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PubMed1994

[Time-resolved fluorometry: principles and applications in clinical biology].

Time-resolved fluorometric assay is based on lanthanide fluorescence. This time-resolved fluorescence has a narrow-band emission line whose wavelength differs from that of emission-pulsed light and has a long decay-time. These characteristics make it possible to avoid background interference from sample constituents (protein, light-scattering particles, etc). Europium and its chelates are the most commonly used lanthanides. The europium-labelling of antigens or antibodies is followed by immunoassay. In the final step, fluorescence is measured, after enhancement, as counts per second. This assay has several advantages, including a wide working range, high sensitivity and good practicability. The method has widespread applications in the field of immunoassays in both clinical and research laboratories. The use of non-radioactive europium-labelled probes and the development of simultaneous multiple tests are possible future orientations.

Europium↗
PubMed1994

Time-resolved fluorometry in the genetic diagnosis of familial defective apolipoprotein B-100.

A novel technique for screening point mutations has been developed for diagnosis of familial defective apolipoprotein (apo) B-100 (FDB). In FDB, an amino acid exchange occurs at position 3500 in apoB-100 due to a point mutation. Polymerase chain reaction (PCR) was performed on the appropriate region of the apoB gene, and the PCR products were hybridized in solution with europium-labeled oligonucleotides, complementary to either the wildtype or the mutant genome. The presence or absence of the apoB-3500 mutation was monitored by time-resolved fluorescence of the europium chelate. The method allows a larger number of samples to be processed simultaneously, and the detection system displays a high level of sensitivity without the hazards connected to the use of radioactivity. When 127 Swedish patients, clinically diagnosed as suffering from heterozygous familial hypercholesterolemia, were screened for the presence of the apoB-3500 mutation, two patients, unrelated to each other, were found to be heterozygotes. These patients are the first reported cases of FDB from Sweden, and the frequency rate observed among hypercholesterolemic patients, 1.6%, is in accordance with the figures reported for several other patient population in Europe and the United States.

Adult↗
PubMedDate not supplied

Cytotoxic T lymphocyte control during ectromelia (mousepox) virus infection: interaction between MHC-restricted cells analyzed by non-radioactive fluorometry.

Cytotoxic T lymphocyte (CTL) activity of draining lymph node (DLN) cells isolated from BALB/c mice infected with ectromelia virus (EV) was examined using a fluorometric cell-mediated cytotoxicity (CMC) assay. Specific lysis of target cells A20 and EMT-6 primed with EV was demonstrated. The classical CD8+ cytolytic pathway dominated (72.7%) as compared to that of CD4+ (27.3%) in the cellular response during acute EV infection. Also an alternative method for determining CMC, employing a bisbenzamide dye for labelling target cells, is described. Coefficient variations of relative fluorescence were below 6%, that makes the method sensitive and reliable.

Animals↗
PubMed1977

Simultaneous determination of captan and captafol in apples and potatoes by thin layer chromatography and in situ fluorometry.

A simple method is described for the simultaneous quantitative determination of captan [N-(trichloromethylthio)-3a,4,7,7a-tetrahydrophthalimide] and captafol [N-(1,1,2,2-tetrachloroethylthio) -3a,4,7,7a - tetrahydrophthalimide] in apples and potatoes. The fungicide residues are extracted with acetonitrile, partitioned into a solution of methylene chloride in petroleum ether, separated from each other and the co-extractives on a thin layer chromatographic plate impregnated with aluminum chloride, sprayed with sodium chlorate, and heated. The fluorescence is then measured directly from the chromatogram. Recoveries were greater than 90% in the test crops at the 0.2 ppm level.

Captan↗
PubMed1999

Mapping retinal fluorescein leakage with confocal scanning laser fluorometry of the human vitreous.

