Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium.

PubMed

Feldman, Tatiana B; Yakovleva, Marina A; Larichev, Andrey V; Arbukhanova, Patimat M; Radchenko, Alexandra Sh; Borzenok, Sergey A; Kuzmin, Vladimir A; Ostrovsky, Mikhail A

2018-05-22

The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.

[Discrimination of Red Tide algae by fluorescence spectra and principle component analysis].

PubMed

Su, Rong-guo; Hu, Xu-peng; Zhang, Chuan-song; Wang, Xiu-lin

2007-07-01

Fluorescence discrimination technology for 11 species of the Red Tide algae at genus level was constructed by principle component analysis and non-negative least squares. Rayleigh and Raman scattering peaks of 3D fluorescence spectra were eliminated by Delaunay triangulation method. According to the results of Fisher linear discrimination, the first principle component score and the second component score of 3D fluorescence spectra were chosen as discriminant feature and the feature base was established. The 11 algae species were tested, and more than 85% samples were accurately determinated, especially for Prorocentrum donghaiense, Skeletonema costatum, Gymnodinium sp., which have frequently brought Red tide in the East China Sea. More than 95% samples were right discriminated. The results showed that the genus discriminant feature of 3D fluorescence spectra of Red Tide algae given by principle component analysis could work well.

Development of a Terbium-Sensitized Fluorescence Method for Analysis of Silibinin.

PubMed

Ershadi, Saba; Jouyban, Abolghasem; Molavi, Ommoleila; Shayanfar, Ali

2017-05-01

Silibinin is a natural flavonoid with potent anticancer properties, as shown in both in vitro and in vivo experiments. Various methods have been used for silibinin analysis. Terbium-sensitized fluorescence methods have been widely used for the determination of drugs in pharmaceutical preparations and biological samples in recent years. The present work is aimed at providing a simple analytical method for the quantitative determination of silibinin in aqueous solutions based on the formation of a fluorescent complex with terbium ion. Terbium concentration, pH, and volume of buffer, the important effective parameters for the determination of silibinin by the proposed method, were optimized using response surface methodology. The fluorescence intensity of silibinin was measured at 545 nm using λex = 334 nm. The developed method was applied for the determination of silibinin in plasma samples after protein precipitation with acetone. Under optimum conditions, the method provided a linear range between 0.10 and 0.50 mg/L, with a coefficient of determination (R2) of 0.997. The LOD and LOQ were 0.034 and 0.112 mg/L, respectively. These results indicate that the developed method is a simple, low-cost, and suitable analytical method for the quantification of silibinin in aqueous solution and plasma samples.

Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements.

PubMed

DeVore, Matthew S; Gull, Stephen F; Johnson, Carey K

2012-04-05

We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

PubMed

Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-15

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

[Determination of H2S in Rat Intestinal Perfusion Solution Based on Fluorescence Analysis].

PubMed

Hou, Jun-feng; Li, Xin-xia; Shen, Xue-ru; Huojia, Miliban; Guan, Ming

2015-08-01

Under alkaline conditions, Fluorescein mercury has strong fluorescence, however, when it met S(2-), its fluorescence would quench, in view of the above, a fluorescence method for determination of H2S in biological samples was established. In the 0.1 mol · L(-1) NaOH dilution, when the concentration of fluorescein Mercury and Na2S was 5.0 × 10(-5) and 1.0 × 10(-5) mol · L(-1) respectively, the fluorescence intensity of system was determined at 522 nm. The results showed that, at the range of 4.0 × 10(-7)~2.0 × 10(-6) mol · L(-1), the concentration decreasing of H2S and fluorescence intensity had good linear relationship, r=0.9980, the RSD of precision test was 4.59% (n=7), the detection limit was 3.5 × 10(-8) mol · L(-1), the content of H2S in the sample were 1.01 × 10(-6) and 1.15 × 10(-6) mol · L(-1), and the recovery rate was 95.8%~101.0%, the method has the advantages of simple operation, high sensitivity, good selectivity, can accurately determine of H2S in intestinal perfused solution, and provides the basis for the determination of endogenous H2S.

The Determination of Metals in Welding Fume by X-RaySpectrometry

NASA Astrophysics Data System (ADS)

Kuznetsova, O. V.; Begunova, L. A.; Romanenko, S. V.; Solodsky, S. A.

2018-01-01

Analysis of the current hygienic situation in the welding production showed that the intensification of welding processes involves the deterioration of air quality, which negatively affects the welders health. Respiratory effects seen in full-time welders have included bronchitis, airway irritation, lung function changes, and a possible increase in the incidence of lung cancer. The metal concentration in the air of the working area have been determined using the photometric method of analysis, which involves the stage of decomposition of the sample material before analysis. However, losses of the analyzed elements are possible when the sample is decomposed. The X-ray fluorescence method of analysis has the advantage of being nondestructive. The investigations shown the data of photometric determination of metals in welding aerosols is 1.5÷2 times lower than the results of X-ray fluorescence analysis.

Application of magnetic carriers to two examples of quantitative cell analysis

NASA Astrophysics Data System (ADS)

Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

2017-04-01

The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

QSPR studies on the photoinduced-fluorescence behaviour of pharmaceuticals and pesticides.

PubMed

López-Malo, D; Bueso-Bordils, J I; Duart, M J; Alemán-López, P A; Martín-Algarra, R V; Antón-Fos, G M; Lahuerta-Zamora, L; Martínez-Calatayud, J

2017-07-01

Fluorimetric analysis is still a growing line of research in the determination of a wide range of organic compounds, including pharmaceuticals and pesticides, which makes necessary the development of new strategies aimed at improving the performance of fluorescence determinations as well as the sensitivity and, especially, the selectivity of the newly developed analytical methods. In this paper are presented applications of a useful and growing tool suitable for fostering and improving research in the analytical field. Experimental screening, molecular connectivity and discriminant analysis are applied to organic compounds to predict their fluorescent behaviour after their photodegradation by UV irradiation in a continuous flow manifold (multicommutation flow assembly). The screening was based on online fluorimetric measurement and comprised pre-selected compounds with different molecular structures (pharmaceuticals and some pesticides with known 'native' fluorescent behaviour) to study their changes in fluorescent behaviour after UV irradiation. Theoretical predictions agree with the results from the experimental screening and could be used to develop selective analytical methods, as well as helping to reduce the need for expensive, time-consuming and trial-and-error screening procedures.

A novel way to monitor urine concentration: fluorescent concentration matrices.

PubMed

Dubayova, Katarina; Luckova, Iveta; Sabo, Jan; Karabinos, Anton

2015-01-01

The amount of water found in urine is important diagnostic information; nevertheless it is not yet directly determined. Indirectly, the water content in urine is expressed by its density (specific gravity). However, without the diuresis value it is not possible to determine whether the increase in density of urine is due to a decrease in water secretion or an increase in the concentration of secreted substances. This problem can be solved by the use of fluorescent concentration 3D-matrices which characterise urine concentration through the pφ (or -logφ) value of the first fluorescence centre. The urine fluorescent concentration 3D-matrix was created by the alignment of the synchronous spectra of the dilution series of urine starting from undiluted (pφ = 0) to 1000-fold diluted urine (pφ = 3). Using the fluorescence concentration 3D-matrix analysis of the urine samples from healthy individuals, a reference range was established for the value pφ, determining the normal, concentrated or diluted type of urine. The diagnostic potential of this approach was tested on urine samples from two patients with a chronic glomerulonephritis. The pφ value of the urine fluorescence concentration 3D-matrix analysis determines whether the urine sample falls within the normal, concentrated or diluted type of urine. This parameter can be directly utilised in sportsmen's hydration state monitoring, as well as in the diagnosis and treatment of serious diseases. An important advantage of this novel diagnostic approach is that a 12/24 h urine collection is not required, which predetermines it for use especially within paediatrics.

Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

PubMed

Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

2014-05-01

The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases

NASA Astrophysics Data System (ADS)

Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

2014-01-01

Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures

PubMed Central

Singh, Pali P.; Hawthorne, Jenci L.; Davis, Christie A.; Quintero, Omar A.

2016-01-01

Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. PMID:27126922

Measuring fluorescence polarization with a dichrometer.

PubMed

Sutherland, John C

2017-09-01

A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer. Copyright © 2017. Published by Elsevier Inc.

Classic Maximum Entropy Recovery of the Average Joint Distribution of Apparent FRET Efficiency and Fluorescence Photons for Single-molecule Burst Measurements

PubMed Central

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2012-01-01

We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions. PMID:22338694

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

PubMed Central

Feinstein, Efraim; Deikus, Gintaras; Rusinova, Elena; Rachofsky, Edward L.; Ross, J. B. Alexander; Laws, William R.

2003-01-01

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay. PMID:12524313

Determining absolute protein numbers by quantitative fluorescence microscopy.

PubMed

Verdaasdonk, Jolien Suzanne; Lawrimore, Josh; Bloom, Kerry

2014-01-01

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition. © 2014 Elsevier Inc. All rights reserved.

Analysis of root surface properties by fluorescence/Raman intensity ratio.

PubMed

Nakamura, Shino; Ando, Masahiro; Hamaguchi, Hiro-O; Yamamoto, Matsuo

2017-11-01

The aim of this study is to evaluate the existence of residual calculus on root surfaces by determining the fluorescence/Raman intensity ratio. Thirty-two extracted human teeth, partially covered with calculus on the root surface, were evaluated by using a portable Raman spectrophotometer, and a 785-nm, 100-mW laser was applied for fluorescence/Raman excitation. The collected spectra were normalized to the hydroxyapatite Raman band intensity at 960 cm -1 . Raman spectra were recorded from the same point after changing the focal distance of the laser and the target radiating angle. In seven teeth, the condition of calculus, cementum, and dentin were evaluated. In 25 teeth, we determined the fluorescence/Raman intensity ratio following three strokes of debridement. Raman spectra collected from the dentin, cementum, and calculus were different. After normalization, spectra values were constant. The fluorescence/Raman intensity ratio of calculus region showed significant differences compared to the cementum and dentin (p < 0.05). The fluorescence/Raman intensity ratio decreased with calculus debridement. For this analysis, the delta value was defined as the difference between the values before and after three strokes, with the final 2 delta values close to zero, indicating a gradual asymptotic curve and the change in intensity ratio approximating that of individual constants. Fluorescence/Raman intensity ratio was effectively used to cancel the angle- and distance-dependent fluctuations of fluorescence collection efficiency during measurement. Changes in the fluorescence/Raman intensity ratio near zero suggested that cementum or dentin was exposed, and calculus removed.

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10*

PubMed Central

Ho, Derek; Lugo, Miguel R.; Merrill, A. Rod

2013-01-01

The pre-channel state of helices 6, 7, and 10 (Val447–Gly475 and Ile508–Ile522) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain. PMID:23264635

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

Critical analysis of commonly used fluorescence metrics to characterize dissolved organic matter.

PubMed

Korak, Julie A; Dotson, Aaron D; Summers, R Scott; Rosario-Ortiz, Fernando L

2014-02-01

The use of fluorescence spectroscopy for the analysis and characterization of dissolved organic matter (DOM) has gained widespread interest over the past decade, in part because of its ease of use and ability to provide bulk DOM chemical characteristics. However, the lack of standard approaches for analysis and data evaluation has complicated its use. This study utilized comparative statistics to systematically evaluate commonly used fluorescence metrics for DOM characterization to provide insight into the implications for data analysis and interpretation such as peak picking methods, carbon-normalized metrics and the fluorescence index (FI). The uncertainty associated with peak picking methods was evaluated, including the reporting of peak intensity and peak position. The linear relationship between fluorescence intensity and dissolved organic carbon (DOC) concentration was found to deviate from linearity at environmentally relevant concentrations and simultaneously across all peak regions. Comparative analysis suggests that the loss of linearity is composition specific and likely due to non-ideal intermolecular interactions of the DOM rather than the inner filter effects. For some DOM sources, Peak A deviated from linearity at optical densities a factor of 2 higher than that of Peak C. For carbon-normalized fluorescence intensities, the error associated with DOC measurements significantly decreases the ability to distinguish compositional differences. An in-depth analysis of FI determined that the metric is mostly driven by peak emission wavelength and less by emission spectra slope. This study also demonstrates that fluorescence intensity follows property balance principles, but the fluorescence index does not. Copyright © 2013 Elsevier Ltd. All rights reserved.

Recent results of synchrotron radiation induced total reflection X-ray fluorescence analysis at HASYLAB, beamline L

NASA Astrophysics Data System (ADS)

Streli, C.; Pepponi, G.; Wobrauschek, P.; Jokubonis, C.; Falkenberg, G.; Záray, G.; Broekaert, J.; Fittschen, U.; Peschel, B.

