent dyes and labeling sites for simple and universal fluorescence anisotropy designs in various applications. PMID:27154716

  2. Molecular Engineering of Thiazole Orange Dye: Change of Fluorescent Signaling from Universal to Specific upon Binding with Nucleic Acids in Bioassay.

    PubMed

    Lu, Yu-Jing; Deng, Qiang; Hou, Jin-Qiang; Hu, Dong-Ping; Wang, Zheng-Ya; Zhang, Kun; Luyt, Leonard G; Wong, Wing-Leung; Chow, Cheuk-Fai

    2016-04-15

    The universal fluorescent staining property of thiazole orange (TO) dye was adapted in order to be specific for G-quadruplex DNA structures, through the introduction of a styrene-like substituent at the ortho-position of the TO scaffold. This extraordinary outcome was determined from experimental studies and further explored through molecular docking studies. The molecular docking studies help understand how such a small substituent leads to remarkable fluorescent signal discrimination between G-quadruplex DNA and other types of nucleic acids. The results reveal that the modified dyes bind to the G-quadruplex or duplex DNA in a similar fashion as TO, but exhibit either enhanced or quenched fluorescent signal, which is determined by the spatial length and orientation of the substituent and has never been known. The new fluorescent dye modified with a p-(dimethylamino)styryl substituent offers 10-fold more selectivity toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based fluorescent probes in bioassays and cell imaging. PMID:26752011

  3. Acid-fast stain

    MedlinePlus

    The acid-fast stain is a laboratory test that determines if a sample of tissue, blood, or other body ... dye. The slide is then washed with an acid solution and a different stain is applied. Bacteria ...

  4. Probing endocytosis from the enterocyte brush border using fluorescent lipophilic dyes: lipid sorting at the apical cell surface.

    PubMed

    Danielsen, E Michael

    2015-05-01

    The small intestinal brush border is a specialized cell membrane that needs to withstand the solubilizing effect of bile salts during assimilation of dietary nutrients and to achieve detergent resistance; it is highly enriched in glycolipids organized in lipid raft microdomains. In the present work, the fluorescent lipophilic probes FM 1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide), TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate), and CellMask Orange plasma membrane stain were used to study endocytosis from the enterocyte brush border of organ-cultured porcine mucosal explants. All the dyes readily incorporated into the brush border but were not detectably endocytosed by 5 min, indicating a slow uptake compared with other cell types. At later time points, FM 1-43 clearly appeared in distinct punctae in the terminal web region, previously shown to represent early endosomes (TWEEs). In contrast, the other dyes were relatively "endocytosis resistant" to varying degrees for periods up to 2 h, indicating an active sorting of lipids in the brush border prior to internalization. For some of the dyes, a diphenylhexatriene motif in the lipophilic tail seemed to confer the relative endocytosis resistance. Lipid sorting by selective endocytosis therefore may be a process in the enterocytes aimed to generate and maintain a unique lipid composition in the brush border. PMID:25526697

  5. Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity

    PubMed Central

    Peck, Evan M.; Liu, Wenqi; Spence, Graeme T.; Shaw, Scott K.; Davis, Anthony P.; Destecroix, Harry; Smith, Bradley D.

    2015-01-01

    A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethyleneglycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with kon ~106–107 M−1s−1. The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ~1000 times higher than in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocyle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethyleneglycol chains that are twenty times shorter. The results are a promising advance towards synthetic mimics of streptavidin/biotin. PMID:26106948

  6. Metal-Enhanced Fluorescence: Ultrafast Energy Transfer from Dyes in a Polymer Film to Metal Nanoparticles.

    PubMed

    Lee, Jaebeom; Pang, Yoonsoo

    2016-02-01

    Fluorescence from dye molecules dispersed in thin polymer layers increases by 20-25 times when a silver island film exists beneath the layer. Polymer layers of <100 nm thick cover the silver island film to minimize emission quenching from direct contact and also keep the dye molecules in close proximity to the metal nanosurface for possible fluorescence enhancements by silver island film. We report an ultrafast radiation process of ~400 ps lifetime from the surface plasmons of silver nanoparticles observed in time-resolved fluorescence of rhodamine 6G and DCM in thin polymer films coated on silver island surface. The ultrafast energy transfer and fluorescence from metal nanoparticles might be strongly related to the efficiency of metal-enhanced fluorescence. PMID:27433635

  7. Photophysics of Laser Dye-Doped Polymer Membranes for Laser-Induced Fluorescence Photogrammetry

    NASA Technical Reports Server (NTRS)

    Dorrington, Adrian A.; Jones, Thomas W.; Danehy, Paul M.

    2004-01-01

    Laser-induced fluorescence target generation in dye-doped polymer films has recently been introduced as a promising alternative to more traditional photogrammetric targeting techniques for surface profiling of highly transparent or reflective membrane structures. We investigate the photophysics of these dye-doped polymers to help determine their long-term durability and suitability for laser-induced fluorescence photogrammetric targeting. These investigations included experimental analysis of the fluorescence emission pattern, spectral content, temporal lifetime, linearity, and half-life. Results are presented that reveal an emission pattern wider than normal Lambertian diffuse surface scatter, a fluorescence time constant of 6.6 ns, a pump saturation level of approximately 20 micro J/mm(exp 2), and a useful lifetime of more than 300,000 measurements. Furthermore, two demonstrations of photogrammetric measurements by laser-induced fluorescence targeting are presented, showing agreement between photogrammetric and physically measured dimensions within the measurement scatter of 100 micron.

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

    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 (ΔF = 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.

  9. Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

    PubMed

    Zhou, Xuan; Hong, Guo-Ying; Huang, Bin-Bin; Duan, Yuan-Meng; Shen, Jia-Yi; Ni, Mao-Wei; Cong, Wei-Tao; Jin, Li-Tai

    2014-05-01

    An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins. PMID:24591039

  10. Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics.

    PubMed

    Seyrig, Gregoire; Stedtfeld, Robert D; Tourlousse, Dieter M; Ahmad, Farhan; Towery, Keara; Cupples, Alison M; Tiedje, James M; Hashsham, Syed A

    2015-12-01

    Loop-mediated isothermal amplification (LAMP) is increasingly used for point-of-care nucleic acid based diagnostics. LAMP can be monitored in real-time by measuring the increase in fluorescence of DNA binding dyes. However, there is little information comparing the effect of various fluorescent dyes on signal to noise ratio (SNR) or threshold time (Tt). This information is critical for implementation with field deployable diagnostic tools that require small, low power consumption, robust, and inexpensive optical components with reagent saving low volume reactions. In this study, SNR and Tt during real-time LAMP was evaluated with eleven fluorescent dyes. Of all dyes tested, SYTO-82, SYTO-84, and SYTOX Orange resulted in the shortest Tt, and SYTO-81 had the widest range of working concentrations. The optimized protocol detected 10 genome copies of Mycobacterium tuberculosis in less than 10 min, 10 copies of Giardia intestinalis in ~20 min, and 10 copies of Staphylococcus aureus or Salmonella enterica in less than 15 min. Results demonstrate that reaction efficiency depends on both dye type and concentration and the selected polymerase. The optimized protocol was evaluated in the Gene-Z™ device, a hand-held battery operated platform characterized via simple and low cost optics, and a multiple assay microfluidic chip with micron volume reaction wells. Compared to the more conventional intercalating dye (SYBR Green), reliable amplification was only observed in the Gene-Z™ when using higher concentrations of SYTO-81. PMID:26554941