OBJECTIVE: To demonstrate an objective, quantitative, and sensitive method of mapping retinal fluorescein leakage into the vitreous while simultaneously imaging the retina. METHODS: A prototype Zeiss confocal scanning laser ophthalmoscope was modified to obtain fluorometric measurements from 18 optical planes across the retina and cortical vitreous, separated from each other by 150 microm, and parallel to the retinal surface. After intravenous administration of fluorescein, an axial graphic of equivalent fluorescein concentration in the vitreous may be obtained from any region of interest. After correcting for fluorescence levels in the retina and choroid and plasma levels of free fluorescein, permeability values of the blood-retinal barrier to fluorescein were obtained from 1512 regions measuring 75 x 75 microm, from a total 3150 x 2700-microm area of the fundus, generating a detailed map of retinal fluorescein leakage. The method was assessed in vitro and in 7 healthy subjects who underwent scans during separate visits. Depth resolution and influence of chorioretinal fluorescence were further tested in 2 patients with multiple drusen and in 2 eyes after vitrectomy. Fourteen eyes from 7 patients with diabetes and nonproliferative retinopathy were also examined. Lateral resolution was tested in 3 diabetic eyes that underwent focal photocoagulation. Four eyes from 2 patients with diabetes and minimal retinopathy were examined at 3-month intervals. All eyes examined had less than 2 diopters of astigmatism. RESULTS: Characteristics of the modified confocal scanning laser fluorometer included a lower limit of detection equal to 0.40 Eq ng/mL and depth precision of +/-15 microm. Values for the blood-retinal barrier permeability index in healthy subjects, measured 30 minutes after a single intravenous pulse of fluorescein (14 mg/kg), ranged from 1.3 +/- 0.4 x 10(-6) cm/s over the foveal avascular zone to 2.2 +/- 0.6 x 10(-6) cm/s over vessels in the retina. Diabetic eyes with retinopathy showed higher values, ranging from 1.4 to 15.0 x 10(-6) cm/s. Vitrectomized eyes and eyes with multiple drusen showed the validity of the correction algorithm demonstrating that measurements of fluorescence in the vitreous are not influenced by the chorioretinal fluorescence level. Argon laser photocoagulation burns placed in the diabetic retina demonstrated a lateral resolution on the order of 75 to 100 microm. Intravisit and intervisit reproducibility was +/-10.2% and +/-13%, respectively. CONCLUSIONS: This new method measures localized alterations of the blood-retinal barrier and allows for direct correlation with retinal anatomy. Its most interesting feature is the ability to map retinal fluorescein leakage while simultaneously imaging the retina. This capability is expected to improve our understanding and management of retinal disease.

Adult↗
PubMed1999

Photosynthetic characteristics of marine phytoplankton from pump-during-probe fluorometry of individual cells at sea.

BACKGROUND: Active fluorescence techniques are becoming commonly used to monitor the state of the photosynthetic apparatus in natural populations of phytoplankton, but at present these are bulk water measurements that average all the fluorescent material in each sample. Here we describe two instruments that combine individual-cell "pump-during-probe" (PDP) measurements of chlorophyll (Chl) fluorescence induction, on the time scale of 30 to 100 micros, with flow cytometric or visual characterization of each cell. METHODS: In the PDP flow cytometer, we measure Chl fluorescence yield as a function of time during a 150 micros excitation flash provided by an argon ion laser; each particle is subsequently classified as in a conventional flow cytometer. In the PDP microfluorometer, individual cells in a sample chamber are visually identified, and fluorescence excitation is provided by a blue light-emitting diode that can be configured to provide a saturating flash and also a subsequent series of short flashlets. This sequence allows both saturation and relaxation kinetics to be monitored. RESULTS: Phytoplankton from natural samples and on-deck iron-enrichment incubation experiments in the Southern Ocean were examined with each PDP instrument, providing estimates of the potential quantum yield of photochemistry and the functional absorption cross section for photosystem 2, for either individuals (for cells larger than a few micrometers) or populations (for smaller cells). CONCLUSIONS: Results from initial field applications indicate that single-cell PDP measurements can be a powerful tool for investigating the nutritional state of phytoplankton cells and the regulation of phytoplankton growth in the sea.