2006-11-01

At the Hamburger Synchrotronstrahlungslabor (HASYLAB), Beamline L, a vacuum chamber for synchrotron radiation-induced total reflection X-ray fluorescence analysis, is now available which can easily be installed using the adjustment components for microanalysis present at this beamline. The detector is now in the final version of a Vortex silicon drift detector with 50-mm 2 active area from Radiant Detector Technologies. With the Ni/C multilayer monochromator set to 17 keV extrapolated detection limits of 8 fg were obtained using the 50-mm 2 silicon drift detector with 1000 s live time on a sample containing 100 pg of Ni. Various applications are presented, especially of samples which are available in very small amounts: As synchrotron radiation-induced total reflection X-ray fluorescence analysis is much more sensitive than tube-excited total reflection X-ray fluorescence analysis, the sampling time of aerosol samples can be diminished, resulting in a more precise time resolution of atmospheric events. Aerosols, directly sampled on Si reflectors in an impactor were investigated. A further application was the determination of contamination elements in a slurry of high-purity Al 2O 3. No digestion is required; the sample is pipetted and dried before analysis. A comparison with laboratory total reflection X-ray fluorescence analysis showed the higher sensitivity of synchrotron radiation-induced total reflection X-ray fluorescence analysis, more contamination elements could be detected. Using the Si-111 crystal monochromator also available at beamline L, XANES measurements to determine the chemical state were performed. This is only possible with lower sensitivity as the flux transmitted by the crystal monochromator is about a factor of 100 lower than that transmitted by the multilayer monochromator. Preliminary results of X-ray absorption near-edge structure measurements for As in xylem sap from cucumber plants fed with As(III) and As(V) are reported. Detection limits of 170 ng/l of As in xylem sap were achieved.

Setting Standards for Reporting and Quantification in Fluorescence-Guided Surgery.

PubMed

Hoogstins, Charlotte; Burggraaf, Jan Jaap; Koller, Marjory; Handgraaf, Henricus; Boogerd, Leonora; van Dam, Gooitzen; Vahrmeijer, Alexander; Burggraaf, Jacobus

2018-05-29

Intraoperative fluorescence imaging (FI) is a promising technique that could potentially guide oncologic surgeons toward more radical resections and thus improve clinical outcome. Despite the increase in the number of clinical trials, fluorescent agents and imaging systems for intraoperative FI, a standardized approach for imaging system performance assessment and post-acquisition image analysis is currently unavailable. We conducted a systematic, controlled comparison between two commercially available imaging systems using a novel calibration device for FI systems and various fluorescent agents. In addition, we analyzed fluorescence images from previous studies to evaluate signal-to-background ratio (SBR) and determinants of SBR. Using the calibration device, imaging system performance could be quantified and compared, exposing relevant differences in sensitivity. Image analysis demonstrated a profound influence of background noise and the selection of the background on SBR. In this article, we suggest clear approaches for the quantification of imaging system performance assessment and post-acquisition image analysis, attempting to set new standards in the field of FI.

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

PubMed Central

Olsen, Katja N.; Budde, Birgitte B.; Siegumfeldt, Henrik; Rechinger, K. Björn; Jakobsen, Mogens; Ingmer, Hanne

2002-01-01

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments. PMID:12147523

Interference techniques in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dogan, Mehmet

We developed a set of interference-based optical microscopy techniques to study biological structures through nanometer-scale axial localization of fluorescent biomarkers. Spectral self-interference fluorescence microscopy (SSFM) utilizes interference of direct and reflected waves emitted from fluorescent molecules in the vicinity of planar reflectors to reveal the axial position of the molecules. A comprehensive calculation algorithm based on Green's function formalism is presented to verify the validity of approximations used in a far-field approach that describes the emission of fluorescent markers near interfaces. Using the validated model, theoretical limits of axial localization were determined with emphasis given to numerical aperture (NA) dependence of localization uncertainty. SSFM was experimentally demonstrated in conformational analysis of nucleoproteins. In particular, interaction between surface-tethered 75-mer double strand DNA and integration host factor (IHF) protein was probed on Si-SiO2 substrates by determining the axial position of fluorescent labels attached to the free ends of DNA molecules. Despite its sub-nanometer precision axial localization capability, SSFM lacks high lateral resolution due to the low-NA requirement for planar reflectors. We developed a second technique, 4Pi-SSFM, which improves the lateral resolution of a conventional SSFM system by an order of magnitude while achieving nanometer-scale axial localization precision. Using two opposing high-NA objectives, fluorescence signal is interferometrically collected and spectral interference pattern is recorded. Axial position of emitters is found from analysis of the spectra. The 4Pi-SSFM technique was experimentally demonstrated by determining the surface profiles of fabricated glass surfaces and outer membranes of Shigella, a type of Gram-negative bacteria. A further discussion is presented to localize surface O antigen, which is an important oligosaccharide structure in the virulence mechanism of the Gram-negative bacteria, including E. coli and Shigella.

A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

PubMed Central

Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

2013-01-01

This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

Kinetic Fluorescence Experiment for the Determination of Thiamine.

ERIC Educational Resources Information Center

Bower, Nathan W.

1982-01-01

Background information, procedures, and typical results are provided for an experiment which integrates principles of fluorescent and kinetic analysis. In the procedure, mecuric chloride is used as a selective oxidizing agent for converting thiamine to thiochrome. The experiment can be completed in a two-hour laboratory period. (Author/JN)

Analysis of plastid number, size, and distribution in Arabidopsis plants by light and fluorescence microscopy.

PubMed

Pyke, Kevin

2011-01-01

Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis.

Forster resonance energy transfer in the system of human serum albumin-xanthene dyes

NASA Astrophysics Data System (ADS)

Kochubey, V. I.; Pravdin, A. B.; Melnikov, A. G.; Konstantinova, I.; Alonova, I. V.

2016-04-01

The processes of interaction of fluorescent probes: eosin and erythrosine with human serum albumin (HSA) were studied by the methods of absorption and fluorescence spectroscopy. Extinction coefficients of probes were determined. Critical transfer radius and the energy transfer efficiency were defined by fluorescence quenching of HSA. Analysis of the excitation spectra of HSA revealed that the energy transfer process is carried out mainly between tryptophanyl and probes.

Fluorescence detection of dental calculus

NASA Astrophysics Data System (ADS)

Gonchukov, S.; Biryukova, T.; Sukhinina, A.; Vdovin, Yu

2010-11-01

This work is devoted to the optimization of fluorescence dental calculus diagnostics in optical spectrum. The optimal wavelengths for fluorescence excitation and registration are determined. Two spectral ranges 620 - 645 nm and 340 - 370 nm are the most convenient for supra- and subgingival calculus determination. The simple implementation of differential method free from the necessity of spectrometer using was investigated. Calculus detection reliability in the case of simple implementation is higher than in the case of spectra analysis at optimal wavelengths. The use of modulated excitation light and narrowband detection of informative signal allows us to decrease essentially its diagnostic intensity even in comparison with intensity of the low level laser dental therapy.

Measuring fluorescence polarization with a dichrometer

DOE PAGES

Sutherland, John C.

2017-04-06

In this article, a method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of amore » polarizer.« less

Time-Gated Raman Spectroscopy for Quantitative Determination of Solid-State Forms of Fluorescent Pharmaceuticals.

PubMed

Lipiäinen, Tiina; Pessi, Jenni; Movahedi, Parisa; Koivistoinen, Juha; Kurki, Lauri; Tenhunen, Mari; Yliruusi, Jouko; Juppo, Anne M; Heikkonen, Jukka; Pahikkala, Tapio; Strachan, Clare J

2018-04-03

Raman spectroscopy is widely used for quantitative pharmaceutical analysis, but a common obstacle to its use is sample fluorescence masking the Raman signal. Time-gating provides an instrument-based method for rejecting fluorescence through temporal resolution of the spectral signal and allows Raman spectra of fluorescent materials to be obtained. An additional practical advantage is that analysis is possible in ambient lighting. This study assesses the efficacy of time-gated Raman spectroscopy for the quantitative measurement of fluorescent pharmaceuticals. Time-gated Raman spectroscopy with a 128 × (2) × 4 CMOS SPAD detector was applied for quantitative analysis of ternary mixtures of solid-state forms of the model drug, piroxicam (PRX). Partial least-squares (PLS) regression allowed quantification, with Raman-active time domain selection (based on visual inspection) improving performance. Model performance was further improved by using kernel-based regularized least-squares (RLS) regression with greedy feature selection in which the data use in both the Raman shift and time dimensions was statistically optimized. Overall, time-gated Raman spectroscopy, especially with optimized data analysis in both the spectral and time dimensions, shows potential for sensitive and relatively routine quantitative analysis of photoluminescent pharmaceuticals during drug development and manufacturing.

The use of wavelength dispersive X-ray fluorescence in the identification of the elemental composition of vanilla samples and the determination of the geographic origin by discriminant function analysis.

PubMed

Hondrogiannis, Ellen; Rotta, Kathryn; Zapf, Charles M

2013-03-01

Sixteen elements found in 37 vanilla samples from Madagascar, Uganda, India, Indonesia (all Vanilla planifolia species), and Papa New Guinea (Vanilla tahitensis species) were measured by wavelength dispersive X-ray fluorescence (WDXRF) spectroscopy for the purpose of determining the elemental concentrations to discriminate among the origins. Pellets were prepared of the samples and elemental concentrations were calculated based on calibration curves created using 4 Natl. Inst. of Standards and Technology (NIST) standards. Discriminant analysis was used to successfully classify the vanilla samples by their species and their geographical region. Our method allows for higher throughput in the rapid screening of vanilla samples in less time than analytical methods currently available. Wavelength dispersive X-ray fluorescence spectroscopy and discriminant function analysis were used to classify vanilla from different origins resulting in a model that could potentially serve to rapidly validate these samples before purchasing from a producer. © 2013 Institute of Food Technologists®

Recent trends in atomic fluorescence spectrometry towards miniaturized instrumentation-A review.

PubMed

Zou, Zhirong; Deng, Yujia; Hu, Jing; Jiang, Xiaoming; Hou, Xiandeng

2018-08-17

Atomic fluorescence spectrometry (AFS), as one of the common atomic spectrometric techniques with high sensitivity, simple instrumentation, and low acquisition and running cost, has been widely used in various fields for trace elemental analysis, notably the determination of hydride-forming elements by hydride generation atomic fluorescence spectrometry (HG-AFS). In recent years, the soaring demand of field analysis has significantly promoted the miniaturization of analytical atomic spectrometers or at least instrumental components. Various techniques have also been developed to approach the goal of portable/miniaturized AFS instrumentation for field analysis. In this review, potentially portable/miniaturized AFS techniques, primarily involving advanced instrumental components and whole instrumentation with references since 2000, are summarized and discussed. The discussion mainly includes five aspects: radiation source, atomizer, detector, sample introduction, and miniaturized atomic fluorescence spectrometer/system. Copyright © 2018 Elsevier B.V. All rights reserved.

The fundamental parameter method applied to X-ray fluorescence analysis with synchrotron radiation

NASA Astrophysics Data System (ADS)

Pantenburg, F. J.; Beier, T.; Hennrich, F.; Mommsen, H.

1992-05-01

Quantitative X-ray fluorescence analysis applying the fundamental parameter method is usually restricted to monochromatic excitation sources. It is shown here, that such analyses can be performed as well with a white synchrotron radiation spectrum. To determine absolute elemental concentration values it is necessary to know the spectral distribution of this spectrum. A newly designed and tested experimental setup, which uses the synchrotron radiation emitted from electrons in a bending magnet of ELSA (electron stretcher accelerator of the university of Bonn) is presented. The determination of the exciting spectrum, described by the given electron beam parameters, is limited due to uncertainties in the vertical electron beam size and divergence. We describe a method which allows us to determine the relative and absolute spectral distributions needed for accurate analysis. First test measurements of different alloys and standards of known composition demonstrate that it is possible to determine exact concentration values in bulk and trace element analysis.

A hybrid segmentation approach for geographic atrophy in fundus auto-fluorescence images for diagnosis of age-related macular degeneration.

PubMed

Lee, Noah; Laine, Andrew F; Smith, R Theodore

2007-01-01

Fundus auto-fluorescence (FAF) images with hypo-fluorescence indicate geographic atrophy (GA) of the retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Manual quantification of GA is time consuming and prone to inter- and intra-observer variability. Automatic quantification is important for determining disease progression and facilitating clinical diagnosis of AMD. In this paper we describe a hybrid segmentation method for GA quantification by identifying hypo-fluorescent GA regions from other interfering retinal vessel structures. First, we employ background illumination correction exploiting a non-linear adaptive smoothing operator. Then, we use the level set framework to perform segmentation of hypo-fluorescent areas. Finally, we present an energy function combining morphological scale-space analysis with a geometric model-based approach to perform segmentation refinement of false positive hypo- fluorescent areas due to interfering retinal structures. The clinically apparent areas of hypo-fluorescence were drawn by an expert grader and compared on a pixel by pixel basis to our segmentation results. The mean sensitivity and specificity of the ROC analysis were 0.89 and 0.98%.

Taking into Account Interelement Interference in X-Ray Fluorescence Analysis of Thin Two-Layer Ti/V Systems

NASA Astrophysics Data System (ADS)

Mashin, N. I.; Razuvaev, A. G.; Cherniaeva, E. A.; Gafarova, L. M.; Ershov, A. V.

2018-03-01

We propose a new method for determining the thickness of layers in x-ray fluorescence analysis of two-layer Ti/V systems, using easily fabricated standardized film layers obtained by sputter deposition of titanium on a polymer film substrate. We have calculated correction factors taking into account the level of attenuation for the intensity of the primary emission from the x-ray tube and the analytical line for the element of the bottom layer in the top layer, and the enhancement of the fluorescence intensity for the top layer by the emission of atoms in the bottom layer.

