Showing papers on "Fluorescence spectrometry published in 1998"

Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis.

Abstract: The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, pb, is in the order of 10-6−10-7 for irradiances below 103 W/cm2. However, a considerable increase of pb for irradiances above this level was observed which can only be described by photobleaching reactions from higher e...

Noninvasive diagnosis of oral neoplasia based on fluorescence spectroscopy and native tissue autofluorescence

Abstract: Objective: To evaluate the clinical potential of fluorescence spectroscopy (a noninvasive technique for assessing the chemical and morphologic composition of tissue) for in vivo detection of oral cavity neoplasia. Design: A fluorescence spectroscopy system recorded spectra from oral cavity sites in 8 healthy volunteers and in 15 patients with premalignant or malignant oral cavity lesions at 337-, 365-, and 410-nm excitation wavelengths in the emission range of 350 to 700 nm. Fluorescence peak intensities and spectral line shapes were compared and diagnostic algorithms were developed to distinguish normal sites from abnormal sites. Setting: The head and neck cancer clinic at a tertiary referral center in Houston, Tex. Results: Differences were found in spectra from normal, dysplastic, and malignant oral mucosa. The fluorescence intensity of normal mucosa was greater than that of abnormal areas. In addition, the ratio of red region (635nm) to blue region (455-490-nm) intensities was greater in abnormal areas. Diagnostic discrimination was achieved when test site spectra were compared with spectra from a normal site in the same patient. One diagnostic algorithm based on spectra at 337 nm gave a sensitivity of 88% and a specificity of 100%. Conclusions: Consistent differences exist between the fluorescence spectra of abnormal and normal oral mucosa. Therefore, fluorescence spectroscopy has the potential to improve the noninvasive diagnosis of oral cavity neoplasia. Further studies will better define the role of this technique in the detection of premalignant and early oral cancer lesions. Arch Otolaryngol Head Neck Surg. 1998;124:1251-1258

Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage.

Abstract: An ultrasensitive assay for measuring DNA base damage is described that couples immunochemical recognition with capillary electrophoresis and laser-induced fluorescence detection The method provides a detection limit of 3 × 10−21 moles, an improvement of four to five orders of magnitude over current methods Induction and repair of thymine glycols were studied in irradiated A549 cells (a human lung carcinoma cell line) Exposure of these cells to a low dose of radiation (025 Gray) 4 hours before a clinically relevant dose (2 Gray) enhanced removal of thymine glycols after the higher dose These data provide evidence for an inducible repair response for radiation-induced damage to DNA bases

Fluorescent Functional Recognition Sites through Molecular Imprinting. A Polymer-Based Fluorescent Chemosensor for Aqueous cAMP.

Abstract: A novel design for template-selective recognition sites in polymers prepared by molecular imprinting is reported. Molecular imprints were prepared against cAMP that contain a fluorescent dye, trans-4-[p-(N,N-dimethylamino)styryl]-N-vinylbenzylpyridinium chloride, as an integral part of the recognition cavity, thus serving as both the recognition element and the measuring element for the fluorescence detection of cAMP in aqueous media. This fluorescent molecularly imprinted polymer displays a quenching of fluorescence in the presence of aqueous cAMP, whereas almost no effect is observed in the presence of the structurally similar molecule, cGMP. The association constant for the binding of cAMP to the imprinted polymer was determined to be in the order of 105 M-1. Such fluorescent molecularly imprinted polymers containing selective sites for cAMP may find applications as fluorescent chemosensors for the aqueous detection of this molecule.

Two-Dimensional Fluorescence Spectroscopy: A New Tool for On-Line Bioprocess Monitoring

Abstract: Two-dimensional fluorescence spectroscopy is presented as a new method for bioprocess monitoring. It covers a wide range of excitation and emission wavelengths and is a further development of the fluorescence measurements performed so far, which concentrated mainly on NAD(P)H culture fluorescence. Biogenic fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively inside and outside the cells. This optical method is noninvasive, suitable for in vivo measurements. One whole spectrum (excitation, 250-550 nm; emission, 260-600 nm) with the described parameters is performed within 1 min, which allows an almost continuous monitoring of the bioprocess. The technique is ideal for on-line, in situ measurements via fiber optical systems. Results are presented for cultivations of Claviceps purpurea, Escherichia coli, Saccharomyces cerevisiae, and Sphingomonas yanoikuyae. Cell growth and the metabolism of the cells (changes from aerobic to anaerobic conditions and uncoupling of the oxidative phosphorylation) could be detected.

Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours

Abstract: We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 micromol/L was used as the cutoff Phe concentration for MS/MS and 258 micromol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.

In situ seagrass photosynthesis measured using a submersible, pulse-amplitude modulated fluorometer

Abstract: Assessments of photosynthetic activity in marine plants can now be made in situ using a newly developed, submersible, pulse-amplitude modulated (PAM) fluorometer: Diving-PAM. PAM fluorometry provides a measure of chlorophyll a fluorescence using rapid-light curves in which the electron-transport rate can be determined for plants exposed to ambient light conditions. This technique was used to compare the photosynthetic responses of seagrasses near Rottnest Island, Western Australia. Several fluorescence parameters were measured as a function of time of day and water depth; electron-transport rate (ETR), quantum yield, photochemical quenching and non-photochemical quenching and Photosystem II (PSII) photochemical efficiency (Fv:Fm ratio) were measured. Results indicate that recent light-history plays a crucial role in seagrass photosynthetic responses. Maximum ETR of Posidonia australis, Amphibolis antarctica and Halophila ovalis is influenced by the irradiance during the diurnal cycle, with low rates at dawn and dusk (<10 μmol electron m−2 s−1), highest rates in late morning (40 to 60 μmol electron m−2 s−1) and a mid-day depression. Maximum ETR and PSII photochemical efficiency varied widely between seagrass species and were not correlated. A comparison of photochemical to non-photochemical quenching indicated that seagrasses in shallow water receiving high light have a high capacity for non-photochemical quenching (e.g. light protection) compared to seagrasses in deep water. These results indicate that in situ measurements of photosynthesis will provide new insights into the mechanisms and adaptive responses of marine plants.

Specific fluorescence assay for advanced glycation end products in blood and urine of diabetic patients

Abstract: Late rearrangement products that accumulate by glycation of proteins, known as advanced glycation end products (AGEs), have been implicated in the pathogenesis of complications related to diabetes. Circulating AGEs, especially in the form of a small peptide (AGE-peptide) of less than 10 kd, increase in the blood of diabetic patients with end-stage renal disease (ESRD). The aim of the study was to evaluate AGE-peptide levels by measuring AGE-specific fluorescence (excitation at 370 nm and emission at 440 nm) and to examine the relationship between AGE-peptide and diabetic nephropathy. AGE-specific fluorescence in serum and urine were examined in diabetic subjects with various levels of renal complications of varying severity: normoalbuminuria (N), microalbuminuria (Mi), macroalbuminuria (Ma), chronic renal failure (C), and hemodialysis (HD). We also assessed correlations among the AGE-peptide level and age, duration of diabetes, hemoglobin A1c (HbA1c), serum creatinine, and creatinine clearance. Serum and urine AGE-peptide levels in C and HD were significantly higher than in N, Mi, and Ma. Serum AGE-peptide levels were significantly correlated with serum creatinine (r=.866, P < .0001) and creatinine clearance (r=-.720, P < .0001) but not with duration of diabetes or age. There was a significant correlation between AGE-peptide levels measured by enzyme-linked immunosorbent assay (ELISA) and levels determined from the specific fluorescence intensity (r=.688, P < .0001). These findings suggest that renal function may play a greater role in the accumulation of AGEs than persistent hyperglycemia in diabetic patients. Measurement of AGE-specific fluorescence (ie, AGE-peptide) may serve as a simple and useful test to assess circulating AGE levels and monitor AGE excretion.

Fluorescent, sequence-selective peptide detection by synthetic small molecules.

