Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “FLUORESCENCE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed1998

Generating green fluorescent mice by germline transmission of green fluorescent ES cells.

Green fluorescent protein (GFP) and its variants currently represent the only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice. or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: 1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage: (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues. and (4) intercrosses of F1 mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence.

Animals↗
PubMed2002

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

Biosensing Techniques↗
PubMed1992

Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.

Animals↗
PubMed1991

Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with fluorescent heterocyclic side chains, that undergo bioreductive binding.

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.

Animals↗
PubMed1996

Arylacetamide-derived fluorescent probes: synthesis, biological evaluation, and direct fluorescent labeling of kappa opioid receptors in mouse microglial cells.

Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3- or 4-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as well as high kappa receptor affinity and selectivity in receptor binding assays. The 4-substituted series (6-9) were substantially less potent than the corresponding 3-substituted compounds. Flow cytometric analysis demonstrated high levels of direct kappa-specific staining of mouse microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a 41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence intensity in the presence of the kappa-selective antagonist, norbinaltorphimine (norBNI). In similar studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report of direct, selective staining of kappa opioid receptors by a fluorescent nonpeptide opioid ligand. The results of the present study illustrate the importance of introducing hydrophilic linkers to reduce nonspecific binding of fluorescent probes for opioid receptors.

Acetamides↗
PubMed1976

Fluorescence-detected magnetic circular dichroism of fluorescent and nonfluorescent molecules.

We have modified a spectrometer for the measurement of fluorescence-detected magnetic (and natural) circular dichroism (FDMCD). The instrument can be operated either in a direct mode in which the average and polarization-induced differential fluorescent intensities are recorded separately or in a mode which records their ratio. We have measured the FDMCD of tryptophan, which is itself fluorescent. With the aid of an added fluorescent molecule we also measured the FDMCD of Fe(III) cytochrome c, which is nonfluorescent. In both cases, the FDMCD agreed with the conventional transmission-detected magnetic circular dichroism within experimental uncertainty. The FDMCD of both fluorescent and nonfluorescent molecules is potentially a useful technique for investigating optically dense materials and biological molecules in their native environments and systems in which there is inter- or intramolecular energy transfer.

Circular Dichroism↗
PubMed2004

Fluorescence resonance energy transfer-based near-infrared fluorescence sensor for glucose monitoring.

A novel near-infrared (NIR) fluorescence affinity sensor for continuous glucose monitoring was developed and characterized. The sensor operates by fluorescence resonance energy transfer between a NIR chromophore linked to concanavalin A (ConA) and an NIR fluorophore linked to free dextran. The binding of dextran with ConA in the absence of glucose results in low fluorescence due to quenching; however, the quenching is reversed by competitive displacement of dextran from ConA by glucose. In order to increase thermodynamic stability and the lifetime of the sensor, ConA was immobilized within a macroporous bead matrix. The sensor was contained within a sealed hollow dialysis fiber (o.d. 215 microm, wall thickness 20 microm), preventing the macromolecules from leaking out and enabling glucose to rapidly enter the fiber lumen. A glucose-insensitive reference fluorophore was also incorporated to allow for ratiometric measurements, resulting in a robust sensor output that is independent of positional and/or light intensity changes. The response of the fluorescence affinity sensor to glucose was tested continuously in an automated test chamber at 37 degrees C. The sensor showed good dynamic range within physiologically relevant glucose concentration range (15% change over 2.5-30 mM, no hysteresis), fast response time (2-4 min), and a remarkable long-term stability (6 months). We interpret the improved longevity of this sensor to be the result of an optimized photo exposure regime and immobilization of ConA to the matrix. Its small size, ratiometric output, and NIR fluorescence make this sensor well suited for dermal implantation and continuous transdermal monitoring.

Automation↗
PubMed1996

Synthesis of a fluorescent 7-methylguanosine analog and a fluorescence spectroscopic study of its reaction with wheatgerm cap binding proteins.