  11. Simultaneous SERS and surface-enhanced fluorescence from dye-embedded metal core-shell nanoparticles.

    PubMed

    Zhou, Yan; Zhang, Peng

    2014-05-21

    We demonstrate a methodology to prepare Au-core-Ag-shell nanoparticles displaying both SERS and surface-enhanced fluorescence (SEF) activities simultaneously by embedding dye molecules between the core and the shell. Polyelectrolytes are used to adjust the spacing and the dye position between the core and the shell. Layer-by-layer polyelectrolyte deposition can serve as an effective and flexible way to introduce various types of dye molecules into the nanostructures. Results from the spectral measurements shed light on the intricacy between SERS and SEF. PMID:24695881

  12. 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 ▿ †

    PubMed Central

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

    2011-01-01

    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

  13. New epifluorescence microscope providing pairs of specific fluorescence images of double-stained cells for simultaneous visual perception and for quantification

    NASA Astrophysics Data System (ADS)

    Heiden, Thomas; Tribukait, Bernhard

    1996-05-01

    A new epi-fluorescence microscope for analysis of cells stained with two fluorochromes which can be spectrally isolated is described. The system makes it possible to perform independent and specific spectral selection of each dye (e.g. DAPI and CY3) while perceiving the two specific images simultaneously by eye. The optics uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm and 546 nm in the excitation light path, 435 - 500 nm and 590 - 750 nm in the emission light beams), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet-excitation/blue-detection and green-excitation/red-detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) the possible separate positioning of lenses for compensation of chromatic aberrations. The system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was attained with good temporal resolution. It has been shown that it is feasible to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. Quantitative and independent measurements of the two fluorescence images by a CCD camera synchronized with the light chopper are feasible. In conclusion, this imaging system is outlined for highly specific visual analysis and exact quantitative measurement of double fluorescence labeled specimens in cytology and histology.

  14. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    PubMed

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

    2016-04-01

    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  15. Highly sensitive fluorescent stain for detecting lipopolysaccharides in sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    PubMed

    Wang, Xu; Zhou, Ayi; Cai, Wanhui; Yu, Dongdong; Zhu, Zhongxin; Jiang, Chengxi; Jin, Litai

    2015-08-01

    A sensitive and simple technique was developed for the visualization of gel-separated lipopolysaccharides by using a hydrazide derivative, UGF202. As low as 0.5-1 ng total LPS could be detected by UGF202 stain, which is 2- and 16-fold more sensitive than that of the commonly used Pro-Q Emerald 300 and Keenan et al. developed silver stain, respectively. The results indicated that UGF202 stain could be a good choice for LPS determination in polyacrylamide gels. PMID:25930092

  16. Testing the Fraunhofer line discriminator by sensing fluorescent dye

    NASA Technical Reports Server (NTRS)

    Stoertz, G. E.

    1969-01-01

    The experimental Fraunhofer Line Discriminator (FLD) has detected increments of Rhodamine WT dye as small as 1 ppb in 1/2 meter depths. It can be inferred that increments considerably smaller than 1 ppb will be detectable in depths considerably greater than 1/2 meter. Turbidity of the water drastically reduces luminescence or even completely blocks the transmission of detectable luminescence to the FLD. Attenuation of light within the water by turbidity and by the dye itself are the major factors to be considered in interpreting FLD records and in relating luminescence coefficient to dye concentration. An airborne test in an H-19 helicopter established feasibility of operating the FLD from the aircraft power supply, and established that the rotor blades do not visibly affect the monitoring of incident solar radiation.

  17. Photosensitive Fluorescent Dye Contributes to Phototoxicity and Inflammatory Responses of Dye-doped Silica NPs in Cells and Mice

    PubMed Central

    Zhao, Yang; Ye, Yan; Zhou, Xikun; Chen, Jiao; Jin, Yuihui; Hanson, Aaron; Zhao, Julia Xiaojun; Wu, Min

    2014-01-01

    Dye-doped fluorescent silica nanoparticles provide highly intense and photostable fluorescence signals. However, some dopant dye molecules are photosensitive. A widely-used photosensitive fluorescent dopant, RuBpy, was chosen to systematically investigate the phototoxicity of the dye-doped silica nanoparticles (NPs). We investigated cell viability, DNA damage, and Reactive Oxygen Species (ROS) levels in alveolar macrophages using the dye-doped NPs with or without irradiation. Our results showed that the RuBpy-doped silica NPs could induce significant amount of ROS, DNA damage, apoptosis and impaired proliferation in MH-S cells. In vivo studies in mice showed that RuBpy-doped silica NPs induced significant inflammatory cytokine production and lowered expression in signaling proteins such as ERK1/2 and NF-κB as well as increased lung injury determined by myeloperoxidase and lipid peroxidation. Strikingly, we also found that both RuBpy alone and NPs induced systemic signaling activation in the kidney compared to the liver and lung where showed highly selective signaling patterns, which is more pronounced than RuBpy-doped silica NPs. Moreover, we discovered a critical biomarker (e.g., HMGB1) for silica NPs-induced stress and toxicity and demonstrated differentially-regulated response patterns in various organs. Our results indicate for the first time that the RuBpy-doped silica NPs may impose less inflammatory responses but stronger thermotherapeutic effects on target cells in animals than naked NPs in a time- and dose-dependent manner. PMID:24578727

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

    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 μM 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 μM 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.

  19. Fluorescence energy transfer in quantum dot/azo dye complexes in polymer track membranes

    NASA Astrophysics Data System (ADS)

    Gromova, Yulia A.; Orlova, Anna O.; Maslov, Vladimir G.; Fedorov, Anatoly V.; Baranov, Alexander V.

    2013-10-01

    Fluorescence resonance energy transfer in complexes of semiconductor CdSe/ZnS quantum dots with molecules of heterocyclic azo dyes, 1-(2-pyridylazo)-2-naphthol and 4-(2-pyridylazo) resorcinol, formed at high quantum dot concentration in the polymer pore track membranes were studied by steady-state and transient PL spectroscopy. The effect of interaction between the complexes and free quantum dots on the efficiency of the fluorescence energy transfer and quantum dot luminescence quenching was found and discussed.

  20. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Reisch, Andreas; Didier, Pascal; Richert, Ludovic; Oncul, Sule; Arntz, Youri; Mély, Yves; Klymchenko, Andrey S.

    2014-06-01

    The current challenge in the field of fluorescent nanoparticles (NPs) for bioimaging is to achieve extreme brightness and external control of their emission using biodegradable materials. Here we propose a new concept of fluorescent polymer NPs, doped with ionic liquid-like salts of a cationic dye (octadecyl rhodamine B) with a bulky hydrophobic counterion (fluorinated tetraphenylborate) that serves as spacer minimizing dye aggregation and self-quenching. The obtained 40-nm poly(D,L-lactide-co-glycolide) NPs containing up to 500 dyes are brighter than quantum dots and exhibit photo-induced reversible on/off fluorescence switching, never reported for dye-doped NPs. We show that this collective switching of hundreds of dyes is due to ultrafast excitation energy transfer and can be used for super-resolution imaging. These NPs, being spontaneously endocytosed by living cells, feature high signal-to-noise ratio and absence of toxicity. The counterion-based concept opens the way to a new class of nanomaterials for sensing, imaging and light harvesting.

  1. Suitability of Mixing Fluorescent Dye in Adulticides and its Impact on Droplet Characteristics and Pesticide Efficacy.