Abstract: Small organic sensor molecules were prepared that bind and signal the presence of unlabeled tripeptides in a sequence-selective manner. Sequence-selective peptide binding is a difficult problem because small peptides are highly flexible and there are no clear rules for designing peptide-binding molecules as there are for the nucleic acids. The signaling system involved the application of fluorescence energy transfer and provided large, real-time fluorescence increases (300 to 500 percent) upon peptide binding. With it, these sensors were sensitive enough to detect unlabeled cognate peptides both in organic solution and in the solid state at low micromolar concentrations.

Kinetic Aspects of Cation-Enhanced Aggregation in Aqueous Humic Acids

Abstract: The cation-enhanced formation of hydrophobic domains in aqueous humic acids has been shown to be a slow process, consistent with the evolution and disintegration of humic acid configurations over p...

Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis.

Abstract: An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2 dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.

Self-assembly in mixtures of poly(ethylene oxide)-graft-poly(ethyleneimine) and alkyl sulfates

Abstract: The complexes formed between the cationic graft copolymer of poly(ethylene oxide) and poly(ethyleneimine) (PEO-g-PEI) and alkyl sulfate surfactants were studied using potentiometric titration, fluorescence spectroscopy, ζ-potential measurement, dynamic light scattering, and electron microscopy. In contrast to the complexes of PEI homopolymer, which phase separate, the PEO-g-PEI complexes are water-soluble over the whole range of compositions of the mixture, including the electroneutral stoichiometric complexes. These species apparently represent micelle-like aggregates with a hydrophobic core from the surfactant-neutralized polyions and a hydrophilic corona from the ethylene oxide chains. This is a very different morphology compared to the previously described complexes of poly(ethylene oxide)-b-polymethacrylate anions with cationic surfactants, which formed vesicles. The difference in the morphology is attributed to the difference in the copolymer architecture (branched vs linear, graft vs block copolyme...

Fluorescence and NMR studies of the effect of a bile acid dimer on the micellization of bile salts

Abstract: The dimerization of bile salts is known to be the critical step in the formation of bile salt micelles. The influence of a sodium cholate dimer, synthesized by linking two cholic acid molecules via a spacer, on the micellization processes of selected bile salts has been investigated. Fluorescence studies of a pyrene probe and NMR spin−lattice relaxation time experiments have been carried out to determine the critical micellar concentration (cmc) of sodium cholate and sodium glycocholate upon the addition of various amounts of the dimer. The cmc's of the bile salts are significantly lowered by the presence of this dimer, which indicates that the sodium cholate dimer can facilitate the micellization of bile salts.

In Vivo Native Cellular Fluorescence and Histological Characteristics of Head and Neck Cancer

Abstract: Native cellular fluorescence (NCF) represents the innate capacity of tissues to absorb and emit light of a specified wavelength. The ability to define the relationship of in vivo NCF with biological characteristics of neoplastic disease may allow for an improved understanding of the clinical course of disease. Head and neck cancers from 35 patients were evaluated in vivo for NCF characteristics using a xenon lamp-based spectrometer coupled to a handheld fiberoptic probe. Spectral assessment was limited to lambda 450-nm emission characteristics, in which tissues were excited at various wavelengths, ranging from lambda 290 nm to lambda 415 nm, and the intensity of lambda 450 nm emission was recorded. Each cancer was subsequently biopsied and assessed for histological differentiation by a pathologist who was blinded to NCF analysis. Considerable variation in spectral characteristics between head and neck cancers was identified, which was determined, in part, by NCF characteristics of the normal mucosa from the same patient. Poorly differentiated tumors were more likely than well- or moderately differentiated tumors to have lower excitation maxima (P < 0.05 by ANOVA). Most significantly, the tumor differentiation status, as well as the probability of demonstrating recurrent disease, could also be related to the NCF characteristics of the patient's normal mucosa from the same site within the upper aerodigestive tract. NCF analysis may represent an effective tool to identify biological characteristics of head and neck tumors in vivo without the need for invasive biopsies. Results suggest the need to explore the determinants of NCF characteristics expressed by clinically normal mucosa.