In the initiation of protein synthesis, the mRNA 5'-terminal 7-methylguanosine cap structure and several recognition proteins play a pivotal role. For the study of this cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose diol-modified fluorescent cap analog, anthraniloyl-m7GTP (Ant-m7GTP), was designed and synthesized for this purpose. This fluorescent cap analog was found to have a high quantum yield, resistance to photobleaching and avoided overlap of excitation and emission wavelengths with those of proteins. The binding of Ant-m7GTP with wheatgerm initiation factors elF-4F and elF-(iso)4F was determined. The fluorescent cap analog and m7GTP had similar interactions with both cap binding proteins. Fluorescence quenching experiments showed that the microenvironment of Ant-m7GTP when bound to protein was hydrophobic.

Guanosine Triphosphate↗
PubMed1991

Analysis of the binding sites of Gd3+ on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum through its effects on fluorescence of tryptophan residues and a covalently attached fluorescent probe N-(1-anilinonaphthyl-4)maleimide.

Interaction of Ca2+ and Gd3+ ions with Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) was analyzed. Binding of Ca2+ to the transport site caused an enhancement of intrinsic fluorescence of SR-ATPase. Gd3+ also induced fluorescence enhancement. However, the effects of Ca2+ and Gd3+ were additive rather than competitive, indicating that the Gd(3+)-binding site responsible for this enhancement is distinct from the Ca(2+)-transport site. Gd3+ ions at concentrations higher than 10 microM caused a marked fluorescence quenching, indicating an additional interaction at low-affinity binding sites. Interaction of Ca2+ with the transport site led to a quenching of fluorescence of N-(1-anilinonaphthyl-4)maleimide (ANM) covalently attached at SHN [as defined in Yasuoka-Yabe, K. & Kawakita, M. (1983) J. Biochem. 94, 665-675]. In this case the effects of Ca2+ and Gd3+ were mutually exclusive, indicating that Ca2+ and Gd3+ were competing for the same binding site (i.e. the transport site) to affect ANM fluorescence. Competition between Ca2+ and Gd3+ for the Ca(2+)-transport site was also demonstrated by direct measurement of Ca(2+)-binding using nitrocellulose membrane filters. Affinity of Gd3+ for the Ca(2+)-transport site was a little lower than that of Ca2+. Based on these results it was concluded that Gd3+ has at least three kinds of binding sites on SR-ATPase, namely the Ca(2+)-transport site, the Gd(3+)-specific high-affinity site, and a number of low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Combination of BUdR-quenched Hoechst fluorescence with DNA-specific ethidium bromide fluorescence for cell cycle analysis with a two-parameter flow cytometer.

Stimulated or asynchronous L-cells were grown in a BUdR-medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a FACS II cell sorter. The u.v. laser line served as a light source for exciting the Hoechst fluorescence, the ethidium bromide fluorescence being excited mainly by energy transfer from the Hoechst dye. The quenched Hoechst fluorescence was analysed between 410 nm and 480 nm, the DNA specific EB fluorescence at beyond 630 nm. Thus, not only the actual location of each cell in the cycle could be determined, but also its initial location at time 0 of the experiment, together with its moment of division (BUdR-quenched Hoechst fluorescence). This method could become a powerful tool in many investigations dealing with cell cycle perturbations in culture.

Animals↗
PubMed1981

Fluorescence and N.M.R. studies of the binding of cholinergic fluorescent probes to horse serum cholinesterase.

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminoaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like carbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Animals↗
PubMed2003

Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand.

For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

Fluorescence Polarization↗
PubMed2005

Improvement of fluorescence characteristics of coumarins: syntheses and fluorescence properties of 6-methoxycoumarin and benzocoumarin derivatives as novel fluorophores emitting in the longer wavelength region and their application to analytical reagents.