    PubMed

    Farooq, Muhammad; Waits, Christy

    2015-12-01

    Fluorescent dyes are commonly used to help visualize insecticidal droplets or to trace movement of insecticides; however, the effect these dyes have on the insecticide's efficacy and droplet characteristics is unknown. This study evaluated the effects of mixing Uvitex OB fluorescent dye with 5 adulticides on their efficacy in a wind tunnel. Efficacy was determined via droplet size characteristics, spray flux (active ingredient [AI] deposition), and female adult Aedes aegypti mortality. Fyfanon® ULV, Anvil® 10+10, Duet™, Aqualuer® 20-20, and Zenivex® E20, diluted with corn oil, were tested with and without the dye at maximum, minimum, and half-minimum label rates. Adulticide droplet size was not affected by the addition of dye to any of the 5 pesticides tested. Mosquito mortality was strongly correlated with AI deposition for all pesticides except Duet. There was no difference among correlation coefficients of the 5 pesticides and between coefficients of any pesticide pairs, indicating that all correlations were similar. The addition of dye slightly but nonsignificantly and nonconsistently affected mortality. It was found that the source of this variability was due to large variation in mortality among different replicates of the same treatment. PMID:26675457

  2. Improvements in laser flare removal for particle image velocimetry using fluorescent dye-doped particles

    NASA Astrophysics Data System (ADS)

    Petrosky, B. J.; Lowe, K. T.; Danehy, P. M.; Wohl, C. J.; Tiemsin, P. I.

    2015-11-01

    Laser flare, or scattering of laser light from a surface, can often be a major issue in particle image velocimetry (PIV) involving solid boundaries in the flow or a gas-liquid interface. The use of fluorescent light from dye-doped particles has been demonstrated in water applications, but reproducing the technique in an airflow is more difficult due to particle size constraints and safety concerns. The following work presents fluorescent Kiton Red 620 (KR620)-doped polystyrene latex microspheres as a solution to this issue. The particles are small and narrowly distributed, with a mean diameter of 0.87 μ \\text{m} and diameter distribution standard deviation of 0.30 μ \\text{m} . Furthermore, the KR620 dye exhibits much lower toxicity than other common fluorescent dyes, and would be safe to use in large flow facilities. The fluorescent signal from the particles is measured on average to be 320  ±  10 times weaker than the Mie scattering signal from the particles. This reduction in signal is counterbalanced by greatly enhanced contrast via optical rejection of the incident laser wavelength. Fluorescent PIV with these particles is shown to eliminate laser flare near surfaces, allowing for velocity measurements as close as 100 μ \\text{m} to the surface. In one case, fluorescent PIV led to velocity vector validation rates more than 20 times that of the Mie scattering results in the boundary layer region of an angled surface.

  3. Optical detection of harmonic oscillations in fluorescent dye-loaded microbubbles ensonified by ultrasound.

    PubMed

    Schutt, Carolyn E; Ibsen, Stuart; Benchimol, Michael; Hsu, Mark; Esener, Sadik

    2015-06-15

    A new optical contrast agent has been developed by exposing dye-loaded microbubbles to a rapidly-cooled thermal treatment to homogenize the dye distribution across the surface. Ultrasound causes these microbubbles to oscillate in size which changes the self-quenching efficiency of the dye molecules creating a "blinking" signal. We demonstrate for the first time that these microbubbles can reproducibly generate second, third, and even fourth harmonic fluorescence intensity modulations, in addition to the fundamental frequency of the driving ultrasound. Detecting these harmonic signals could produce a higher signal-to-noise ratio for fluorescence imaging in medical applications by allowing fundamental frequency interference and artifacts to be filtered out. PMID:26076274

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

    NASA Astrophysics Data System (ADS)

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

    1996-01-01

    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.

  5. Highly sensitive turn-on biosensors by regulating fluorescent dye assembly on liposome surfaces.

    PubMed

    Seo, Sungbaek; Kwon, Min Sang; Phillips, Andrew W; Seo, Deokwon; Kim, Jinsang

    2015-06-25

    We developed a new self-signaling sensory system built on phospholipid liposomes having H-aggregated R6G dyes on their surface. Selective molecular recognition of a target by the phospholipid displaces R6G from the liposome surface to turn on fluorescence signal. Selective and sensitive detection of neomycin down to 2.3 nM is demonstrated. PMID:26022090

  6. Development of Thermally Stable and Highly Fluorescent IR Dyes

    NASA Technical Reports Server (NTRS)

    Bu, Xiu R.

    2004-01-01

    Fluorophores are the core component in various optical applications such as sensors and probes. Fluorphores with low-energy or long wavelength emission, in particular, in NIR region, possess advantages of low interference and high sensitivity. In this study, we has explored several classes of imidazole-based compounds for NIR fluorescent properties and concluded: (1) thiazole-based imidazole compounds are fluorescent; (2) emission energy is tunable by additional donor groups; (3) they also possess impressive two- photon absorption properties; and (4) fluorescence emission can be induced by two- photon input. This report summarizes (1) synthesis of new series of fluorophore; (2) impact of electron-withdrawing groups on fluorescent property; (3) unique property of two-photon absorption; and (4) on-going development.

  7. Supramolecular Recognition Induces Nonsynchronous Change of Dye Fluorescence Properties.

    PubMed

    Pereira-Vilar, Alba; Martin-Pastor, Manuel; Pessêgo, Marcia; García-Río, Luis

    2016-08-01

    Fluorescence behavior of 8-anilino-1-naphthalenesulfonate (ANS) reflects a blue-shift and fluorescence enhancement on decreasing solvent polarity, with both properties affected in a synchronous way in solvent mixtures where ANS senses a homogeneous solvation shell. ANS complexation by cyclodextrins or bovine serum albumin (BSA) results in a nonhomogeneous solvation shell that is reflected by nonsynchronous variation of fluorescence properties. Steady-state fluorescence and saturation transfer difference NMR experiments allow us to conclude the formation of 1:1 and 2:1 host/guest complexes through the aniline or naphthalene moieties of ANS with cyclodextrins. This nonhomogeneous solvation shell has been ignored in studies using ANS to sense the microenvironment of proteins, micelles, bilayers, and other organized systems. ANS interaction with BSA reflects the existence of a large number of binding pockets in the surface of the protein that can be classified into two well-differentiated categories. PMID:27385129

  8. Interactions of dissolved humic substances with oppositely charged fluorescent dyes for tracer techniques.

    PubMed

    Hafuka, Akira; Ding, Qing; Yamamura, Hiroshi; Yamada, Koji; Satoh, Hisashi

    2015-11-15

    To investigate interactions between oppositely charged fluorescent dyes and dissolved humic substances, fluorescence quenching of fluorescein and rhodamine 6G with dissolved humic substances was performed. Binding coefficients were obtained by the Stern-Volmer equation. The fluorescence of rhodamine 6G was largely quenched by the addition of humic acid and a non-linear Stern-Volmer plot was obtained. This strong quenching may be caused by the electrostatic interaction between cationic rhodamine 6G and humic acid and strengthened by the hydrophobic repulsion. In contrast, the quenching and interactive effects of dissolved humic substances for fluorescein were relatively weak. PMID:26318652

  9. Multifunctional particles: Magnetic nanocrystals and gold nanorods coated with fluorescent dye-doped silica shells

    SciTech Connect

    Heitsch, Andrew T.; Smith, Danielle K.; Patel, Reken N.; Ress, David; Korgel, Brian A.