Argon content of the Martian atmosphere at the Viking 1 landing site - Analysis by X-ray fluorescence spectroscopy

NASA Technical Reports Server (NTRS)

Clark, B. C.; Toulmin, P., III; Rose, H. J., Jr.; Baird, A. K.; Keil, K.

1976-01-01

Spectra provided by the Viking 1 X-ray fluorescence spectrometer operating in the calibration mode (without a soil sample in the analysis chamber) were analyzed to determine the argon content of the Martian atmosphere at the landing site. This was found to be less than or equal to 0.15 millibar, or not more than 2% by volume, consistent with data obtained by the entry mass spectrometer and by the mass spectrometer on the lander. It is anticipated that analysis of the K content of surface samples using X-ray fluorescence data will provide information on the evolution of the atmosphere, since most atmospheric argon is apparently produced by decay of K-40.

The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors.

PubMed

Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias; Stummer, Walter

2016-03-01

Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. Age, tumor volume, and F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased Ki-67/MIB-1 index and high-grade pathology. Whether fluorescence in grade II gliomas identifies a subtype with worse prognosis remains to be determined.

Time resolved fluorescence of cow and goat milk powder

NASA Astrophysics Data System (ADS)

Brandao, Mariana P.; de Carvalho dos Anjos, Virgílio; Bell., Maria José V.

2017-01-01

Milk powder is an international dairy commodity. Goat and cow milk powders are significant sources of nutrients and the investigation of the authenticity and classification of milk powder is particularly important. The use of time-resolved fluorescence techniques to distinguish chemical composition and structure modifications could assist develop a portable and non-destructive methodology to perform milk powder classification and determine composition. This study goal is to differentiate milk powder samples from cows and goats using fluorescence lifetimes. The samples were excited at 315 nm and the fluorescence intensity decay registered at 468 nm. We observed fluorescence lifetimes of 1.5 ± 0.3, 6.4 ± 0.4 and 18.7 ± 2.5 ns for goat milk powder; and 1.7 ± 0.3, 6.9 ± 0.2 and 29.9 ± 1.6 ns for cow's milk powder. We discriminate goat and cow powder milk by analysis of variance using Fisher's method. In addition, we employed quadratic discriminant analysis to differentiate the milk samples with accuracy of 100%. Our results suggest that time-resolved fluorescence can provide a new method to the analysis of powder milk and its composition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutherland, John C.

In this article, a method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of amore » polarizer.« less

X-ray Fluorescence Holography: Principles, Apparatus, and Applications

NASA Astrophysics Data System (ADS)

Hayashi, Kouichi; Korecki, Pawel

2018-06-01

X-ray fluorescence holography (XFH) is an atomic structure determination technique that combines the capabilities of X-ray diffraction and X-ray fluorescence spectroscopy. It provides a unique means of gaining fully three-dimensional information about the local atomic structure and lattice site positions of selected elements inside compound samples. In this work, we discuss experimental and theoretical aspects that are essential for the efficient recording and analysis of X-ray fluorescence holograms and review the most recent advances in XFH. We describe experiments performed with brilliant synchrotron radiation as well as with tabletop setups that employ conventional X-ray tubes.

Fluorescence-based enhanced reality (FLER) for real-time estimation of bowel perfusion in minimally invasive surgery

NASA Astrophysics Data System (ADS)

Diana, Michele

2016-03-01

Pre-anastomotic bowel perfusion is a key factor for a successful healing process. Clinical judgment has limited accuracy to evaluate intestinal microperfusion. Fluorescence videography is a promising tool for image-guided intraoperative assessment of the bowel perfusion at the future anastomotic site in the setting of minimally invasive procedures. The standard configuration for fluorescence videography includes a Near-Infrared endoscope able to detect the signal emitted by a fluorescent dye, more frequently Indocyanine Green (ICG), which is administered by intravenous injection. Fluorescence intensity is proportional to the amount of fluorescent dye diffusing in the tissue and consequently is a surrogate marker of tissue perfusion. However, fluorescence intensity alone remains a subjective approach and an integrated computer-based analysis of the over-time evolution of the fluorescence signal is required to obtain quantitative data. We have developed a solution integrating computer-based analysis for intra-operative evaluation of the optimal resection site, based on the bowel perfusion as determined by the dynamic fluorescence intensity. The software can generate a "virtual perfusion cartography", based on the "fluorescence time-to-peak". The virtual perfusion cartography can be overlapped onto real-time laparoscopic images to obtain the Enhanced Reality effect. We have defined this approach FLuorescence-based Enhanced Reality (FLER). This manuscript describes the stepwise development of the FLER concept.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M; Finlay, J; Zhu, T

Purpose: Photosensitizer concentration during photodynamic therapy (PDT) is an important parameter for accurate dosimetry. Fluorescence signal can be used as a measure of photosensitizer concentration. Two methods of data acquisition were compared to an ex vivo study both for in vivo and phantom models. Methods: Fluorescence signal of commonly used photosensitizer benzoporphyrin derivative monoacid ring A (BPD) was obtained in phantoms and mouse tumors using an excitation light of 405 nm. Interstitial fluorescence signal was obtained using a side-cut fiber inserted into the tumor tissue of interest. Using a previously developed multi-fiber probe, tumor surface fluorescence measurements were also collected.more » Signals were calibrated according to optical phantoms with known sensitizer fluorescence. Optical properties for each sample were determined and the influence of different absorption and scattering properties on the fluorescence signals was investigated. Using single value decomposition of the spectra, the sensitizer concentration was determined using the two different measurement geometries. An ex vivo analysis was also performed for tumor samples to determine the sensitizer concentration. Results: The two fluorescence signals obtained from the surface multi-fiber probe and the interstitial measurements were compared and were corresponding for both phantoms and mouse models. The values obtained were comparable to the ex vivo measurements as well. Despite the difference in geometry, the surface probe measurements can still be used as a metric for determining the presence of sensitizer in small volume tumors. Conclusion: The multi-fiber contact probe can be used as a tool to measure fluorescence at the surface of the treatment area for PDT and predict sensitizer concentration throughout the tumor. This is advantageous in that the measurement does not damage any tissue. Future work will include investigating the dependence of these results on intratumor sensitizer distribution.« less

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging.

PubMed

Lin, Xiaolin; Jia, Junshuang; Qin, Yujuan; Lin, Xia; Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

2015-11-17

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging

PubMed Central

Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

2015-01-01

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study. PMID:26472024

Determination of the botanical origin of honey by front-face synchronous fluorescence spectroscopy.

PubMed

Lenhardt, Lea; Zeković, Ivana; Dramićanin, Tatjana; Dramićanin, Miroslav D; Bro, Rasmus

2014-01-01

Front-face synchronous fluorescence spectroscopy combined with chemometrics is used to classify honey samples according to their botanical origin. Synchronous fluorescence spectra of three monofloral (linden, sunflower, and acacia), polyfloral (meadow mix), and fake (fake acacia and linden) honey types (109 samples) were collected in an excitation range of 240-500 nm for synchronous wavelength intervals of 30-300 nm. Chemometric analysis of the gathered data included principal component analysis and partial least squares discriminant analysis. Mean cross-validated classification errors of 0.2 and 4.8% were found for a model that accounts only for monofloral samples and for a model that includes both the monofloral and polyfloral groups, respectively. The results demonstrate that single synchronous fluorescence spectra of different honeys differ significantly because of their distinct physical and chemical characteristics and provide sufficient data for the clear differentiation among honey groups. The spectra of fake honey samples showed pronounced differences from those of genuine honey, and these samples are easily recognized on the basis of their synchronous fluorescence spectra. The study demonstrated that this method is a valuable and promising technique for honey authentication.

Confocal fluorescence techniques in industrial application

NASA Astrophysics Data System (ADS)

Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

2003-06-01

The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

Multi-Channel Hyperspectral Fluorescence Detection Excited by Coupled Plasmon-Waveguide Resonance

PubMed Central

Du, Chan; Liu, Le; Zhang, Lin; Guo, Jun; Guo, Jihua; Ma, Hui; He, Yonghong

2013-01-01

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes. PMID:24129023

Feasibility of the determination of polycyclic aromatic hydrocarbons in edible oils via unfolded partial least-squares/residual bilinearization and parallel factor analysis of fluorescence excitation emission matrices.

PubMed

Alarcón, Francis; Báez, María E; Bravo, Manuel; Richter, Pablo; Escandar, Graciela M; Olivieri, Alejandro C; Fuentes, Edwar

2013-01-15

The possibility of simultaneously determining seven concerned heavy polycyclic aromatic hydrocarbons (PAHs) of the US-EPA priority pollutant list, in extra virgin olive and sunflower oils was examined using unfolded partial least-squares with residual bilinearization (U-PLS/RBL) and parallel factor analysis (PARAFAC). Both of these methods were applied to fluorescence excitation emission matrices. The compounds studied were benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-c,d]-pyrene. The analysis was performed using fluorescence spectroscopy after a microwave assisted liquid-liquid extraction and solid-phase extraction on silica. The U-PLS/RBL algorithm exhibited the best performance for resolving the heavy PAH mixture in the presence of both the highly complex oil matrix and other unpredicted PAHs of the US-EPA list. The obtained limit of detection for the proposed method ranged from 0.07 to 2 μg kg(-1). The predicted U-PLS/RBL concentrations were satisfactorily compared with those obtained using high-performance liquid chromatography with fluorescence detection. A simple analysis with a considerable reduction in time and solvent consumption in comparison with chromatography are the principal advantages of the proposed method. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Single-molecule detection: applications to ultrasensitive biochemical analysis

NASA Astrophysics Data System (ADS)

Castro, Alonso; Shera, E. Brooks

1995-06-01

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.

Specimen preparation for x-ray fluorescence analysis of solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eksperiandova, L.P.; Spolnik, Z.M.; Blank, A.B.

1995-12-31

Specimens for x-ray fluorescence analysis (XRFA) were prepared by adding dry gelatine (10%) to the analysis solution, homogenizing the mixture and cooling for 20 minutes. Thus, a compact resilient mass could be formed with the required shape and size; the roughness of the surface was determined by the roughness of the surface on which the specimen was formed, much the same as highly polished. Various calibration methods can be applied in the XRFA of a variety of materials if such specimens are used. 12 refs., 1 fig., 2 tabs.

Sensitive determination of endogenous hydroxyl radical in live cell by a BODIPY based fluorescent probe.

PubMed

Lei, Kepeng; Sun, Mingtai; Du, Libo; Zhang, Xiaojie; Yu, Huan; Wang, Suhua; Hayat, Tasawar; Alsaedi, Ahmed

2017-08-01

The sensitive and selective fluorescence probe for hydroxyl radical analysis is of significance because hydroxyl radical plays key roles in many physiological and pathological processes. In this work, a novel organic fluorescence molecular probe OHP for hydroxyl radical is synthesized by a two-step route. The probe employs 4-bora-3a,4a-diaza-s-indacene (difluoroboron dipyrromethene, BODIPY) as the fluorophore and possesses relatively high fluorescence quantum yields (77.14%). Hydroxyl radical can rapidly react with the probe and quench the fluorescence in a good linear relationship (R 2 =0.9967). The limit of detection is determined to be as low as 11nM. In addition, it has been demonstrated that the probe has a good stability against pH and light illumination, low cytotoxicity and high biocompatibility. Cell culture experimental results show that the probe OHP is sensitive and selective for imaging and tracking endogenous hydroxyl radical in live cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Laser fluorescence fluctuation excesses in molecular immunology experiments

NASA Astrophysics Data System (ADS)

Galich, N. E.; Filatov, M. V.

2007-04-01

A novel approach to statistical analysis of flow cytometry fluorescence data have been developed and applied for population analysis of blood neutrophils stained with hydroethidine during respiratory burst reaction. The staining based on intracellular oxidation hydroethidine to ethidium bromide, which intercalate into cell DNA. Fluorescence of the resultant product serves as a measure of the neutrophil ability to generate superoxide radicals after induction respiratory burst reaction by phorbol myristate acetate (PMA). It was demonstrated that polymorphonuclear leukocytes of persons with inflammatory diseases showed a considerably changed response. Cytofluorometric histograms obtained have unique information about condition of neutrophil population what might to allow a determination of the pathology processes type connecting with such inflammation. A novel approach to histogram analysis is based on analysis of high-momentum dynamic of distribution. The features of fluctuation excesses of distribution have unique information about disease under consideration.

Fluorescence lifetime measurements in flow cytometry

NASA Astrophysics Data System (ADS)

Beisker, Wolfgang; Klocke, Axel

1997-05-01

Fluorescence lifetime measurements provide insights int eh dynamic and structural properties of dyes and their micro- environment. The implementation of fluorescence lifetime measurements in flow cytometric systems allows to monitor large cell and particle populations with high statistical significance. In our system, a modulated laser beam is used for excitation and the phase shift of the fluorescence signal recorded with a fast computer controlled digital oscilloscope is processed digitally to determine the phase shift with respect to a reference beam by fast fourier transform. Total fluorescence intensity as well as other parameters can be determined simultaneously from the same fluorescence signal. We use the epi-illumination design to allow the use of high numerical apertures to collect as much light as possible to ensure detection of even weak fluorescence. Data storage and processing is done comparable to slit-scan flow cytometric data using data analysis system. The results are stored, displayed, combined with other parameters and analyzed as normal listmode data. In our report we discuss carefully the signal to noise ratio for analog and digital processed lifetime signals to evaluate the theoretical minimum fluorescence intensity for lifetime measurements. Applications to be presented include DNA staining, parameters of cell functions as well as different applications in non-mammalian cells such as algae.