New Fluorimetric Alkali and Alkaline Earth Metal Cation Sensors Based on Noncyclic Crown Ethers by Means of Intramolecular Excimer Formation of Pyrene

Abstract: New fluorescent reagents, 2,2‘-[oxybis(3-oxapentamethyleneoxy)]-bis[N-(1-pyrenylmethyl)benzamide)] (4) and its analogues (2, 3, and 5) which have two pyrenes at the both terminals of polyoxyethylene compounds, were synthesized, and their complexation behavior with alkaline earth cations was investigated by fluorescence spectrometry, fluorescence lifetimes, and 1H NMR spectrometry. These reagents (3−5) showed strong intramolecular excimer emissions around at 480 nm in the fluorescence spectra. On the complexation with alkaline earth metal cations, the increase of monomer emission around at 400 nm accompanied by the disappearance of intramolecular excimer emission of free reagents was observed. These reagents formed a 1:1 complex, and the order of complex formation constants was Ca2+ ≅ Sr2+ > Ba2+ > Mg2+ > Li+ for all reagents. 1H NMR spectra of these complexes with alkaline earth cations suggested the helical structures of the complexes. Fluorescence spectral changes at the formation of complexes depended ...

Fluorescence endoscopy of gastrointestinal diseases : basic principles, techniques, and clinical experience

Abstract: Visual detection of tumors, especially during the early stages, is expected to be improved considerably by examining the fluorescence either of tumor-selective exogenous dyes such as protoporphyrin IX, induced by 5-aminolevulinic acid, or by analyzing the autofluorescent properties of healthy and neoplastic tissue. The present paper describes technical devices using light sources for fluorescence excitation, and sensitive detection systems such as intensified cameras and optical multichannel analyzers used for fiber-assisted point measurements. In the discussion of these systems, special consideration is given to their commercial availability and potential for endoscopic applications in the gastrointestinal tract. In this clinical discipline, the major interest lies in the ability to locate malignancies in the esophagus and colon. In recent years, there has been increasing clinical experience in this area, particularly in detecting adenocarcinoma in Barrett's esophagus and malignant alterations in the colon, such as in ulcerative colitis and polyps. Although several research groups have reported sensitivities and specificities for fluorescence gastroscopy of more than 80%, the potential benefits of the technique to patients need to be evaluated in further clinical studies.

Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2

Abstract: Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators...

Comparative study of the luminescent properties of europium and terbium coordinated with thenoyltrifluoroacetone or pyridine-2,6-dicarboxylic acid in aqueous solutions

Abstract: The chelates formed with europium(III) and terbium(III) coordinated with either thenoyltrifluoroacetone or pyridine-2,6-dicarboxylic acid were studied. The formation and the composition of the chelates were compared with respect to the number of donor groups of the ligand and the coordination number of the lanthanide ion. The luminescent properties of the chelates were investigated and compared in terms of the relative luminescence intensity, the linear dynamic range and the detection limit. The results are discussed with respect to the stoichiometry of the chelate, the lifetime of the emitting level and the energy gap between the donor level of the ligand and the emitting level of the ion.

Ensembles of radial basis function networks for spectroscopic detection of cervical precancer

Abstract: The mortality related to cervical cancer can be substantially reduced through early detection and treatment. However, current detection techniques, such as Pap smear and colposcopy, fail to achieve a concurrently high sensitivity and specificity. In vivo fluorescence spectroscopy is a technique which quickly, noninvasively and quantitatively probes the biochemical and morphological changes that occur in precancerous tissue. A multivariate statistical algorithm was used to extract clinically useful information from tissue spectra acquired from 361 cervical sites from 95 patients at 337-, 380-, and 460-nm excitation wavelengths. The multivariate statistical analysis was also employed to reduce the number of fluorescence excitation-emission wavelength pairs required to discriminate healthy tissue samples from precancerous tissue samples. The use of connectionist methods such as multilayered perceptrons, radial basis function (RBF) networks, and ensembles of such networks was investigated. RBF ensemble algorithms based on fluorescence spectra potentially provide automated and near real-time implementation of precancer detection in the hands of nonexperts. The results are more reliable, direct, and accurate than those achieved by either human experts or multivariate statistical algorithms.

Single-Molecule Identification in Flowing Sample Streams by Fluorescence Burst Size and Intraburst Fluorescence Decay Rate

Abstract: We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.