    2008-07-15

    Multifunctional colloidal core-shell nanoparticles of magnetic nanocrystals (of iron oxide or FePt) or gold nanorods encapsulated in silica shells doped with the fluorescent dye, Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy) were synthesized. The as-prepared magnetic nanocrystals are initially hydrophobic and were coated with silica using a microemulsion approach, while the as-prepared gold nanorods are hydrophilic and were coated with silica using a Stoeber type of process. Each approach yielded monodisperse nanoparticles with uniform fluorescent dye-doped silica shells. These colloidal heterostructures have the potential to be used as dual-purpose tags-exhibiting a fluorescent signal that could be combined with either dark-field optical contrast (in the case of the gold nanorods), or enhanced contrast in magnetic resonance images (in the case of magnetic nanocrystal cores). The optical and magnetic properties of the fluorescent silica-coated gold nanorods and magnetic nanocrystals are reported. - Graphical abstract: Colloidal gold nanorods and iron platinum and iron oxide nanocrystals were encapsulated with fluorescent dye-doped silica shells using a generic coating strategy. These heterostructures are promising contrast agents for dual-mode medical imaging. Their optical and magnetic properties were studied and are reported here.

  10. Miniaturized side-viewing imaging probe for fluorescence lifetime imaging (FLIM): validation with fluorescence dyes, tissue structural proteins and tissue specimens

    NASA Astrophysics Data System (ADS)

    Elson, D. S.; Jo, J. A.; Marcu, L.

    2007-05-01

    We report a side viewing fibre-based endoscope that is compatible with intravascular imaging and fluorescence lifetime imaging microscopy (FLIM). The instrument has been validated through testing with fluorescent dyes and collagen and elastin powders using the Laguerre expansion deconvolution technique to calculate the fluorescence lifetimes. The instrument has also been tested on freshly excised unstained animal vascular tissues.

  11. Identification of the mineralization front: comparison of a modified toluidine blue stain with tetracycline fluorescence.

    PubMed

    Villanueva, A R; Kujawa, M; Mathews, C H; Parfitt, A M

    A modified toluidine blue method for identification of the mineralization front at the zone of demarcation between bone and osteoid in undecalcified, plastic-embedded sections of bone is described. The intensity of staining is increased both by increasing the pH of the solution and by increasing the duration of staining. The method is reproducible, since measurements of the extent of mineralization front on two nonadjacent sections from the same biopsy in 20 cases had a correlation coefficient of 0.98. The identification of the mineralization front by toluidine blue agrees closely with identification by means of in vivo tetracycline uptake, with a correlation coefficient of 0.97 between measurements of its extent using the two methods on adjacent sections from the same biopsy. It is likely that both toluidine blue staining at pH 6.5 and tetracycline uptake depend on some property of the most recently deposited bone mineral. With either method we find low values for the extent of mineralization front as a fraction of osteoid surface in many patients with osteoporosis. The uniformly normal values for this quantity in osteoporosis reported by other investigators may reflect different criteria for distinguishing osteoid-covered from quiescent bone surfaces. PMID:6200748

  12. Photoactivatable Synthetic Dyes for Fluorescence Imaging at the Nanoscale.

    PubMed

    Raymo, Françisco M

    2012-09-01

    The transition from conventional to photoactivatable fluorophores can bring the resolution of fluorescence images from the micrometer to the nanometer level. Indeed, fluorescence photoactivation can overcome the limitations that diffraction imposes on the resolution of optical microscopes. Specifically, distinct fluorophores positioned within the same subdiffraction volume can be resolved only if their emissions are activated independently at different intervals of time. Under these conditions, the sequential localization of multiple probes permits the reconstruction of images with a spatial resolution that is otherwise impossible to achieve with conventional fluorophores. The irreversible photolysis of protecting groups or the reversible transformations of photochromic compounds can be employed to control the emission of appropriate fluorescent chromophores and allow the implementation of these ingenious operating principles for superresolution imaging. Such molecular constructs enable the spatiotemporal control that is required to avoid diffraction and can become invaluable analytical tools for the optical visualization of biological specimens and nanostructured materials with unprecedented resolution. PMID:26292118

  13. Polar diketopyrrolopyrrole-imidazolium salts as selective probes for staining mitochondria in two-photon fluorescence microscopy.

    PubMed

    Grzybowski, Marek; Glodkowska-Mrowka, Eliza; Hugues, Vincent; Brutkowski, Wojciech; Blanchard-Desce, Mireille; Gryko, Daniel T

    2015-06-15

    Three rationally designed polar derivatives of diketopyrrolopyrrole consisting of 1,3-dimethylimidazolium cationic units and benzene, thiophene, or furan rings as π spacers were synthesized and thoroughly studied. The obtained salts are soluble in polar organic solvents and show satisfactory solubility in water, which makes them suitable for the applications in bioimaging. Photophysical measurements revealed that the obtained derivatives are characterized by strong absorption and good fluorescence quantum yields. The corresponding two-photon properties were also examined and showed that the synthesized salts exhibit large two-photon absorption cross-sections reaching 4000 GM (GM=Goeppert-Mayer unit, 1 GM=10(-50)  cm(4)  s photon(-1) ) and very high two-photon brightness values exceeding 2000 GM. It was demonstrated that these salts can be safely applied in two-photon fluorescence microscopy for selective staining of mitochondria in living cells. PMID:25966282

  14. Effect of Fluorescent Dyes on In Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

    PubMed Central

    Gebru, Tamirat; Mordmüller, Benjamin

    2014-01-01

    Plasmodium falciparum gametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates of P. falciparum with a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly high in vitro activity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds. PMID:25267675

  15. Self-assembled hydrophobin for producing water-soluble and membrane permeable fluorescent dye.

    PubMed

    Wang, Kunpeng; Xiao, Yunjie; Wang, Yanyan; Feng, Yaqing; Chen, Cheng; Zhang, Jie; Zhang, Qian; Meng, Shuxian; Wang, Zefang; Yang, Haitao

    2016-01-01

    Low water solubility and poor membrane permeability are major disadvantages that compromise applications of most fluorescent dyes. To resolve these problems, herein, using Boron-dipyrromethene (BODIPY) as a model fluorescent dye, for the first time, we provide a new strategy for the rapid and efficient production of a water-soluble and membrane-permeable dye by mixing with an amphiphilic protein named hydrophobin. Data shows BODIPY could be effectively solubilized and dispersed in 200 μg/mL hydrophobin by simple mixing and sonication. Subsequent experiments indicated that hydrophobin self-assembled into a protein film on the surface of BODIPY forming stable hydrophobin-BODIPY complexes with a size range of 10-30 nm. Furthermore, we demonstrated hydrophobin-functionalized BODIPY are toxicity free to cells. The hydrophobin-BODIPY complex could pass through both the cell plasma membrane and nuclear membrane efficiently. Our work opens a novel route to modify and functionalize fluorescent dyes and may be developed as a general strategy for broadening their applications. PMID:26976627

  16. Self-assembled hydrophobin for producing water-soluble and membrane permeable fluorescent dye

    PubMed Central

    Wang, Kunpeng; Xiao, Yunjie; Wang, Yanyan; Feng, Yaqing; Chen, Cheng; Zhang, Jie; Zhang, Qian; Meng, Shuxian; Wang, Zefang; Yang, Haitao

    2016-01-01

    Low water solubility and poor membrane permeability are major disadvantages that compromise applications of most fluorescent dyes. To resolve these problems, herein, using Boron-dipyrromethene (BODIPY) as a model fluorescent dye, for the first time, we provide a new strategy for the rapid and efficient production of a water-soluble and membrane-permeable dye by mixing with an amphiphilic protein named hydrophobin. Data shows BODIPY could be effectively solubilized and dispersed in 200 μg/mL hydrophobin by simple mixing and sonication. Subsequent experiments indicated that hydrophobin self-assembled into a protein film on the surface of BODIPY forming stable hydrophobin-BODIPY complexes with a size range of 10–30 nm. Furthermore, we demonstrated hydrophobin-functionalized BODIPY are toxicity free to cells. The hydrophobin-BODIPY complex could pass through both the cell plasma membrane and nuclear membrane efficiently. Our work opens a novel route to modify and functionalize fluorescent dyes and may be developed as a general strategy for broadening their applications. PMID:26976627