Two-photon optical imaging, spectral and fluorescence lifetime analysis to discriminate urothelial carcinoma grades.

PubMed

Pradère, B; Poulon, F; Compérat, E; Lucas, I; Bazin, D; Doizi, S; Cussenot, O; Traxer, O; Abi Haidar, D

2018-05-28

In the framework of urologic oncology, mini-invasive procedures have increased in the last few decades particularly for urothelial carcinoma. One of the essential elements in the management of this disease is still the diagnosis, which strongly influences the choice of treatment. The histopathologic evaluation of the tumor grade is a keystone of diagnosis, and tumor characterization is not possible with just a macroscopic evaluation. Even today intraoperative evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore. This study assessed an optical multimodal technique based on endogenous fluorescence, combining qualitative and quantitative analysis, for the diagnostic of urothelial carcinoma. It was found that the combination of two photon fluorescence, second harmonic generation microscopy, spectral analysis and fluorescence lifetime imaging were all able to discriminate tumor from healthy tissue, and to determine the grade of tumors. Spectral analysis of fluorescence intensity and the redox ratio used as quantitative evaluations showed statistical differences between low grade and high grade tumors. These results showed that multimodal optical analysis is a promising technology for the development of an optical fiber setup designed for an intraoperative diagnosis of urothelial carcinoma in the area of endourology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Determination of the thermal, oxidative and photochemical degradation rates of scintillator liquid by fluorescence EEM spectroscopy.

PubMed

Andrews, N L P; Fan, J Z; Forward, R L; Chen, M C; Loock, H-P

2016-12-21

The thermal, oxidative and photochemical stability of the scintillator liquid proposed for the SNO+ experiment has been tested experimentally using accelerated aging methods. The stability of the scintillator constituents was determined through fluorescence excitation emission matrix (EEM) spectroscopy and absorption spectroscopy, using parallel factor analysis (PARAFAC) as an multivariate analysis tool. By exposing the scintillator liquid to a well-known photon flux at 365 nm and by measuring the decay rate of the fluorescence shifters and the formation rate of their photochemical degradation products, we can place an upper limit on the acceptable photon flux as 1.38 ± 0.09 × 10 -11 photon mol L -1 . Similarly, the oxidative stability of the scintillator liquid was determined by exposure to air at several elevated temperatures. Through measurement of the corresponding activation energy it was determined that the average oxygen concentration would have to be kept below 4.3-7.1 ppb w (headspace partial pressure below 24 ppm v ). On the other hand, the thermal stability of the scintillator cocktail in the absence of light and oxygen was remarkable and poses no concern to the SNO+ experiment.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Polydopamine nanotube mediated fluorescent biosensor for Hg(ii) determination through exonuclease III-assisted signal amplification.

PubMed

A, Ravikumar; P, Panneerselvam

2018-05-29

We describe a highly sensitive fluorescence biosensor incorporating polydopamine nanotubes (PDNTs) based on the mechanism of exonuclease III (Exo III) assisted signal amplification for the determination of Hg2+ in aqueous solution. Fluorescent probes of FAM labeled ssDNA (FAM-ssDNA) adsorbed on the PDNTs act as an efficient quencher. In the presence of Hg2+, the FAM-ssDNA can bind to Hg2+ to form double stranded DNA (dsDNA) via the formation of T-Hg2+-T base pairs. Then, the dsDNA was removed from the surface of the PDNTs to restore the fluorescence. The release of the dsDNA was triggered by Exo III digestion. At the same time, the liberated Hg2+ mediates a new cycle of digestion. This assay is ultrasensitive for the selective recognition of Hg2+, and a detection limit as low as 10 pM was achieved. In addition, the fluorescent biosensing system also displays remarkable specificity to Hg2+ in the presence of other possible competing ions. This approach was applied to the determination of Hg2+ in real water samples with good recovery and high efficiency for practical analysis.

Characterization of brightness and stoichiometry of bright particles by flow-fluorescence fluctuation spectroscopy.

PubMed

Johnson, Jolene; Chen, Yan; Mueller, Joachim D

2010-11-03

Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Highly selective and sensitive determination of Cu2+ in drink and water samples based on a 1,8-diaminonaphthalene derived fluorescent sensor

NASA Astrophysics Data System (ADS)

Sun, Tao; Li, Yang; Niu, Qingfen; Li, Tianduo; Liu, Yan

2018-04-01

A new simple and efficient fluorescent sensor L based on 1,8-diaminonaphthalene Schiff-base for highly sensitive and selective determination of Cu2+ in drink and water has been developed. This Cu2+-selective detection over other tested metal ions displayed an obvious color change from blue to colorless easily detected by naked eye. The detection limit is determined to be as low as 13.2 nM and the response time is very fast within 30 s. The 1:1 binding mechanism was well confirmed by fluorescence measurements, IR analysis and DFT calculations. Importantly, this sensor L was employed for quick detection of Cu2+ in drink and environmental water samples with satisfactory results, providing a simple, rapid, reliable and feasible Cu2+-sensing method.

In situ optical measurements for characterization of flame species and remote sensing

NASA Astrophysics Data System (ADS)

Cullum, Brian Michael

1998-12-01

The following dissertation describes the use of spectroscopic techniques for both characterization of combustion intermediates and remote chemical sensing. The primary techniques that have been used for these measurements include, laser-induced fluorescence (LIF), time resolved LIF, resonance enhanced multiphoton ionization (REMPI) and Raman spectroscopy. A simple and quantitative means of measuring the efficiency of halogenated flame retardants is described, using laser-induced fluorescence (LIF). Intensity based LIF measurements of OH radical have been used to quantitatively measure the efficacy of halogenated flame retardant/polymer plaques. Temporally resolved LIF has been used to determine the extent to which the chemical kinetic theory of flame retardation applies to the effect of these compounds on combustion. We have shown that LIF of OH radicals is a very sensitive means of measuring the efficiency of these flame retardants as well as the giving information about the nature of flame retardation. In addition, we have developed a technique for the introduction of insoluble polymer plaques into a flame for fluorescence analysis. A high power pulsed Nd:YAG laser is used to ablate the sample into the flame while a second pulse from a dye laser is used to measure the LIF of OH radicals. Spectroscopic techniques are also very useful for trace remote analysis of environmental pollutants via optical fibers. A simple fiber-optic probe suitable for remote analysis using resonance enhanced multiphoton ionization (REMPI) has been developed for this purpose and is used to determine the toluene/gasoline concentration in water samples via a headspace measurement. The limit of detection for toluene in water using this probe is 0.54 ppb (wt/wt) with a sample standard deviation of 0.02 ppb (wt/wt). Another technique that has great potential for optical sensing is fluorescence lifetime imaging. A new method for measuring fluorescence lifetime images of quickly decaying species has been developed. This method employs a high powered pulsed laser that excites the fluorescent species in a dual pulse manner, and a non-gated charge coupled device (CCD) for detection of the fluorescence. Unlike other fluorescence lifetime imaging methods, this technique has the potential of monitoring fluorescent species with picosecond lifetimes.

Quinoline containing acetyl hydrazone: An easily accessible switch-on optical chemosensor for Zn2 +

NASA Astrophysics Data System (ADS)

Wu, Wei-Na; Mao, Pan-Dong; Wang, Yuan; Zhao, Xiao-Lei; Xu, Zhou-Qing; Xu, Zhi-Hong; Xue, Yuan

2018-01-01

A simple chemosensor, namely, N-((quinolin-8-yl)methylene)acetohydrazide (1) was synthesized and used as an off-on fluorescence sensor, which exhibits high selectivity toward Zn2 + in aqueous media. The probe has large Stokes shift of > 200 nm, and its detection limit for Zn2 + is 89.3 nM. The binding process was confirmed through UV-vis absorption analysis, fluorescence measurements, mass spectroscopy study, 1H NMR spectra and density functional theory calculation. The crystal structures of Zn2 +, Ni2 +, and Cu2 + complexes based on 1 were determined through X-ray crystallographic analysis. The fluorescent probe was then applied to monitor intracellular Zn2 + in HeLa cells.

Determination of minor and trace elements in kidney stones by x-ray fluorescence analysis

NASA Astrophysics Data System (ADS)

Srivastava, Anjali; Heisinger, Brianne J.; Sinha, Vaibhav; Lee, Hyong-Koo; Liu, Xin; Qu, Mingliang; Duan, Xinhui; Leng, Shuai; McCollough, Cynthia H.

2014-03-01

The determination of accurate material composition of a kidney stone is crucial for understanding the formation of the kidney stone as well as for preventive therapeutic strategies. Radiations probing instrumental activation analysis techniques are excellent tools for identification of involved materials present in the kidney stone. In particular, x-ray fluorescence (XRF) can be very useful for the determination of minor and trace materials in the kidney stone. The X-ray fluorescence measurements were performed at the Radiation Measurements and Spectroscopy Laboratory (RMSL) of department of nuclear engineering of Missouri University of Science and Technology and different kidney stones were acquired from the Mayo Clinic, Rochester, Minnesota. Presently, experimental studies in conjunction with analytical techniques were used to determine the exact composition of the kidney stone. A new type of experimental set-up was developed and utilized for XRF analysis of the kidney stone. The correlation of applied radiation source intensity, emission of X-ray spectrum from involving elements and absorption coefficient characteristics were analyzed. To verify the experimental results with analytical calculation, several sets of kidney stones were analyzed using XRF technique. The elements which were identified from this techniques are Silver (Ag), Arsenic (As), Bromine (Br), Chromium (Cr), Copper (Cu), Gallium (Ga), Germanium (Ge), Molybdenum (Mo), Niobium (Nb), Rubidium (Rb), Selenium (Se), Strontium (Sr), Yttrium (Y), Zirconium (Zr). This paper presents a new approach for exact detection of accurate material composition of kidney stone materials using XRF instrumental activation analysis technique.

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

PubMed Central

Axelrod, D; Koppel, D E; Schlessinger, J; Elson, E; Webb, W W

1976-01-01

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G. PMID:786399

Determination of 2-alkylcyclobutanones by combining precolumn derivatization with 1-naphthalenyl hydrazine and ultra-performance liquid chromatography with fluorescence detection.

PubMed

Meng, Xiangpeng; Tong, Tong; Wang, Lianrong; Liu, Hanxia; Chan, Wan

2016-05-01

2-Alkylcyclobutanones (2-ACBs) are uniquely formed when triglycerides-containing food products are exposed to ionizing radiation. Thus, 2-ACBs have been used as marker molecules to identify irradiated food. Most methods to determine 2-ACBs involve mass spectrometric detection after chromatographic separation. The spectrofluorometer is rarely used to determine 2-ACBs because these molecules do not fluoresce. In this study, we developed an ultra-performance liquid chromatography (UPLC) method to determine 2-ACBs. 2-ACBs were converted into fluorophores after reacting with 1-naphthalenyl hydrazine to facilitate their sensitive and selective detection using a fluorescence detector (FLD). Analysis of 2-ACBs using our developed UPLC-FLD method allows sensitive determination of 2-ACBs at a detection limit of 2 ng 2-ACBs per g of fat (30 pg/injection), which is significantly lower than that of existing analytical methods. After validation for trueness and precision, the method was applied to γ-irradiated chicken samples to determine their 2-ACB content. Comparative studies employing liquid chromatography-tandem mass spectrometric method revealed no systematic difference between the two methods, thereby demonstrating that the proposed UPLC-FLD method can be suitably used to determine 2-ACBs in irradiated foodstuffs. Graphical Abstract Determination of radiation-induced food-borne 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone by combining 1-naphthalenyl hydrazine derivatization and ultra-performance liquid chromatography with fluorescence detection.

A coumarin-based "turn-on" fluorescent sensor for the determination of Al3+: single crystal X-ray structure and cell staining properties.

PubMed

Guha, Subarna; Lohar, Sisir; Sahana, Animesh; Banerjee, Arnab; Safin, Damir A; Babashkina, Maria G; Mitoraj, Mariusz P; Bolte, Michael; Garcia, Yann; Mukhopadhyay, Subhra Kanti; Das, Debasis

2013-07-28

An efficient Al(3+) receptor, 6-(2-hydroxybenzylideneamino)-2H-chromen-2-one (HBC), has been synthesized by condensing salicylaldehyde with 6-aminocoumarin. The molecular structure of HBC has been determined by a single crystal X-ray analysis. It was established that in the presence of Al(3+), HBC shows 25 fold enhancement of fluorescence intensity which might be attributed to the chelation-enhanced fluorescence (CHEF) process. HBC binds Al(NO3)3 in a 1 : 1 stoichiometry with a binding constant (K) of 7.9 × 10(4) M(-1). Fe(3+) and Mn(2+) quench the emission intensity of the [HBC + Al(3+)] system to an insignificant extent at a concentration 10 times higher compared to that of Al(3+). HBC is highly efficient in the detection of intracellular Al(3+) under a fluorescence microscope.

A rapid fluorescence assay for danofloxacin in beef muscle: effect of muscle type on limit of quantitation.