Cell Count↗
PubMedDate not supplied

Time-resolved fluorometry in end-point and real-time PCR quantification of nucleic acids.

Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5'-->3' exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 x 10(6) and 10-5 x 10(7) input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.

Base Sequence↗
PubMed2002

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies.

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.

Aspergillus oryzae↗
PubMed1995

Measurement of basal tear turnover using a standardized protocol. European concerted action on ocular fluorometry.

BACKGROUND: The aim of this study was to compare basal tear turnover values of healthy volunteers in different countries. METHODS: Healthy volunteers aged between 20 and 70 years were selected in three European cities. Basal tear turnover values were calculated according to a standardized protocol from the decay of the fluorescein concentration in tears after instillation of 1-microliters drop of fluorescein in the conjunctival sac. Fluorescein concentration was measured with identical commercial fluorophotometers. A mono-exponential decay of fluorescein was assumed to represent basal tear flow. RESULTS: The mean tear turnover values were 13.1%/min +/- 4.6 SD (n = 4), 16.0%/min +/- 5.2 SD (n = 24) and 17.5%/min +/- 3.4 SD (n = 20) in Clermont-Ferrand (France), Leiden (The Netherlands) and Madrid (Spain), respectively. The differences between the values were not significant (Mann-Whitney test P > 0.09). CONCLUSIONS: The tear turnover in the different cities was similar. The methods used were simple and the software easy to use.

Adult↗
PubMed1988

The application of immunoassays and fluorometry to the detection of polycyclic hydrocarbon-macromolecular adducts and anti-adduct antibodies in humans.

The metabolic activation of polycyclic aromatic hydrocarbons (PAH) to chemical species that form covalent adducts with cellular macromolecules (DNA and protein) is central to theories of carcinogenesis. Assays are currently being developed that will accurately reflect human macromolecular exposure to these carcinogens. Immunoassays are capable of detecting low levels of PAH-DNA adducts and antibodies directed against these adducts in humans and HPLC/spectrophotofluorimetry allows the detection of carcinogen-DNA or carcinogen-protein adducts in human peripheral blood. Both types of method have inherent advantages and disadvantages, and the use of more than one type of corroborative assay is a feature in our work. Simplified but highly specific synchronous fluorescence spectra have been obtained for BP-tetrols after mild acid hydrolysis and HPLC of sample materials. When using a wavelength difference of 34 nm to drive the excitation and emission monochromators simultaneously, the pyrene fluorophore, when present, has a signature peak at 345 nm (excitation). The results of immunoassays demonstrate human exposure to PAH as a class of carcinogen, since serological cross-reactivity of antibodies does not limit detection in this system to a single chemical compound. In addition the formation in humans of anti-PAH-DNA antibodies has been shown, presumably in response to both past and present exposure to the parent compounds. In summary the results of each assay can indicate human exposure to PAH and have the potential for molecular dosimetry of this exposure.

Antibodies↗
PubMed1981

A flexible system for microscope fluorometry served by a personal computer.

An automated microscope fluorometer based on a modified Leitz MPV I system is described. Automation is mainly attained by the use of a cheap, commercially available personal computer system programmed in extended BASIC supplemented by a few assembly-level subroutines. The automation has resulted in simplified measuring procedures and increased measuring speed. The statistical precision is enhanced because greater cell populations can be analysed within reasonable time. Programs for several biological applications are described. Routines for cell population analysis, including storage of data on disk files, has proved especially useful. Programs for recording of corrected fluorescence emission spectra and of time-dependent fluorescence variables have also been developed. A high flexibility is achieved, as the BASIC programs can readily be modified for specific purposes. Some data on the sensitivity and reproducibility of the instrument alone, and in combination with a staining method, are also presented.