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

    PubMed

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

    2014-07-01

    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. PMID:23281280

  18. Using Fluorescent Dyes to Demonstrate Solution-Mixing Techniques.

    ERIC Educational Resources Information Center

    Shmaefsky, Brian; Shmaefsky, Mary Jo

    1994-01-01

    Describes a demonstration using a variety of clear solutions in which the instructor asks students whether the solutions appear homogeneous or inadequately mixed. The solutions are then induced to fluoresce with ultraviolet light to provide visible evidence of homogeneity or nonhomogeneity. (PR)

  19. New pyridinium-based fluorescent dyes: A comparison of symmetry and side-group effects on G-Quadruplex DNA binding selectivity and application in live cell imaging.

    PubMed

    Lu, Yu-Jing; Hu, Dong-Ping; Zhang, Kun; Wong, Wing-Leung; Chow, Cheuk-Fai

    2016-07-15

    A series of C1-, C2-and C3-symmetric pyridinium conjugates with different styrene-like side groups were synthesized and were utilized as G-quadruplex selective fluorescent probes. The new compounds were well-characterized. Their selectivity, sensitivity, and stability towards G-quadruplex were studied by fluorescence titration, native PAGE experiments, FRET and circular dichroism (CD) analyses. These new compounds investigated in the fluorescence assays were preferentially bound with G-quadruplex DNA compared with other type of nucleic acids and it is fascinating to realize the effects of molecular symmetry and associated side groups showing unexpectedly great influence on the fluorescent signal enhancement for the discrimination of G-quadruplexes DNA from other nucleic acids. This may correlate with the pocket symmetry and shape of the G-quadruplex DNA inherently. Among the compounds, a C2-symmetric dye (2,6-bis-((E)-2-(1H-indol-3-yl)-vinyl)-1-methylpyridin-1-ium iodide) with indolyl-groups substituted was screened out from the series giving the best selectivity and sensitivity towards G-quadruplexes DNA, particularly telo21, due to its high equilibrium binding constant (K=2.17×10(5)M(-1)). In addition, the limit of detection (LOD) of the dye to determine telo21 DNA in bioassays was found as low as 33nM. The results of the study give insight and certain crucial factors, such as molecular symmetry and the associated side groups, on developing of effective fluorescent dyes for G-quadruplex DNA applications including G-quadruplex structure stabilization, biosensing and clinical applications. The compound was also demonstrated as a very selective G-quadruplex fluorescent agent for living cell staining and imaging. PMID:26994364

  20. pI-Control in Comparative Fluorescence Gel Electrophoresis (CoFGE) using amphoteric azo dyes.

    PubMed

    Hanneken, Marina; Šlais, Karel; König, Simone

    2015-06-01

    Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates. PMID:26217748

  1. pI-Control in Comparative Fluorescence Gel Electrophoresis (CoFGE) using amphoteric azo dyes

    PubMed Central

    Hanneken, Marina; Šlais, Karel; König, Simone

    2015-01-01

    Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates. PMID:26217748

  2. Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye

    NASA Astrophysics Data System (ADS)

    Patra, Digambara; Barakat, Christelle

    2011-09-01

    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 Δ λ = 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 λSFSmax 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 λSFSmax vs. π* scale of solvent polarity was found compared to λabsmax or λemmax 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.

  3. A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

    PubMed

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

    2015-10-01

    In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and β-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research. PMID:26177935

  4. Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

    PubMed

    Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

    2011-05-01

    To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

  5. Selective fluorescence functionalization of dye-doped polymerized structures fabricated by direct laser writing (DLW) lithography

    NASA Astrophysics Data System (ADS)

    de Miguel, Gustavo; Vicidomini, Giuseppe; Duocastella, Martí; Diaspro, Alberto

    2015-11-01

    The continuous development of the vast arsenal of fabrication techniques is a pivotal factor in the breakthrough of nanotechnology. Although the broad interest is generally focused on the reduction of the dimensions of the fabricated structures, localized functionalization of the nanomaterials emerges as a key factor closely linked to their potential applications. In particular, fabrication of spatially selective fluorescence nanostructures is highly demanded in nanophotonics, as for example in three-dimensional (3D) optical data storage (ODS), where massive storage capacity and fast writing-reading processes are promised. We have developed an innovative method to control the location and intensity of the fluorescence signal in dye-doped photopolymerized structures fabricated with Direct Laser Writing (DLW) lithography. Well-defined fluorescent pixels (area = 0.24 μm2) were written inside a polymer matrix with the help of a femtosecond pulsed laser (multiphoton absorption) via a thermally-induced di-aggregation of a fluorescent dye. Moreover, we have accomplished a fine control of the fluorescence intensity which can increase the storage capacity of ODS systems fabricated with this approach.The continuous development of the vast arsenal of fabrication techniques is a pivotal factor in the breakthrough of nanotechnology. Although the broad interest is generally focused on the reduction of the dimensions of the fabricated structures, localized functionalization of the nanomaterials emerges as a key factor closely linked to their potential applications. In particular, fabrication of spatially selective fluorescence nanostructures is highly demanded in nanophotonics, as for example in three-dimensional (3D) optical data storage (ODS), where massive storage capacity and fast writing-reading processes are promised. We have developed an innovative method to control the location and intensity of the fluorescence signal in dye-doped photopolymerized structures fabricated

  6. Benchtop Antigen Detection Technique using Nanofiltration and Fluorescent Dyes

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2009-01-01

    The designed benchtop technique is primed to detect bacteria and viruses from antigenic surface marker proteins in solutions, initially water. This inclusive bio-immunoassay uniquely combines nanofiltration and near infrared (NIR) dyes conjugated to antibodies to isolate and distinguish microbial antigens, using laser excitation and spectrometric analysis. The project goals include detecting microorganisms aboard the International Space Station, space shuttle, Crew Exploration Vehicle (CEV), and human habitats on future Moon and Mars missions, ensuring astronaut safety. The technique is intended to improve and advance water contamination testing both commercially and environmentally as well. Lastly, this streamlined technique poses to greatly simplify and expedite testing of pathogens in complex matrices, such as blood, in hospital and laboratory clinics.

  7. Sulfone-Rhodamines: A New Class of Near-Infrared Fluorescent Dyes for Bioimaging.

    PubMed

    Liu, Jing; Sun, Yuan-Qiang; Zhang, Hongxing; Shi, Heping; Shi, Yawei; Guo, Wei

    2016-09-01

    Given the wavelength dependence of tissue transparency and the requirement for sufficiently low background autofluorescence, the development of fluorescent dyes with excitation and emission maxima beyond 700 nm is highly desired, but it is a challenging task. Herein, a new class of fluorescent dyes, named sulfone-rhodamines (SO2Rs), was developed on the basis of the one-atom replacement of the rhodamine 10-position O atom by a sulfone group. Such a modification makes their absorption and emission maxima surprisingly reach up to 700-710 and 728-752 nm, respectively, much longer than their O-, C-, and Si-rhodamine analogs, due to the unusual d*-π* conjugation. Among these dyes, SO2R4 and SO2R5, bearing disubstituted meso-phenyl groups, show the greatest potentials for bioimaging applications in view of their wide pH range of application, high photostability, and big extinction coefficients and fluorescence quantum yields. They could quickly penetrate cells to give stable NIR fluorescence, even after continuous irradiation by a semiconductor laser, making them suitable candidates for time-lapse and long-term bioimaging applications. Moreover, they could specifically localize in lysosomes independent of alkylmorpholine targeted group, thus avoiding the problematic alkalization effect suffered by most LysoTrackers. Further imaging assays of frozen slices of rat kidney reveal that their tissue imaging depth is suprior to the widely used NIR labeling agent Cy5.5. PMID:27548811

  8. Localized surface plasmon-influenced fluorescence decay in dye-doped metallo-dielectric opals

    NASA Astrophysics Data System (ADS)

    Rout, Dipak; Vijaya, R.