PubMed

Schneider, Marilyn J

2008-08-01

A simple, rapid fluorescence screening assay was applied to the analysis of beef muscle for danofloxacin at the U.S. tolerance level of 200 ng/g. Muscle samples were homogenized in acetic acid-acetonitrile, the resultant mixture centrifuged, and fluorescence of the supernatants was then measured. The significant difference between the fluorescence of control muscle sample extracts and extracts of samples fortified at 200 ng/g allowed for successful discrimination between the samples. Setting a threshold level at the average 200 ng/g fortified sample extract fluorescence -3sigma allowed for identification of potentially violative samples. Successful analysis of a group of blind fortified samples over a range of concentrations was accomplished in this manner, without any false-negative results. The limits of quantitation for danofloxacin, as well as enrofloxacin, using this assay were determined in three types of beef muscle (hanging tenderloin, neck, and eye round steak), as well as in serum. Significant differences in limits of quantitation were found among the three different muscle types examined, with hanging tenderloin muscle providing the lowest value. This work not only shows the potential for use of the fluorescence screening assay as an alternative to currently used microbial or antibody-based assays for the analysis of danofloxacin in beef muscle, but also suggests that assays using beef muscle may vary in performance depending on the specific muscle selected for analysis.

Polar plot representation of time-resolved fluorescence.

PubMed

Eichorst, John Paul; Wen Teng, Kai; Clegg, Robert M

2014-01-01

Measuring changes in a molecule's fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscillation in the emitted fluorescence of the sample in response to repetitively modulated excitation light. In either the time or frequency domain, the analysis of data to extract lifetimes can be computationally intensive. For example, a variety of iterative fitting algorithms already exist to determine lifetimes from samples that contain multiple fluorescing species. However, recently a method of analysis referred to as the polar plot (or phasor plot) is a graphical tool that projects the time-dependent features of the sample's fluorescence in either the time or frequency domain into the Cartesian plane to characterize the sample's lifetime. The coordinate transformations of the polar plot require only the raw data, and hence, there are no uncertainties from extensive corrections or time-consuming fitting in this analysis. In this chapter, the history and mathematical background of the polar plot will be presented along with examples that highlight how it can be used in both cuvette-based and imaging applications.

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A

PubMed Central

Gururani, Mayank Anand; Venkatesh, Jelli; Ganesan, Markkandan; Strasser, Reto Jörg; Han, Yunjeong; Kim, Jeong-Il; Lee, Hyo-Yeon; Song, Pill-Soon

2015-01-01

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions. PMID:26010864

Fluorescence fluctuation spectroscopy for clinical applications

NASA Astrophysics Data System (ADS)

Olson, Eben

Fluorescence correlation spectroscopy (FCS) and the related techniques of brightness analysis have become standard tools in biological and biophysical research. By analyzing the statistics of fluorescence emitted from a restricted volume, a number of parameters including concentrations, diffusion coefficients and chemical reaction rates can be determined. The single-molecule sensitivity, spectral selectivity, small sample volume and non-perturbative measurement mechanism of FCS make it an excellent technique for the study of molecular interactions. However, its adoption outside of the research laboratory has been limited. Potential reasons for this include the cost and complexity of the required apparatus. In this work, the application of fluorescence fluctuation analysis to several clinical problems is considered. Optical designs for FCS instruments which reduce the cost and increase alignment tolerance are presented. Brightness analysis of heterogenous systems, with application to the characterization of protein aggregates and multimer distributions, is considered. Methods for FCS-based assays of two clinically relevant proteins, von Willebrand factor and haptoglobin, are presented as well.

Analysis of particle in liquid using excitation-fluorescence spectral flow cytometer

NASA Astrophysics Data System (ADS)

Takenaka, Kei; Togashi, Shigenori

2018-01-01

We have developed a new flow cytometer that can measure the excitation-fluorescence spectra of a single particle. This system consists of a solution-transmitting unit and an optical unit. The solution-transmitting unit allows a sample containing particles to flow through the center of a flow cell by hydrodynamic focusing. The optical unit irradiates particles with dispersed white light (wavelength band: 400-650 nm) along the flow direction and measures their fluorescence spectra (wavelength band: 400-700 nm) using a spectroscopic photodetector array. The fluorescence spectrum of a particle changes with the shift of the wavelength of the excitation light. Using this system, the excitation-fluorescence spectra of a fluorescent particle were measured. Additionally, a homogenized tomato suspension and a homogenized spinach suspension were measured using the system. Measurement results show that it is possible to determine the components of vegetables by comparing measured fluorescence spectra of particles in a vegetable suspension.

[Sensitive Determination of Chondroitin Sulfate by Fluorescence Recovery of an Anionic Aluminum Phthalocyanine-Cationic Surfactant Ion-Association Complex Used as a Fluorescent Probe Emitting at Red Region].

PubMed

Chen, Lin; Huang, Ping; Yang, Hui-qing; Deng, Ya-bin; Guo, Meng-lin; Li, Dong-hui

2015-08-01

Determination of chondroitin sulfate in the biomedical field has an important value. The conventional methods for the assay of chondroitin sulfate are still unsatisfactory in sensitivity, selectivity or simplicity. This work aimed at developing a novel method for sensitive and selective determination of chondroitin sulfate by fluorimetry. We found that some kinds of cationic surfactants have the ability to quench the fluorescence of tetrasulfonated aluminum phthalocyanine (AlS4Pc), a strongly fluorescent compound which emits at red region, with high efficiency. But, the fluorescence of the above-mentioned fluorescence quenching system recovered significantly when chondroitin sulfate (CS) exits. Tetradecyl dimethyl benzyl ammonium chloride(TDBAC) which was screened from all of the candidates of cationic surfactants was chosen as the quencher because it shows the most efficient quenching effect. It was found that the fluorescence of AlS4Pc was extremely quenched by TDBAC because of the formation of association complex between AlS4Pc and TDBAC. Fluorescence of the association complex recovered dramatically after the addition of chondroitin sulfate (CS) due to the ability of chondroitin sulfate to shift the association equilibrium of the association, leading to the release of AlS4Pc, thus resulting in an increase in the fluorescence of the reaction system. Based on this phenomenon, a novel method with simplicity, accuracy and sensitivity was developed for quantitative determination of CS. Factors including the reaction time, influencing factors and the effect of coexisting substances were investigated and discussed. Under optimum conditions the linear range of the calibration curve was 0.20~10.0 μg · mL(-1). The detection limit for CS was 0.070 μg · mL(-1). The method has been applied to the analysis of practical samples with satisfied results. This work expands the applications of AlS4Pc in biomedical area.

Determination of Ivermectin in Medicated Feeds by Liquid Chromatography with Fluorescence Detection

PubMed Central

2013-01-01

A labour- and time-effective analytical procedure for determination of ivermectin in medicated feed at recommended level of 2.0 mg kg−1 has been developed and validated. The analyte was extracted from grinded feed samples with acetonitrile and derivatisated with N-methylimidazole and trifluoracetic anhydride. The fluorescent derivatives were analysed by liquid chromatography method using C8 column. The isocratic conditions using acetonitrile, methanol, water, and tetrahydrofuran were applied. Fluorescence detection was performed at 365 nm (excitation) and 475 nm (emission) wavelengths. The total analysis time was 10 min. The validation results of the method (within-laboratory reproducibility 4.0% CV, mean recovery 100.1%) confirm the appropriate precision and accuracy of the developed method. PMID:24453835

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Analysis of tincal ore waste by energy dispersive X-ray fluorescence (EDXRF) Technique

NASA Astrophysics Data System (ADS)

Kalfa, Orhan Murat; Üstündağ, Zafer; Özkırım, Ilknur; Kagan Kadıoğlu, Yusuf

2007-01-01

Etibank Borax Plant is located in Kırka-Eskişehir, Turkey. The borax waste from this plant was analyzed by means of energy dispersive X-ray fluorescence (EDXRF). The standard addition method was used for the determination of the concentration of Al, Fe, Zn, Sn, and Ba. The results are presented and discussed in this paper.

Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

NASA Astrophysics Data System (ADS)

Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

2014-03-01

It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

Analysis of short tandem repeat polymorphisms using infrared fluorescence with M18 tailed primers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oetting, W.S.; Wiesner, G.; Laken, S.

The use of short tandem repeat polymorphisms (STRPs) are becoming increasingly important as markers for linkage analysis due to their large numbers of the human genome and their high degree of polymorphism. Fluorescence based detection of the STRP pattern using the LI-COR model 4000S automated DNA sequencer eliminates the need for radioactivity and produces a digitized image that can be used for the analysis of the polymorphisms. In an effort to reduce the cost of STRP analysis, we have synthesized primers with a 19 bp extension complementary to the sequence of the M13 primer on the 5{prime} end of onemore » of the two primers used in the amplification of the STRP instead of using primers with direct conjugation of the infrared fluorescent dye. Up to 5 primer pairs can be multiplexed together with the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye. Comparisons between primers that have been directly conjugated to the fluor with those having the M13 sequence extension show no difference in the ability to determine the STRP pattern. At present, the entire Weber 4A set of STRP markers is available with the M13 5{prime} extension. We are currently using this technique for linkage analysis of familial breast cancer and asthma. The combination of STRP analysis using fluorescence detection will allow this technique to be fully automated for allele scoring and linkage analysis.« less

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1993-01-01

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

Glycosylatable GFP as a compartment-specific membrane topology reporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hunsang; Min, Jisoo; Heijne, Gunnar von

2012-11-02

Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present amore » robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.« less

Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching, UV/vis and FTIR spectroscopic techniques.

PubMed

Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

2016-03-01

The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non-fluorescent CF-CFA and CF-CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (KSV), quenching rate constant (kq), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (KSV) between CF and CGA, CFA are 1.84 × 10(4) and 1.04 × 10(4) L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA. Copyright © 2015 John Wiley & Sons, Ltd.

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC-Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma.

PubMed

El-Zaher, Asmaa A; Mahrouse, Marianne A

2013-01-01

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min(-1). A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20-200 ng mL(-1). The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC—Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma

PubMed Central

El-Zaher, Asmaa A.; Mahrouse, Marianne A.

2013-01-01

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min−1. A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20–200 ng mL−1. The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference. PMID:23700362

Sensitive determination of enoxacin in pharmaceutical formulations by its quench effect on the fluorescence of glutathione-capped CdTe quantum dots.

PubMed

Yang, Qiong; Tan, Xuanping; Yang, Jidong

2016-02-01

A sensitive and simple method for the determination of enoxacin (ENX) was developed based on the fluorescence quenching effect of ENX for glutathione (GSH)-capped CdTe quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 4.333 × 10(-9)  mol⋅L(-1) to 1.4 × 10(-5)  mol⋅L(-1) with a correlation coefficient (R) of 0.9987, and the detection limit (3σ/K) was 1.313 × 10(-9)  mol⋅L(-1). The corresponding mechanism has been proposed on the basis of electron transfer supported by ultraviolet-visible (UV) light absorption, fluorescence spectroscopy, and the measurement of fluorescence lifetime. The method has been applied to the determination of ENX in pharmaceutical formulations (enoxacin gluconate injections and commercial tablets) with satisfactory results. The proposed method manifested several advantages such as high sensitivity, short analysis time, low cost and ease of operation. Copyright © 2015 John Wiley & Sons, Ltd.

Validated stability-indicating spectrofluorimetric methods for the determination of ebastine in pharmaceutical preparations

PubMed Central

2011-01-01

Two sensitive, selective, economic, and validated spectrofluorimetric methods were developed for the determination of ebastine (EBS) in pharmaceutical preparations depending on reaction with its tertiary amino group. Method I involves condensation of the drug with mixed anhydrides (citric and acetic anhydrides) producing a product with intense fluorescence, which was measured at 496 nm after excitation at 388 nm. Method (IIA) describes quantitative fluorescence quenching of eosin upon addition of the studied drug where the decrease in the fluorescence intensity was directly proportional to the concentration of ebastine; the fluorescence quenching was measured at 553 nm after excitation at 457 nm. This method was extended to (Method IIB) to apply first and second derivative synchronous spectrofluorimetric method (FDSFS & SDSFS) for the simultaneous analysis of EBS in presence of its alkaline, acidic, and UV degradation products. The proposed methods were successfully applied for the determination of the studied compound in its dosage forms. The results obtained were in good agreement with those obtained by a comparison method. Both methods were utilized to investigate the kinetics of the degradation of the drug. PMID:21385439

Determination of Components in Beverages by Thin-Layer Chromatography.

ERIC Educational Resources Information Center

Ma, Yinfa; Yeung, Edward S.

1990-01-01

Described is a simple and interesting chromatography experiment using three different fluorescence detection principles for the determination of caffeine, saccharin and sodium benzoate in beverages. Experimental procedures and an analysis and discussion of the results are included. (CW)

Solid-phase microextraction of benzimidazole fungicides in environmental liquid samples and HPLC-fluorescence determination.

PubMed

López Monzón, A; Vega Moreno, D; Torres Padrón, M E; Sosa Ferrera, Z; Santana Rodríguez, J J

2007-03-01

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) with fluorescence detection was optimized for extraction and determination of four benzimidazole fungicides (benomyl, carbendazim, thiabendazole, and fuberidazole) in water. We studied extraction and desorption conditions, for example fiber type, extraction time, ionic strength, extraction temperature, and desorption time to achieve the maximum efficiency in the extraction. Results indicate that SPME using a Carboxen-polydimethylsiloxane 75 microm (CAR-PDMS) fiber is suitable for extraction of these types of compound. Final analysis of benzimidazole fungicides was performed by HPLC with fluorescence detection. Recoveries ranged from 80.6 to 119.6 with RSDs below 9% and limits of detection between 0.03 and 1.30 ng mL-1 for the different analytes. The optimized procedure was applied successfully to the determination of benzimidazole fungicides mixtures in environmental water samples (sea, sewage, and ground water).