Animals↗
PubMed1977

DNA-fluorometry of mammalian sperm.

The Dna-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescene distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.

Animals↗
PubMed1997

Permeability of the blood-retinal barrier in healthy humans. European Concerted Action on Ocular Fluorometry.

BACKGROUND: The aim of this study was to compare the inward permeability of the blood-retinal barrier in healthy subjects from six European cities. METHODS: Seventy-two healthy subjects (age 20-70 years) were selected. At 30 min and 60 min after fluorescein injection, fluorescein mass in vitreous was calculated from the concentrations measured along the optical axis of the eye. Non-protein-bound fluorescein (NPBF) concentrations were measured in plasma prepared from blood samples taken 7, 15 and 55 min after injection. Blood-retinal barrier permeability (PBRB) was calculated from the vitreous fluorescein mass and the time integral of NPBF and was corrected for the autofluorescence of ocular tissue and for lenticular light transmittance. RESULTS: Mean PBRB values +/- SD (nm.s-1) were 2.07 +/- 0.54 (Coimbra), 2.01 +/- 0.43 (Frankfurt), 2.24 +/- 0.50 (Ghent), 2.37 +/- 0.56 (Herlev), 1.89 +/- 0.44 (Leiden) and 1.74 +/- 0.38 (Porto). Differences between centers were not significant (P > 0.35). Measurements were reproducible and independent of the time after fluorescein injection (P > 0.50). A PBRB higher than 3.16 nm.s-1 or a value which had increased by 32% was considered abnormal (P < 0.05). CONCLUSION: PBRB values were similar in all centers. The results demonstrate that this is a highly sensitive and reliable method for measuring the permeability of the blood-retinal barrier.

Adult↗
PubMed1992

Interactions between volatile anesthetics and dipalmitoyl phosphatidylcholine liposomes as studied by fluorometry with a thiacarbocyanine dye.

The effects of volatile anesthetics on the properties of dipalmitoyl phosphatidylcholine liposome were investigated by fluorescence spectroscopy with a thiacarbocyanine dye (3,3"-dioctadecyl-2,2"-thiacarbocyanine) which is sensitive to the viscosity and the dielectric constant of the environment. Seven volatile anesthetics, halothane, enflurane, isoflurane, methoxyflurane, sevoflurane, diethylether and chloroform were used. All anesthetics decreased the phase transition temperature of the liposome and increased the effective dielectric constant of the water-liposome interface. The increase of the effective dielectric constant was attributed to the release of the hydrated water molecules from the membrane surface. The increment of the effective dielectric constant depended on the thermodynamic activity of anesthetics in the solution, and was not affected seriously by the kind of anesthetics. On the other hand, the degree of the depression of the phase transition temperature depended on the molar concentrations of anesthetics. Considering from the Ferguson's report, which is dealt with the relationship between the physiological effect and the thermodynamic activity, the effect of anesthetics on the effective dielectric constant of the membrane surface is more correlated to the anesthetic action than the effect on the phase transition temperature.

Journal Article↗
PubMed2005

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part I--Immunometric assays.

Seven different two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) and a frequently used TAMRA label were conjugated to mouse IgG against alpha-fetoprotein in variable substitution degrees. Altogether 40 IgG conjugates were prepared, and studied with respect to one-photon absorption and emission properties, and two-photon fluorescence efficiency using 1064 nm laser as illumination source. Performance of the IgG conjugates as tracers in a separation-free immunometric assay of alpha-fetoprotein was evaluated using two-photon excitation assay technology, ArcDia TPX. The results show that the dipyrrylmethene-BF(2) labels provide subpicomolar sensitivity, which is an order of magnitude better than that of TAMRA label. The effect of chromophore structure and substitution degree of IgG-label conjugates on the assay performance is discussed.

Journal Article↗
PubMed2005

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part II--Nucleic acid hybridization assays.

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF(2) labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.

Journal Article↗