    2016-01-01

    Well-ordered opaline photonic crystals are grown by inward growing self-assembly method from Rhodamine B dye-doped polystyrene colloids. Subsequent to self-assembly, the crystals are infiltrated with gold nanoparticles of 40 nm diameter. Measurements of the stopband features and photoluminescence intensity from these crystals are supplemented by fluorescence decay time analysis. The fluorescence decay times from the dye-doped photonic crystals before and after the infiltration are dramatically different from each other. A lowered fluorescence decay time was observed for the case of gold infiltrated crystal along with an enhanced emission intensity. Double-exponential decay nature of the fluorescence from the dye-doped crystal gets converted into single-exponential decay upon the infiltration of gold nanoparticles due to the resonant radiative process resulting from the overlap of the surface plasmon resonance with the emission spectrum. The influence of localized surface plasmon due to gold nanoparticles on the increase in emission intensity and decrease in decay time of the emitters is established.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    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 μmol.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.

  10. Fluorescence lifetimes of molecular dye ensembles near interfaces

    SciTech Connect

    Danz, Norbert; Heber, Joerg; Braeuer, Andreas; Kowarschik, Richard

    2002-12-01

    Fluorescence lifetimes of thin, rhodamine 6G-doped polymer layers in front of a mirror have been determined as a function of the emitter-mirror separation and the conditions of excitation and observation. Lifetime is well known to depend on the spatial emitter-mirror separation. The explanation of experimental data needs to consider direction, polarization, and numerical aperture of the experimental system. As predicted theoretically, experimental results depend on the conditions of illumination and observation, because of the different lifetimes of emitters aligned horizontally or vertically with respect to the plane of interfaces and their selection by the experimental system. This effect is not observable when ions are used as a source of fluorescence, because ensemble averaging depends on the properties of sources.

  11. An artery-specific fluorescent dye for studying neurovascular coupling

    PubMed Central

    Shen, Zhiming; Lu, Zhongyang; Chhatbar, Pratik Y; O’Herron, Philip; Kara, Prakash

    2012-01-01

    We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus–evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments. PMID:22266543

  12. Synthesis and Characterization of Far-Red/NIR-Fluorescent BODIPY Dyes, Solid-State Fluorescence, and Application as Fluorescent Tags Attached to Carbon Nano-onions.

    PubMed

    Bartelmess, Juergen; Baldrighi, Michele; Nardone, Valentina; Parisini, Emilio; Buck, David; Echegoyen, Luis; Giordani, Silvia

    2015-06-26

    A series of π-extended distyryl-substituted boron dipyrromethene (BODIPY) derivatives with intense far-red/near-infrared (NIR) fluorescence was synthesized and characterized, with a view to enhance the dye's performance for fluorescence labeling. An enhanced brightness was achieved by the introduction of two methyl substituents in the meso positions on the phenyl group of the BODIPY molecule; these substituents resulted in increased structural rigidity. Solid-state fluorescence was observed for one of the distyryl-substituted BODIPY derivatives. The introduction of a terminal bromo substituent allows for the subsequent immobilization of the BODIPY fluorophore on the surface of carbon nano-onions (CNOs), which leads to potential imaging agents for biological and biomedical applications. The far-red/NIR-fluorescent CNO nanoparticles were characterized by absorption, fluorescence, and Raman spectroscopies, as well as by thermogravimetric analysis, dynamic light scattering, high-resolution transmission electron microscopy, and confocal microscopy. PMID:26015289

  13. Critical tonicity determination of sperm using fluorescent staining and flow cytometry

    SciTech Connect

    Noiles, E.E.; Ruffing, N.A.; Kleinhans, F.W.; Mark, L.A.; Watson, P.F.; Critser, J.K. ); Horstman, L. . School of Veterinary Medicine); Mazur, P. )

    1990-01-01

    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.

  14. Enhancement of the fluorescence of triphenylmethane dyes caused by their interaction with nanoparticles from β-diketonate complexes

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

    We have studied the absorption and fluorescence spectra of Malachite Green and Crystal Violet in aqueous and alcoholic-aqueous solutions in which nanoparticles from Ln(III) and Sc(III) diketonates are formed at concentrations of complexes in a solution of 5-30 μM. We have shown that, if the concentrations of the dyes in the solution are lower than 0.5 μM, dye molecules are incorporated completely into nanoparticles or are precipitated onto their surface. The fluorescence intensity of these incorporated and adsorbed Malachite Green and Crystal Violet molecules increases by several orders of magnitude compared to the solution, which takes place because of a sharp increase in the fluorescence quantum yields of these dyes and at the expense of the sensitization of their fluorescence upon energy transfer from β-diketonate complexes entering into the composition of nanoparticles. We have shown that, if there is no concentration quenching, the values of the fluorescence quantum yield of the Crystal Violet dye incorporated into nanoparticles and adsorbed on their surface vary from 0.06 to 0.13, i.e., are close to the fluorescence quantum yield of this dye in solid solutions of sucrose acetate at room temperature. The independence of the fluorescence quantum yield of Crystal Violet on the morphology of nanoparticles testifies to a high binding constant of complexes and the dye. The considerable fluorescence quantum yields of triphenylmethane dyes in nanoparticles and sensitization of their fluorescence by nanoparticle-forming complexes make it possible to determine the concentration of these dyes in aqueous solutions by the luminescent method in the range of up to 1 nM.

  15. Analyzing Cell Death by Nuclear Staining with Hoechst 33342.

    PubMed

    Crowley, Lisa C; Marfell, Brooke J; Waterhouse, Nigel J

    2016-01-01

    The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes. PMID:27587774

  16. Gram stain

    MedlinePlus

    A Gram stain is a test used to identify bacteria. It is one of the most common ways to ... Urethral discharge Gram stain; Feces Gram stain; Stool Gram stain; Joint fluid Gram stain; Pericardial fluid Gram stain; Gram stain of ...

  17. Hierarchical growth of fluorescent dye aggregates in water by fusion of segmented nanostructures.

    PubMed

    Zhang, Xin; Görl, Daniel; Stepanenko, Vladimir; Würthner, Frank

    2014-01-27

    Dye aggregates are becoming increasingly attractive for diverse applications, in particular as organic electronic and sensor materials. However, the growth processes of such aggregates from molecular to small assemblies up to nanostructures is still not properly understood, limiting the design of materials' functional properties. Here we elucidate the supramolecular growth process for an outstanding class of functional dyes, perylene bisimides (PBIs), by transmission electron microscopy (TEM), cryogenic scanning electron microscopy (cryo-SEM), and atomic force microscopy (AFM). Our studies reveal a sequential growth of amphiphilic PBI dyes from nanorods into nanoribbons in water by fusion and fission processes. More intriguingly, the fluorescence observed for higher hierarchical order nanoribbons was enhanced relative to that of nanorods. Our results provide insight into the relationship between molecular, morphological, and functional properties of self-assembled organic materials. PMID:24352910

  18. Targeting tumor hypoxia: a third generation 2-nitroimidazole-indocyanine dye-conjugate with improved fluorescent yield.