Rapid determination of ampicillin in bovine milk by liquid chromatography with fluorescence detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ang, C.Y.W.; Luo, Wenhong

1997-01-01

A rapid and sensitive liquid chromatographic (LC) method was developed for the determination of ampicillin residues in raw bovine milk, processed skim milk, and pasteurized, homogenized whole milk with vitamin D. Milk samples were deproteinized with trichloroacetic acid (TCA) and acetonictrile. After centrifugation, the clear supernatant was reacted with formaldehyde and TCA under heat. The major fluorescent derivative of ampicillin was then determined by reversed-phase LC with fluorescence detection. Average recoveries of ampicillin fortified at 5, 10, and 20 ppb (ng/mL) were all >85% with coefficients of variation <10%. Limits of detection ranged from 0.31 to 0.51 ppb and limitsmore » of quantitation, from 0.66 to 1.2 ppb. After appropriate validation, this method should be suitable for rapid analysis of milk for ampicillin residues at the tolerance level of 10 ppb. 16 refs., 4 figs., 3 tabs.« less

Excitation-emission matrix fluorescence spectroscopy in conjunction with multiway analysis for PAH detection in complex matrices.

PubMed

Nahorniak, Michelle L; Booksh, Karl S

2006-12-01

A field portable, single exposure excitation-emission matrix (EEM) fluorometer has been constructed and used in conjunction with parallel factor analysis (PARAFAC) to determine the sub part per billion (ppb) concentrations of several aqueous polycyclic aromatic hydrocarbons (PAHs), such as benzo(k)fluoranthene and benzo(a)pyrene, in various matrices including aqueous motor oil extract and asphalt leachate. Multiway methods like PARAFAC are essential to resolve the analyte signature from the ubiquitous background in environmental samples. With multiway data and PARAFAC analysis it is shown that reliable concentration determinations can be achieved with minimal standards in spite of the large convoluting fluorescence background signal. Thus, rapid fieldable EEM analyses may prove to be a good screening method for tracking pollutants and prioritizing sampling and analysis by more complete but time consuming and labor intensive EPA methods.

Determination of Zn/Cu ratio and oligoelements in serum samples by total reflection X-ray fluorescence spectrometry for cancer diagnosis

NASA Astrophysics Data System (ADS)

Marcó P., L. M.; Jiménez, E.; Hernández C., E. A.; Rojas, A.; Greaves, E. D.

2001-11-01

The method of quantification using the Compton peak as an internal standard, developed in a previous work, was applied to the routine determination of Fe, Cu, Zn and Se in serum samples from normal individuals and cancer patients by total reflection X-ray fluorescence spectrometry. Samples were classified according to age and sex of the donor, in order to determine reference values for normal individuals. Results indicate that the Zn/Cu ratio and the Cu concentration could prove to be useful tools for cancer diagnosis. Significant differences in these parameters between the normal and cancer group were found for all age ranges. The multielemental character of the technique, coupled with the small amounts of sample required and the short analysis time make it a valuable tool in clinical analysis.

Array biosensor for detection of toxins

NASA Technical Reports Server (NTRS)

Ligler, Frances S.; Taitt, Chris Rowe; Shriver-Lake, Lisa C.; Sapsford, Kim E.; Shubin, Yura; Golden, Joel P.

2003-01-01

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Gadolinium concentration analysis in brain phantom by X-ray fluorescence.

PubMed

Almalki, Musaed; Majid, Samir Abdul; Butler, Philip H; Reinisch, Lou

2010-06-01

We have measured the X-ray fluorescence from gadolinium as a function of concentration and position in tumors of different sizes and shapes in a head phantom. The gadolinium fluorescence was excited with a 36 GBq Am-241 source. The fluorescence signal was detected with a CdTe detector and a multi-channel analyzer. The fluorescence peak was clearly separated from the scattered X-rays. Concentrations of 5.62-78.63 mg/ml of Gd ion were used in 1, 2, and 3 cm diameter spherical tumors and a 2x4 cm oblate spheroid tumor. The data show trends approaching saturation for the highest concentrations, probably due to reabsorption in the tumor. A comparison of X-ray photographic imaging and densitometer measurements to determine concentration is also presented.

Determination of tannin in green tea infusion by flow-injection analysis based on quenching the fluorescence of 3-aminophthalate.

PubMed

Chen, Richie L C; Lin, Chun-Hsun; Chung, Chien-Yu; Cheng, Tzong-Jih

2005-11-02

A flow-injection analytical system was developed to determine tannin content in green tea infusions. The flow-injection system is based on measuring the quenching effect of tannin on the fluorescence of 3-aminophthalate. Fluorophore was obtained by auto-oxidation of luminol during solution preparation. System performance was satisfactory for routine analysis (sample throughput >20 h(-1); linear dynamic range for tannic acid, 0.005-0.3 mg/mL; linear dynamic range for green tea tannin, 0.02-1.0 mg/mL; CV < 3%). The flow-injection method is immune from interference by coexisting ascorbate in green tea infusion. Analytical results were verified by the ferrous tartrate method, the Japanese official analytical method.

Classification of corn kernels contaminated with aflatoxins using fluorescence and reflectance hyperspectral images analysis

NASA Astrophysics Data System (ADS)

Zhu, Fengle; Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Brown, Robert; Bhatnagar, Deepak; Cleveland, Thomas

2015-05-01

Aflatoxins are secondary metabolites produced by certain fungal species of the Aspergillus genus. Aflatoxin contamination remains a problem in agricultural products due to its toxic and carcinogenic properties. Conventional chemical methods for aflatoxin detection are time-consuming and destructive. This study employed fluorescence and reflectance visible near-infrared (VNIR) hyperspectral images to classify aflatoxin contaminated corn kernels rapidly and non-destructively. Corn ears were artificially inoculated in the field with toxigenic A. flavus spores at the early dough stage of kernel development. After harvest, a total of 300 kernels were collected from the inoculated ears. Fluorescence hyperspectral imagery with UV excitation and reflectance hyperspectral imagery with halogen illumination were acquired on both endosperm and germ sides of kernels. All kernels were then subjected to chemical analysis individually to determine aflatoxin concentrations. A region of interest (ROI) was created for each kernel to extract averaged spectra. Compared with healthy kernels, fluorescence spectral peaks for contaminated kernels shifted to longer wavelengths with lower intensity, and reflectance values for contaminated kernels were lower with a different spectral shape in 700-800 nm region. Principal component analysis was applied for data compression before classifying kernels into contaminated and healthy based on a 20 ppb threshold utilizing the K-nearest neighbors algorithm. The best overall accuracy achieved was 92.67% for germ side in the fluorescence data analysis. The germ side generally performed better than endosperm side. Fluorescence and reflectance image data achieved similar accuracy.

Analysis of surgical margins in oral cancer using in situ fluorescence spectroscopy.

PubMed

Francisco, Ana Lucia Noronha; Correr, Wagner Rafael; Pinto, Clóvis Antônio Lopes; Gonçalves Filho, João; Chulam, Thiago Celestino; Kurachi, Cristina; Kowalski, Luiz Paulo

2014-06-01

Oral cancer is a public health problem with high prevalence in the population. Local tumor control is best achieved by complete surgical resection with adequate margins. A disease-free surgical margin correlates with a lower rate of local recurrence and a higher rate of disease-free survival. Fluorescence spectroscopy is a noninvasive diagnostic tool that can aid in real-time cancer detection. The technique, which evaluates the biochemical composition and structure of tissue fluorescence, is relatively simple, fast and, accurate. This study aimed to compare oral squamous cell carcinoma lesions to surgical margins and the mucosa of healthy volunteers by fluorescence spectroscopy. The sample consisted of 56 individuals, 28 with oral squamous cell carcinoma and 28 healthy volunteers with normal oral mucosa. Thirty six cases (64.3%) were male and the mean age was 60.9 years old. The spectra were classified and compared to histopathology to determine fluorescence efficiency for diagnostic discrimination of tumors. In the analysis of the other cases we observed discrimination between normal mucosa, injury and margins. At two-year follow up, three individuals had local recurrence, and in two cases investigation fluorescence in the corresponding area showed qualitative differences in spectra between the recurrence area and the area without recurrence at the same anatomical site in the same patient. In situ analysis of oral mucosa showed the potential of fluorescence spectroscopy as a diagnostic tool that can aid in discrimination of altered mucosa and normal mucosa. Copyright © 2014 Elsevier Ltd. All rights reserved.

Laser diagnostics of an evaporating electrospray

NASA Astrophysics Data System (ADS)

Yi, Tongxun

2014-01-01

An electrospray atomizer generates monodisperse, dilute sprays when working in the cone-jet mode. Evolution of an electrospray with droplet diameter below 10 μm is studied with phase Doppler particle analyzer (PDPA) and the exciplex-PLIF technique. The evaporation rate constant is determined from droplet velocity and diameter measured with a PDPA and is found to sharply increase with the velocity slip and the coflow temperature. Fluorescence around 400 nm, usually referred to as TMPD fluorescence, is calibrated with a heated, laminar, coflow vapor jet diluted with nitrogen. The TMPD fluorescence yield nonlinearly increases with temperature up to 538 K and then declines. Single-shot images show that fluorescence around 400 nm is mainly generated from TMPD vapor and that from droplets can be neglected as a first analysis; however, fluorescence around 490 nm, usually referred to as exciplex fluorescence, is generated from both droplets and fuel vapor immediately around droplets. Exciplex fluorescence is correlated with PDPA measurements and TMPD fluorescence. Effects of temperature, fuel composition, overlap of fluorescent spectra, and chemical equilibrium for exciplex formation are discussed. Technical challenges for quantitative exciplex-PLIF measurements are highlighted.

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.

On-line analysis of algae in water by discrete three-dimensional fluorescence spectroscopy.

PubMed

Zhao, Nanjing; Zhang, Xiaoling; Yin, Gaofang; Yang, Ruifang; Hu, Li; Chen, Shuang; Liu, Jianguo; Liu, Wenqing

2018-03-19

In view of the problem of the on-line measurement of algae classification, a method of algae classification and concentration determination based on the discrete three-dimensional fluorescence spectra was studied in this work. The discrete three-dimensional fluorescence spectra of twelve common species of algae belonging to five categories were analyzed, the discrete three-dimensional standard spectra of five categories were built, and the recognition, classification and concentration prediction of algae categories were realized by the discrete three-dimensional fluorescence spectra coupled with non-negative weighted least squares linear regression analysis. The results show that similarities between discrete three-dimensional standard spectra of different categories were reduced and the accuracies of recognition, classification and concentration prediction of the algae categories were significantly improved. By comparing with that of the chlorophyll a fluorescence excitation spectra method, the recognition accuracy rate in pure samples by discrete three-dimensional fluorescence spectra is improved 1.38%, and the recovery rate and classification accuracy in pure diatom samples 34.1% and 46.8%, respectively; the recognition accuracy rate of mixed samples by discrete-three dimensional fluorescence spectra is enhanced by 26.1%, the recovery rate of mixed samples with Chlorophyta 37.8%, and the classification accuracy of mixed samples with diatoms 54.6%.

Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR

PubMed Central

Cutrale, Francesco; Salih, Anya; Gratton, Enrico

2013-01-01

The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein. PMID:24040513

Fast and accurate automated cell boundary determination for fluorescence microscopy

NASA Astrophysics Data System (ADS)

Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

2013-07-01

Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

Mercury mass measurement in fluorescent lamps via neutron activation analysis

NASA Astrophysics Data System (ADS)

Viererbl, L.; Vinš, M.; Lahodová, Z.; Fuksa, A.; Kučera, J.; Koleška, M.; Voljanskij, A.

2015-11-01

Mercury is an essential component of fluorescent lamps. Not all fluorescent lamps are recycled, resulting in contamination of the environment with toxic mercury, making measurement of the mercury mass used in fluorescent lamps important. Mercury mass measurement of lamps via instrumental neutron activation analysis (NAA) was tested under various conditions in the LVR-15 research reactor. Fluorescent lamps were irradiated in different positions in vertical irradiation channels and a horizontal channel in neutron fields with total fluence rates from 3×108 cm-2 s-1 to 1014 cm-2 s-1. The 202Hg(n,γ)203Hg nuclear reaction was used for mercury mass evaluation. Activities of 203Hg and others induced radionuclides were measured via gamma spectrometry with an HPGe detector at various times after irradiation. Standards containing an Hg2Cl2 compound were used to determine mercury mass. Problems arise from the presence of elements with a large effective cross section in luminescent material (europium, antimony and gadolinium) and glass (boron). The paper describes optimization of the NAA procedure in the LVR-15 research reactor with particular attention to influence of neutron self-absorption in fluorescent lamps.

Fluorescence guided surgery and tracer-dose, fact or fiction?