To improve the fluorescence characteristics, especially emission wavelength, of coumarins, various 3-substituted-6-methoxycoumarin derivatives were synthesized, and then benzocoumarin derivatives were also synthesized in expectation of the shift to the longer wavelength region by the extension of the conjugated system. Their fluorescence properties were investigated spectrophotometrically in acetonitrile and evaluated from the viewpoint of the intramolecular charge transfer (ICT) between push- and pull-substituents in the ground and the excited states. Among them, benzocoumarin derivatives especially fluoresced in the longer wavelength around 540 nm with remarkably large Stokes shifts beyond 10,000 cm(-1). Using such fluorophores, some novel fluorescence derivatization reagents for carboxylic acids, alcohols, phenols, and amines were preliminarily prepared as an example, and their derivatized products were also found to fluoresce in the longer wavelength region with large Stokes shifts.

Coumarins↗
PubMed1987

Changes in fluorescence energy transfer between sulfhydryl fluorescent residues during ouabain sensitive Na+,K+-ATP hydrolysis.

Na+,K+-ATPase from pig kidney was sequentially modified with two different sulfhydryl fluorescent reagents, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) and N-[7-dimethylamino 4-coumarinyl]maleimide (DACM). The preparation thus obtained contained 3 and 2 moles of each residue in the alpha-chain. When the BIPM residues were excited at 313 nm, ouabain sensitive decrease and increase in the fluorescence intensity at not only 365 nm (BIPM fluorescence) but also 455 nm (DACM fluorescence) were observed, which were dependent on the amounts of reaction intermediates accumulated. When DACM residues were excited directly at 390 nm, only the decrease in the fluorescence intensity was observed irrespective of the intermediates accumulated. The data suggest that at least two DACM residues which differently change their microenvironments during ouabain sensitive Na+,K+-ATPase reaction are present. One is located close enough and the other is located too far to accept the energy from BIPM residue(s) in the three dimensional structure of Na+,K+-ATPase. Addition of sodium dodecyl sulfate (SDS) remarkably inhibited the energy transfer from BIPM to DACM residues. Limited proteolysis suggested that BIPM residues are located mainly in the peptides which are assumed to contain ATP binding sites and that DACM residues are located near the phosphorylation sites.

Adenosine Triphosphate↗
PubMed2006

Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not.

Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells.

Animals↗
PubMed1996

Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.

The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.

DNA Mutational Analysis↗
PubMedDate not supplied

[After effect of heat shock on induction of fluorescence and low temperature fluorescence spectra of wheat leaves].

The dynamics of restoration of variable and millisecond delayed fluorescence of chlorophyll a, as well as low-temperature fluorescence spectra at 77 K in leaves of 11-day-old wheat seedlings subjected to heat shock (41.5 degrees C, 20 min) and cultivated for 4 days under white light of different intensity was investigated. A comparative analysis of changes in variable, delayed, and low-temperature fluorescence of chlorophyll a depending on light intensity and the proteolitic activity in leaf homogenates during the restoration of the seedlings was carried out. It follows from the data that the restoration of fluorescence includes several phases and is stimulated by light. A possible mechanism of restoration of fluorescence and photochemical activity of photosystem II is discussed.

Cold Temperature↗
PubMed2003

[Potential and limitations of fluorescence diagnosis and photodynamic therapy. Part I: Fluorescence diagnosis].

Fluorescence diagnostic methods enable tumor visualization via specific excitation of endogenous or exogenous tumor-selective fluorescent markers and subsequent optical measurement of the induced fluorescence signals. In the following review, the reader will be given a clear and extensive understanding of all aspects of these methods relevant for ENT specialists. Apart from a demonstration of the clinical difficulties in the diagnosis of malignant tumors of the upper aerodigestive tract, a historical synopsis of fluorescence diagnosis and an explanation of the biophysical basics, this publication focuses mainly on a detailed discussion of the current status of research and a critical consideration of the commercially available systems for fluorescence diagnosis in ENT oncology. The topic is rounded off by a short account of possible future activities in this field.

Bronchoscopes↗