    PubMed

    Zhou, Feifei; Zanganeh, Saeid; Mohammad, Innus; Dietz, Christopher; Abuteen, Akram; Smith, Michael B; Zhu, Quing

    2015-12-14

    Tumor hypoxia is associated with the rapid proliferation and growth of malignant tumors, and the ability to detect tumor hypoxia is important for predicting tumor response to anti-cancer treatments. We have developed a class of dye-conjugates that are related to indocyanine green (ICG, ) to target tumor hypoxia, based on in vivo infrared fluorescence imaging using nitroimidazole moieties linked to indocyanine fluorescent dyes. We previously reported that linking 2-nitroimidazole to an indocyanine dicarboxylic acid dye derivative () using an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG, ), led to a dye-conjugate that gave promising results for targeting cancer hypoxia in vivo. Structural modification of the dye conjugate replaced the ethanolamine unit with a piperazineacetyl unit and led a second generation dye conjugate, piperzine-2-nitroimidazole-ICG (). This second generation dye-conjugate showed improved targeting of tumor hypoxia when compared with . Based on the hypothesis that molecules with more planar and rigid structures have a higher fluorescence yield, as they could release less absorbed energy through molecular vibration or collision, we have developed a new 2-nitroimidazole ICG conjugate, , with two carbon atoms less in the polyene linker. Dye-conjugate was prepared from our new dye (), and coupled to 2-nitroimidazole using a piperazine linker to produce this third-generation dye-conjugate. Spectral measurements showed that the absorption/emission wavelengths of 657/670 were shifted ∼100 nm from the second-generation hypoxia dye of 755/780 nm. Its fluorescence quantum yield was measured to be 0.467, which is about 5 times higher than that of (0.083). In vivo experiments were conducted with balb/c mice and showed more than twice the average in vivo fluorescence intensity in the tumor beyond two hours post retro-orbital injection as compared with . These initial results suggest that may significantly improve in vivo tumor hypoxia targeting

  19. Imaging fluorescence correlation spectroscopy studies of dye diffusion in self-assembled organic nanotubes.

    PubMed

    Xu, Hao; Nagasaka, Shinobu; Kameta, Naohiro; Masuda, Mitsutoshi; Ito, Takashi; Higgins, Daniel A

    2016-06-22

    The rate and mechanism of diffusion by anionic sulforhodamine B (SRB) dye molecules within organic nanotubes self-assembled from bolaamphiphile surfactants were investigated by imaging fluorescence correlation spectroscopy (imaging-FCS). The inner and outer surfaces of the nanotubes are terminated with amine and glucose groups, respectively; the former allow for pH-dependent manipulation of nanotube surface charge while the latter enhance their biocompatibility. Wide-field fluorescence video microscopy was used to locate and image dye-doped nanotubes dispersed on a glass surface. Imaging-FCS was then used to spatially resolve the SRB transport dynamics. Mobilization of the dye molecules was achieved by immersion of the nanotubes in buffer solution. Experiments were performed in pH 6.4, 7.4 and 8.4 buffers, at ionic strengths ranging from 1.73 mM to 520 mM. The results show that coulombic interactions between cationic ammonium ions on the inner nanotube surface and the anionic SRB molecules play a critical role in governing mass transport of the dye. The apparent dye diffusion coefficient, D, was found to generally increase with increasing ionic strength and with increasing pH. The D values obtained were found to be invariant along the nanotube length. Mass transport of the SRB molecules within the nanotubes is concluded to occur by a desorption-mediated Fickian diffusion mechanism in which dye motion is slowed by its coulombic interactions with the inner surfaces of the nanotubes. The results of these studies afford information essential to the use of organic nanotubes in controlled drug release applications. PMID:27271313

  20. Measurement of atmospheric OH by titration of near-IR fluorescent dyes

    NASA Technical Reports Server (NTRS)

    Betterton, Eric A.; Gast, Karl

    1994-01-01

    Recent research has shown that certain polymethine dyes can be detected at ultratrace levels (greater than or equal to 6x10(exp -14) M) in solution by fluorimetry. These detection limits are possible because of the inherent sensitivity of fluorescence techniques, because the dyes fluoresce in the near infrared region where background interference is negligible, and because powerful infrared diode lasers are now available to improve the signal to noise ratio. Other work has shown that the hydroxyl radical destroys the ability of polymethine dyes to fluoresce. These observations form the basis for a new hydroxyl radical detector that is essentially a fluorometric titrator. Theoretically, the detector should show an acceptable sensitivity and response time. Assuming that the atmospheric HO concentration is about 10(exp -11) moles m(exp -3) (i.e. 10(exp 6) molecules cm(exp -3)), then 10 L of air 'titrated' with 20 mL of 10(exp -11) M dye solution (an easily detected concentration) should result in a drop in the fluorescent signal of 50 percent - a readily detectable change. At a flow rate of 3 L min(exp -1) the sampling time would be 3 minutes. The biggest potential problem is selectivity: other oxidants may also cause the fluorescence signal to be lost. The chemistry of polymethine dyes has not been studied in detail and so no quantitative data are available. However, a survey of the literature suggests that in general HO should react up to six orders of magnitude faster than HO2 and other radicals such as RO2 and RO. It should also react much more rapidly than H2O2 and O3. Thus it may be possible to discriminate kinetically against potential interfering substances. It was shown in the laboratory that 10(exp -4) M H2O2 has little effect on the absorption spectrum of the dye IR125 over a period of hours but that the band at 780 nm is slowly lost in water over a period of days even under argon in the dark. By contrast, DMSO solutions of IR125 are stable.

  1. Dicyanostilbene-derived two-photon fluorescence dyes with large two-photon absorption cross sections

    NASA Astrophysics Data System (ADS)

    Huang, Chibao; Lin, Changhua; Ren, Anxiang; Yang, Nianfa

    2011-12-01

    Four dicyanostilbene-derived two-photon fluorescence (TPF) dyes were synthesized as the model compounds to systematically study the effect of the dicyano and the terminal substituent on the two-photon absorption (TPA). These four compounds ( DSO, DCY, DTO and DPH) exhibit very large two-photon absorption cross sections ( δ). DCY (A- π-A) with the terminal cyano group has especially high fluorescence quantum yield (0.71) and relatively large δ (1480 GM), while DPH (D- π-A) with the substitutedamino group at its terminus possesses the largest δ (2800 GM) and the longest emission wavelength (620 nm). The idealest terminal substituent should not be the alkoxy group but the substitutedamino group. This class of dicyanostilbene dyes possess small molecule size, large δ (830-2800 GM), long-wavelength emission (459-620 nm) and large Stokes shift (80-206 nm), and are ideal chromophores for TPF labels and probes.

  2. Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity.