PubMed

KleinJan, Gijs H; Bunschoten, Anton; van den Berg, Nynke S; Olmos, Renato A Valdès; Klop, W Martin C; Horenblas, Simon; van der Poel, Henk G; Wester, Hans-Jürgen; van Leeuwen, Fijs W B

2016-09-01

Fluorescence guidance is an upcoming methodology to improve surgical accuracy. Challenging herein is the identification of the minimum dose at which the tracer can be detected with a clinical-grade fluorescence camera. Using a hybrid tracer such as indocyanine green (ICG)-(99m)Tc-nanocolloid, it has become possible to determine the accumulation of tracer and correlate this to intraoperative fluorescence-based identification rates. In the current study, we determined the lower detection limit of tracer at which intraoperative fluorescence guidance was still feasible. Size exclusion chromatography (SEC) provided a laboratory set-up to analyze the chemical content and to simulate the migratory behavior of ICG-nanocolloid in tissue. Tracer accumulation and intraoperative fluorescence detection findings were derived from a retrospective analysis of 20 head-and-neck melanoma patients, 40 penile and 20 prostate cancer patients scheduled for sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. In these patients, following tracer injection, single photon emission computed tomography fused with computed tomography (SPECT/CT) was used to identify the SN(s). The percentage injected dose (% ID), the amount of ICG (in nmol), and the concentration of ICG in the SNs (in μM) was assessed for SNs detected on SPECT/CT and correlated with the intraoperative fluorescence imaging findings. SEC determined that in the hybrid tracer formulation, 41 % (standard deviation: 12 %) of ICG was present in nanocolloid-bound form. In the SNs detected using fluorescence guidance a median of 0.88 % ID was present, compared to a median of 0.25 % ID in the non-fluorescent SNs (p-value < 0.001). The % ID values could be correlated to the amount ICG in a SN (range: 0.003-10.8 nmol) and the concentration of ICG in a SN (range: 0.006-64.6 μM). The ability to provide intraoperative fluorescence guidance is dependent on the amount and concentration of the fluorescent dye accumulated in the lesion(s) of interest. Our findings indicate that intraoperative fluorescence detection with ICG is possible above a μM concentration.

Acute toxicity of excess mercury on the photosynthetic performance of cyanobacterium, S. platensis--assessment by chlorophyll fluorescence analysis.

PubMed

Lu, C M; Chau, C W; Zhang, J H

2000-07-01

Measurement of chlorophyll fluorescence has been shown to be a rapid, non-invasive, and reliable method to assess photosynthetic performance in a changing environment. In this study, acute toxicity of excess Hg on the photosynthetic performance of the cyanobacterium S. platensis, was investigated by use of chlorophyll fluorescence analysis after cells were exposed to excess Hg (up to 20 microM) for 2 h. The results determined from the fast fluorescence kinetics showed that Hg induced a significant increase in the proportion of the Q(B)-non-reducing PSII reaction centers. The fluorescence parameters measured under the steady state of photosynthesis demonstrated that the increase of Hg concentration led to a decrease in the maximal efficiency of PSII photochemistry, the efficiency of excitation energy capture by the open PSII reaction centers, and the quantum yield of PSII electron transport. Mercury also resulted in a decrease in the coefficients of photochemical and non-photochemical quenching. Mercury may have an acute toxicity on cyanobacteria by inhibiting the quantum yield of photosynthesis sensitively and rapidly. Such changes occurred before any other visible damages that may be evaluated by other conventional measurements. Our results also demonstrated that chlorophyll fluorescence analysis can be used as a useful physiological tool to assess early stages of change in photosynthetic performance of algae in response to heavy metal pollution.

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

PubMed

Lyon, E; Millson, A; Lowery, M C; Woods, R; Wittwer, C T

2001-05-01

Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

DNA Encapsulation of Ten Silver Atoms Produces a Bright, Modulatable, Near Infrared-Emitting Cluster

PubMed Central

Petty, Jeffrey T.; Fan, Chaoyang; Story, Sandra P.; Sengupta, Bidisha; Iyer, Ashlee St. John; Prudowsky, Zachary; Dickson, Robert M.

2010-01-01

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively that is stabilized within C3AC3AC3TC3A. Following chromatographic resolution of the near infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster’s 30% fluorescence quantum yield and 180,000 M−1cm−1 extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength co-illumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity can be realized through Synchronously Amplified Fluorescence Image Recovery (SAFIRe), which further differentiates this new fluorophore. PMID:21116486

Application of normal fluorescence and stability-indicating derivative synchronous fluorescence spectroscopy for the determination of gliquidone in presence of its fluorescent alkaline degradation product

NASA Astrophysics Data System (ADS)

El-ghobashy, Mohamed R.; Yehia, Ali M.; Helmy, Aya H.; Youssef, Nadia F.

2018-01-01

Simple, smart and sensitive normal fluorescence and stability-indicating derivative synchronous spectrofluorimetric methods have been developed and validated for the determination of gliquidone in the drug substance and drug product. Normal spectrofluorimetric method of gliquidone was established in methanol at λ excitation 225 nm and λ emission 400 nm in concentration range 0.2-3 μg/ml with LOD equal 0.028. The fluorescence quantum yield of gliquidone was calculated using quinine sulfate as a reference and found to be 0.542. Stability-indicating first and third derivative synchronous fluorescence spectroscopy were successfully utilized to overcome the overlapped spectra in normal fluorescence of gliquidone and its alkaline degradation product. Derivative synchronous methods are based on using the synchronous fluorescence of gliquidone and its degradation product in methanol at Δ λ50 nm. Peak amplitude in the first derivative of synchronous fluorescence spectra was measured at 309 nm where degradation product showed zero-crossing without interference. The peak amplitudes in the third derivative of synchronous fluorescence spectra, peak to trough were measured at 316,329 nm where degradation product showed zero-crossing. The different experimental parameters affecting the normal and synchronous fluorescence intensity of gliquidone were studied and optimized. Moreover, the cited methods have been validated as per ICH guidelines. The peak amplitude-concentration plots of the derivative synchronous fluorescence were linear over the concentration range 0.05-2 μg/ml for gliquidone. Limits of detection were 0.020 and 0.022 in first and third derivative synchronous spectra, respectively. The adopted methods were successfully applied to commercial tablets and the results demonstrated that the derivative synchronous fluorescence spectroscopy is a powerful stability-indicating method, suitable for routine use with a short analysis time. Statistical comparison between the results obtained by normal fluorescence and derivative synchronous methods and the official one using student's t-test and F-ratio showed no significant difference regarding accuracy and precision.

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF.

PubMed

Yan, Weiying; Colyer, Christa L

2005-08-01

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.

Novel Spectrofluorimetric Method for the Determination of Perindopril Erbumine Based on Fluorescence Quenching of Rhodamine B.

PubMed

Fael, Hanan; Sakur, Amir Al-Haj

2015-11-01

A novel, simple and specific spectrofluorimetric method was developed and validated for the determination of perindopril erbumine (PDE). The method is based on the fluorescence quenching of Rhodamine B upon adding perindopril erbumine. The quenched fluorescence was monitored at 578 nm after excitation at 500 nm. The optimization of the reaction conditions such as the solvent, reagent concentration, and reaction time were investigated. Under the optimum conditions, the fluorescence quenching was linear over a concentration range of 1.0-6.0 μg/mL. The proposed method was fully validated and successfully applied to the analysis of perindopril erbumine in pure form and tablets. Statistical comparison of the results obtained by the developed and reference methods revealed no significant differences between the methods compared in terms of accuracy and precision. The method was shown to be highly specific in the presence of indapamide, a diuretic that is commonly combined with perindopril erbumine. The mechanism of rhodamine B quenching was also discussed.

Excited-state absorption and fluorescence dynamics of Er3+:KY3F10

NASA Astrophysics Data System (ADS)

Labbé, C.; Doualan, J. L.; Moncorgé, R.; Braud, A.; Camy, P.

2018-05-01

We report here on a complete investigation of the excited-state absorption and fluorescence dynamics of Er3+ doped KY3F10 single crystals versus dopant concentrations and optical excitation conditions. Radiative and effective (including non-radiative relaxations) emission lifetimes and branching ratios are determined from a Judd-Ofelt analysis of the absorption spectra and via specific fluorescence experiments using wavelength selective laser excitations. Excited-state absorption and emission spectra are registered within seven spectral domains, i.e. 560 nm, 650 nm, 710 nm, 810 nm, 970 nm, 1550 nm and 2750 nm. A maximum gain cross-section of 0.93 × 10-21 cm2 is determined at the potential laser wavelength of 2.801 μm for a population ratio of 0.48. Saturation of fluorescence intensities and variations of population ratios versus pumping rates are registered and confronted with a rate equation model to derive the rates of the most important up-conversion and cross-relaxation energy transfers occurring at high dopant concentrations.

Upconversion fluorescent strip sensor for rapid determination of Vibrio anguillarum

NASA Astrophysics Data System (ADS)

Zhao, Peng; Wu, Yuanyuan; Zhu, Yihua; Yang, Xiaoling; Jiang, Xin; Xiao, Jingfan; Zhang, Yuanxing; Li, Chunzhong

2014-03-01

Here, we report a simple and ultrasensitive upconversion fluorescent strip sensor based on NaYF4:Yb,Er nanoparticles (NPs) and the lateral flow immunochromatographic assay (LFIA). Carboxyl-modified β-NaYF4:Yb,Er NPs were successfully synthesized by a facile one-pot solvothermal approach, upon further coupling with monoclonal antibody, the resultant UCNPs-antibody conjugates probes were used in LFIA and served as signal vehicles for the fluorescent reporters. V. anguillarum was used as a model analyte to demonstrate the use of this strip sensor. The limit of the detection for the fluorescent strip was determined as 102 CFU mL-1, which is 100 times lower than those displayed by enzyme-linked immunosorbent assays, while the time needed for the detection was only 15 min. Furthermore, no cross-reaction with other eight pathogens was found, indicating the good specificity of the strip. This developed LFIA would offer the potential as a useful tool for the quantification of pathogens analysis in the future.

Combined Raman spectroscopy and autofluoresence imaging method for in vivo skin tumor diagnosis

NASA Astrophysics Data System (ADS)

Zakharov, V. P.; Bratchenko, I. A.; Myakinin, O. O.; Artemyev, D. N.; Khristoforova, Y. A.; Kozlov, S. V.; Moryatov, A. A.

2014-09-01

The fluorescence and Raman spectroscopy (RS) combined method of in vivo detection of malignant human skin cancer was demonstrated. The fluorescence analysis was used for detection of abnormalities during fast scanning of large tissue areas. In suspected cases of malignancy the Raman spectrum analysis of biological tissue was performed to determine the type of neoplasm. A special RS phase method was proposed for in vivo identification of skin tumor. Quadratic Discriminant Analysis was used for tumor type classification on phase planes. It was shown that the application of phase method provides a diagnosis of malignant melanoma with a sensitivity of 89% and a specificity of 87%.

[Development of selective determination methods for quinones with fluorescence and chemiluminescence detection and their application to environmental and biological samples].

PubMed

Kishikawa, Naoya

2010-10-01

Quinones are compounds that have various characteristics such as a biological electron transporter, an industrial product and a harmful environmental pollutant. Therefore, an effective determination method for quinones is required in many fields. This review describes the development of sensitive and selective determination methods for quinones based on some detection principles and their application to analyses in environmental, pharmaceutical and biological samples. Firstly, a fluorescence method was developed based on fluorogenic derivatization of quinones and applied to environmental analysis. Secondly, a luminol chemiluminescence method was developed based on generation of reactive oxygen species through the redox cycle of quinone and applied to pharmaceutical analysis. Thirdly, a photo-induced chemiluminescence method was developed based on formation of reactive oxygen species and fluorophore or chemiluminescence enhancer by the photoreaction of quinones and applied to biological and environmental analyses.

Determination of the implantation dose in silicon wafers by X-ray fluorescence analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klockenkaemper, R.; Becker, M.; Bubert, H.

1990-08-01

The ion dose implanted in silicon wafers was determined by X-ray fluorescence analysis after the implantation process. As only near-surface layers below 1-{mu}m thickness were considered, the calibration could be carried out with external standards consisting of thin films of doped gelatine spread on pure wafers. Dose values for Cr and Co were determined between 4 {times} 10{sup 15} and 2 {times} 10{sup 17} atoms/cm{sup 2}, the detection limits being about 3 {times} 10{sup 14} atoms/cm{sup 2}. The results are precise and accurate apart from a residual scatter of less than 7%. This was confirmed by flame atomic absorption spectrometrymore » after volatilization of the silicon matrix as SiF{sub 4}. It was found that ion-current measurements carried out during the implantation process can have considerable systematic errors.« less

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2011-10-01

study! of! the! pathogenesis! of! breast! cancer.! These! include! analysis ! of! intracellular! signaling!by!Western!blotting,! determination!of! cell...proliferation!by! sulforhodamine!B! staining,! fluorescence: activated!cell!sorting!(FACS)! analysis ,!stable!cell!line!generation,!production!of!and...transduction!using!retroviral! and!lentiviral!supernatants,! immunocytochemistry!and!confocal! laser!microscopy,! immunohistochemistry,!and! analysis

Sensitivity and specificity of indocyanine green near-infrared fluorescence imaging in detection of metastatic lymph nodes in colorectal cancer: Systematic review and meta-analysis.