    PubMed

    Peck, Evan M; Liu, Wenqi; Spence, Graeme T; Shaw, Scott K; Davis, Anthony P; Destecroix, Harry; Smith, Bradley D

    2015-07-15

    A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethylene glycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with k(on) ≈ 10(6)-10(7) M(-1) s(-1). The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ∼1000 times higher than those in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocycle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethylene glycol chains that are 20 times shorter. The results are a promising advance toward synthetic mimics of streptavidin/biotin. PMID:26106948

  3. Switch-On Fluorescence of a Perylene-Dye-Functionalized Metal-Organic Framework through Postsynthetic Modification.

    PubMed

    Dietl, Christian; Hintz, Henrik; Rühle, Bastian; Schmedt Auf der Günne, Jörn; Langhals, Heinz; Wuttke, Stefan

    2015-07-20

    A perylene dye was introduced directly as a linker into a metal-organic framework (MOF) during synthesis. Depending on the dye concentration in the MOF synthesis mixture, different fluorescent materials were generated. The successful incorporation of the dye was proven by using (13) C and (27) Al MAS NMR spectroscopy, by solution NMR spectroscopy after digestion of the MOF sample, and by synthesizing a reference dye without connecting groups, which could coordinate on the metal-oxo cluster inside the MOF. Fluorescence quenching effects of the MOF linker, 2-aminoterephthalate, were observed and overcome by postsynthetic modification with acetic anhydride. We show here for the first time that amino groups, which can be used as anchoring points for covalent attachment of other molecules, are responsible for fluorescence quenching. Thus, a very promising strategy to implement switchable fluorescence into MOFs is shown here. PMID:26037475

  4. Influence of Solvation and Structural Contributions on Fluorescence of Dipyrrine Dyes.

    PubMed

    Ksenofontov, A A; Guseva, G B; Antina, E V; Vyugin, A I; Nuraneeva, E N

    2015-11-01

    The results of quantum-chemical and spectral researches of zinc((II)) complexes with alkylated dipyrrine and 3,3'-, 2,3'- and 2,2'-bis(dipyrrine)s in non-polar and polar solvents and their binary mixtures are presented. It was investigated the efficiency of the fluorescence quenching of fluorophores depending on of the solvation and structural contributions. Found that 3,3'-bis(dipyrrinato)zinc((II)) demonstrates the highest sensitivity of the fluorescence to the presence of the electron-donor component compared with the studied complexes. The obtained results allow to offer dipyrrine and bis(dipyrrine) zinc((II)) complexes as new, highly sensitive and selective fluorescent sensors of the N- and O-containing toxicants. Graphical Abstract Influence of solvation and structural contributions on fluorescence of dipyrrine dyes. PMID:26439931

  5. Dye-biomolecule conjugates and NIR-fluorescent particles for targeting of disease-related biomarkers

    NASA Astrophysics Data System (ADS)

    Pauli, J.; Brehm, R.; Grabolle, M.; Behnke, T.; Mathejczyk, J.; Hamann, F.; Alves, F.; Hilger, I.; Resch-Genger, U.

    2011-03-01

    Indispensable for fluorescence imaging are highly specific and sensitive molecular probes that absorb and emit in the near infrared (NIR) spectral region and respond to or target molecular species or processes. Here, we present approaches to targeted fluorescent probes for in vivo imaging in the intensity and lifetime domain exploiting NIR dyes. Screening schemes for the fast identification of suitable fluorophores are derived and design criteria for highly emissive optical probes. In addition, as a signal amplification strategy that enables also the use of hydrophobic NIR fluorophores as fluorescent reporters, first steps towards versatile strategies for the preparation of NIR-fluorescent polymeric particles are presented that can be utilized also for the design of targeted and analyte-responsive probes.

  6. Fluorescence quenching and photocatalytic degradation of textile dyeing waste water by silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Kavitha, S. R.; Umadevi, M.; Janani, S. R.; Balakrishnan, T.; Ramanibai, R.

    2014-06-01

    Silver nanoparticles (Ag NPs) of different sizes have been prepared by chemical reduction method and characterized using UV-vis spectroscopy and transmission electron microscopy (HRTEM). Fluorescence spectral analysis showed that the quenching of fluorescence of textile dyeing waste water (TDW) has been found to decrease with decrease in the size of the Ag NPs. Experimental results show that the silver nanoparticles can quench the fluorescence emission of adsorbed TDW effectively. The fluorescence interaction between Ag NPs (acceptor) and TDW (donor) confirms the Förster Resonance Energy Transfer (FRET) mechanism. Long range dipole-dipole interaction between the excited donor and ground state acceptor molecules is the dominant mechanism responsible for the energy transfer. Furthermore, photocatalytic degradation of TDW was measured spectrophotometrically by using silver as nanocatalyst under UV light illumination. The kinetic study revealed that synthesized Ag NPs was found to be effective in degrading TDW.

  7. Study of excitation transfer in laser dye mixtures by direct measurement of fluorescence lifetime

    NASA Technical Reports Server (NTRS)

    Lin, C.; Dienes, A.

    1973-01-01

    By directly measuring the donor fluorescence lifetime as a function of acceptor concentration in the laser dye mixture Rhodamine 6G-Cresyl violet, we found that the Stern-Volmer relation is obeyed, from which the rate of excitation transfer is determined. The experimental results indicate that the dominant mechanism responsible for the efficient excitation transfer is that of resonance transfer due to long range dipole-dipole interaction.

  8. Charged solvatochromic dyes as signal transducers in pH independent fluorescent and colorimetric ion selective nanosensors.

    PubMed

    Xie, Xiaojiang; Gutiérrez, Agustín; Trofimov, Valentin; Szilagyi, Istvan; Soldati, Thierry; Bakker, Eric

    2015-10-01

    Ionophore-based ion selective optical nanosensors that operate independently of the sample pH are developed here by the use of electrically charged solvatochromic dyes as signal transducers. A series of dye molecules with a D-π-A structure was synthesized and characterized in various solvents and incorporated into ion selective nanospheres for K(+), Na(+), and H(+). Since dye leakage was greatly suppressed when the solvatochromic dyes were encapsulated in the nanosphere core, ion sensing nanospheres were explored for cellular ion imaging in Dictyostelium discoideum live cells but spontaneous dye loss resulted in undesired staining of cells. The in vitro analysis of potassium in human plasma was successfully demonstrated with this approach. A theoretical model was developed for the response of the ion selective nanosensors containing charged solvatochromic dyes. The nanosensors exhibited a tunable response range, high sensitivity, and good stability. PMID:26352133

  9. Design and synthesis of efficient fluorescent dyes for incorporation into DNA backbone and biomolecule detection.

    PubMed

    Wang, Wei; Li, Alexander D Q

    2007-01-01

    We report here the design and synthesis of a series of pi-conjugated fluorescent dyes with D-A-D (D, donor; A, acceptor), D-pi-D, A-pi-A, and D-pi-A for applications as the signaling motif in biological-synthetic hybrid foldamers for DNA detection. The Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation were demonstrated as the optimum ways for construction of long pi-conjugated systems. Such rodlike chromophores have distinct advantages, as their fluorescence properties are not quenched by the presence of DNA. To be incorporated into the backbone of DNA, the chromophores need to be reasonably soluble in organic solvent for solid-phase synthesis, and therefore a strategy of using flexible tetraethylene glycol (TEG) linkers at either end of these rodlike dyes was developed. The presence of TEG facilitates the protection of the chain-growing hydroxyl group with DMTrCl (dimethoxytrityl chloride) as well as the activation of the coupling step with phosphoramidite chemistry on an automated DNA synthesizer. To form fluorescence resonance energy transfer (FRET) pairs, six synthetic chromophores with blue to red fluorescence have been developed, and those with orthogonal fluorescent emission were chosen for incorporation into DNA-chromophore hybrid foldamers. PMID:17508711

  10. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

    PubMed Central

    Ju, J; Ruan, C; Fuller, C W; Glazer, A N; Mathies, R A

    1995-01-01

    Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods. Images Fig. 4 Fig. 5 PMID:7753809