PubMed

Emile, Sameh H; Elfeki, Hossam; Shalaby, Mostafa; Sakr, Ahmad; Sileri, Pierpaolo; Laurberg, Søren; Wexner, Steven D

2017-11-01

This review aimed to determine the overall sensitivity and specificity of indocyanine green (ICG) near-infrared (NIR) fluorescence in sentinel lymph node (SLN) detection in Colorectal cancer (CRC). A systematic search in electronic databases was conducted. Twelve studies including 248 patients were reviewed. The median sensitivity, specificity, and accuracy rates were 73.7, 100, and 75.7. The pooled sensitivity and specificity rates were 71% and 84.6%. In conclusion, ICG-NIR fluorescence is a promising technique for detecting SLNs in CRC. © 2017 Wiley Periodicals, Inc.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Feasibility of the simultaneous determination of polycyclic aromatic hydrocarbons based on two-dimensional fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Renjie; Dong, Guimei; Sun, Xueshan; Yang, Yanrong; Yu, Yaping; Liu, Haixue; Zhang, Weiyu

2018-02-01

A new approach for quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in environment was proposed based on two-dimensional (2D) fluorescence correlation spectroscopy in conjunction with multivariate method. 40 mixture solutions of anthracene and pyrene were prepared in the laboratory. Excitation-emission matrix (EEM) fluorescence spectra of all samples were collected. And 2D fluorescence correlation spectra were calculated under the excitation perturbation. The N-way partial least squares (N-PLS) models were developed based on 2D fluorescence correlation spectra, showing a root mean square error of calibration (RMSEC) of 3.50 μg L- 1 and root mean square error of prediction (RMSEP) of 4.42 μg L- 1 for anthracene and of 3.61 μg L- 1 and 4.29 μg L- 1 for pyrene, respectively. Also, the N-PLS models were developed for quantitative analysis of anthracene and pyrene using EEM fluorescence spectra. The RMSEC and RMSEP were 3.97 μg L- 1 and 4.63 μg L- 1 for anthracene, 4.46 μg L- 1 and 4.52 μg L- 1 for pyrene, respectively. It was found that the N-PLS model using 2D fluorescence correlation spectra could provide better results comparing with EEM fluorescence spectra because of its low RMSEC and RMSEP. The methodology proposed has the potential to be an alternative method for detection of PAHs in environment.

A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

PubMed

Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

2017-09-15

Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective Determination of Trinitrotoluene Based on Energy Transfer between Carbon Dots and Gold Nanoparticles.

PubMed

Oskoei, Yones Mosaei; Fattahi, Hassan; Hassanzadeh, Javad; Azar, Ali Mousavi

2016-01-01

A fluorescence resonance energy transfer (FRET) system between carbon dots (C-dots) and amine-capped gold nanoparticles (AuNPs) was developed for the selective determination of 2,4,6-trinitrotoluene (TNT). C-dots have an intrinsic florescence emission depending on their exciting wavelength. In the presence of AuNPs, C-dots adsorb on the Au surfaces, and NPs treat as energy acceptor, which can receive light emitted by C-dots, leading to decrease the fluorescence intensity of C-dots. Furthermore, it is observed that nitroaromatic compounds, especially TNT, could restore this fluorescence due to selective interaction with AuNPs via amine groups, and so releasing the C-dots. Based on this effect, a sensitive and selective fluorescence turn-on probe was designed for the determination of TNT. Some important factors including AuNPs and C-dot concentrations and media pH, which would affect the efficiency of the probe, were optimized. Under the optimum experimental conditions, good linear relationships in the range of 7 - 250 nmol L(-1) TNT with the detection limit of 2.2 nmol L(-1) were obtained. The proposed method was satisfactorily applied to the determination of TNT in the environmental water samples. Compared with previous reports, the developed method has relatively high sensitivity, short analysis time, low cost and ease of operation.

Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP)

PubMed Central

Day, Charles A.; Kraft, Lewis J.; Kang, Minchul; Kenworthy, Anne K.

2012-01-01

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection and analysis. In this review we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. PMID:23042527

Quinoline containing acetyl hydrazone: An easily accessible switch-on optical chemosensor for Zn2.

PubMed

Wu, Wei-Na; Mao, Pan-Dong; Wang, Yuan; Zhao, Xiao-Lei; Xu, Zhou-Qing; Xu, Zhi-Hong; Xue, Yuan

2018-01-05

A simple chemosensor, namely, N-((quinolin-8-yl)methylene)acetohydrazide (1) was synthesized and used as an off-on fluorescence sensor, which exhibits high selectivity toward Zn 2+ in aqueous media. The probe has large Stokes shift of >200nm, and its detection limit for Zn 2+ is 89.3nM. The binding process was confirmed through UV-vis absorption analysis, fluorescence measurements, mass spectroscopy study, 1 H NMR spectra and density functional theory calculation. The crystal structures of Zn 2+ , Ni 2+ , and Cu 2+ complexes based on 1 were determined through X-ray crystallographic analysis. The fluorescent probe was then applied to monitor intracellular Zn 2+ in HeLa cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of carrier yields for neutron activation analysis using energy dispersive X-ray spectrometry

USGS Publications Warehouse

Johnson, R.G.; Wandless, G.A.

1984-01-01

A new method is described for determining carrier yield in the radiochemical neutron activation analysis of rare-earth elements in silicate rocks by group separation. The method involves the determination of the rare-earth elements present in the carrier by means of energy-dispersive X-ray fluorescence analysis, eliminating the need to re-irradiate samples in a nuclear reactor after the gamma ray analysis is complete. Results from the analysis of USGS standards AGV-1 and BCR-1 compare favorably with those obtained using the conventional method. ?? 1984 Akade??miai Kiado??.

The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors

PubMed Central

Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias

2015-01-01

BACKGROUND: Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. OBJECTIVE: The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [18F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. METHODS: Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and 18F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. RESULTS: Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and 18F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. CONCLUSION: Age, tumor volume, and 18F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased Ki-67/MIB-1 index and high-grade pathology. Whether fluorescence in grade II gliomas identifies a subtype with worse prognosis remains to be determined. ABBREVIATIONS: 5-ALA, 5-aminolevulinic acid CRT, classification and regression tree 18F-FET PET, 18F-fluoroethyl tyrosine positron emission tomography FLAIR, fluid-attenuated inversion recovery GBM, glioblastoma multiforme O6-MGMT, methylguanine DNA methyltransferase ROC, receiver-operating characteristic SUV, standardized uptake value WHO, World Health Organization PMID:26366972

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

NASA Astrophysics Data System (ADS)

Rolinski, Olaf J.; McLaughlin, Damien; Birch, David J. S.; Vyshemirsky, Vladislav

2016-09-01

The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the conventional approach to modelling intrinsic florescence intensity decay in protein where the key factors determining fluorescence decay, i.e. the excited-state depopulation and the dielectric relaxation (Toptygin and Brand 2000 Chem. Phys. Lett. 322 496-502), are represented by the individual relaxation functions. This allows quantification of both effects separately by determining their parameters from the global analysis of a series of fluorescence intensity decays measured at different detection wavelengths. Moreover, certain pairs of the recovered parameters of tryptophan were found to be correlated, indicating the influence of the dielectric relaxation on the transient rate of the electronic transitions. In this context the potential for the dual excited state depopulation /dielectric relaxation fluorescence lifetime sensing is discussed.

Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

PubMed Central

Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

2011-01-01

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers. PMID:22194797

Fluorescence fingerprint as an instrumental assessment of the sensory quality of tomato juices.

PubMed

Trivittayasil, Vipavee; Tsuta, Mizuki; Imamura, Yoshinori; Sato, Tsuneo; Otagiri, Yuji; Obata, Akio; Otomo, Hiroe; Kokawa, Mito; Sugiyama, Junichi; Fujita, Kaori; Yoshimura, Masatoshi

2016-03-15

Sensory analysis is an important standard for evaluating food products. However, as trained panelists and time are required for the process, the potential of using fluorescence fingerprint as a rapid instrumental method to approximate sensory characteristics was explored in this study. Thirty-five out of 44 descriptive sensory attributes were found to show a significant difference between samples (analysis of variance test). Principal component analysis revealed that principal component 1 could capture 73.84 and 75.28% variance for aroma category and combined flavor and taste category respectively. Fluorescence fingerprints of tomato juices consisted of two visible peaks at excitation/emission wavelengths of 290/350 and 315/425 nm and a long narrow emission peak at 680 nm. The 680 nm peak was only clearly observed in juices obtained from tomatoes cultivated to be eaten raw. The ability to predict overall sensory profiles was investigated by using principal component 1 as a regression target. Fluorescence fingerprint could predict principal component 1 of both aroma and combined flavor and taste with a coefficient of determination above 0.8. The results obtained in this study indicate the potential of using fluorescence fingerprint as an instrumental method for assessing sensory characteristics of tomato juices. © 2015 Society of Chemical Industry.

A novel immobilization multienzyme glucose fluorescence capillary biosensor.

PubMed

Li, Yong-Sheng; Du, Yun-Dong; Chen, Ting-Mei; Gao, Xiu-Feng

2010-02-15

Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, beta-D-glucose reacts with dissolved oxygen to form gluconic acid-delta-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320 nm and 410 nm, respectively. Fluorescence of the dimer is proportional to the concentration of the beta-D-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the L-tyrosine used as fluorescence reagent was 0.15 mol L(-1), the active concentrations of the GOD and the HRP for the immobilization were 15 kU L(-1) and 8 kU L(-1), respectively. Consumptions of the sample and reagents in one determination were 5.0 microL and 15 microL, respectively. Quantitative range of the biosensor for the glucose was in the range 1-10 micromol L(-1), its relative standard deviation was less than 4.9%, and its detection limit was 0.62 micromol L(-1). The biosensor's recovery was in the range 96-105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes. Copyright 2009 Elsevier B.V. All rights reserved.

Determination of curcumin in biologically active supplements and food spices using a mesofluidic platform with fluorescence detection.

PubMed

Petrova, Anastasiia; Dar'in, Dmitriy; Ivanov, Aleхander; Moskvin, Leonid; Ishimatsu, Ryoichi; Nakano, Koji; Imato, Toshihiko; Bulatov, Andrey

2016-10-01

A mesofluidic platform (MP) with fluorescence detection based on a stepwise injection analysis (SWIA) was used for the determination of curcumin in biologically active supplements and food spices. The main units of the MP are a mixing chamber (MC) and an optical channel with a quartz capillary inside. The MC provides rapid and complete mixing solutions by gas bubbling. The proposed method is based on the new rapid and sensitive reaction of curcumin with a fluorescence reagent - 4-(2,3,3-trimethyl-3H-indolium-1-yl)butane-1-sulfonate (TIBS). The fluorescence intensity of TIBS is greatly quenched in the presence of curcumin in an alkaline medium. The linear range was from 1 to 10µM of curcumin, and the limit of detection, calculated as 3σ of a blank test (n=5), was found to be 0.3µM. The sample throughput was 24h(-1). The proposed method was successfully applied for the determination of curcumin in biologically active supplements and samples of food spices. The obtained data were in good agreement with those measured by a HPLC-UV method. Copyright © 2016 Elsevier B.V. All rights reserved.

Organic petrology of selected oil shale samples from lower Carboniferous Albert Formation, New Brunswick, Canada

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalkreuth, W.; Macauley, G.

1984-04-01

Incident light microscopy was used to describe maturation and composition of organic material in oil shale samples from the Lower Carboniferous Albert Formation of New Brunswick. The maturation level was determined in normal (white) light by measuring vitrinite reflectance and in fluorescent light by measuring fluorescence spectral of alginite B. Results indicate low to intermediate maturation for all of the samples. Composition was determined by maceral analysis. Alginite B is the major organic component in all samples having significant oil potential. Oil yields obtained from the Fischer Assay process, and oil and gas potentials from Rock-Eval analyses correlate to themore » amounts of alginite B and bituminite determined in the samples. In some of the samples characterized by similar high concentrations of alginite B, decrease in Fischer Assay yields and oil and gas potentials is related to an increase in maturation, as expected by increase in the fluorescence parameter lambda/sub max/ and red/green quotient of alginite B. Incident light microscopy, particularly with fluorescent light, offers a valuable tool for the identification of the organic matter in oil shales and for the evaluation of their oil and gas potentials.« less

Stoichiometry determination of (Pb,La)(Zr,Ti)O3-type nano-crystalline ferroelectric ceramics by wavelength-dispersive X-ray fluorescence spectrometry.

PubMed

Sitko, Rafał; Zawisza, Beata; Kita, Andrzej; Płońska, Małgorzata

2006-07-01

Analysis of small samples of lanthanum-doped lead zirconate titanate (PLZT) by wavelength-dispersive X-ray fluorescence spectrometry (WDXRF) is presented. The powdered material in ca. 30 mg was suspended in water and collected on the membrane filter. The pure oxide standards (PbO, La2O3, ZrO2 and TiO2) were used for calibration. The matrix effects were corrected using a theoretical influence coefficients algorithm for intermediate-thickness specimens. The results from XRF method were compared with the results from the inductively coupled plasma optical emission spectrometry (ICP-OES). Agreement between XRF and ICP-OES analysis was satisfactory and indicates the usefulness of XRF method for stoichiometry determination of PLZT.

Evaluation of CDOM sources and their links with water quality in the lakes of Northeast China using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Ying; Song, Kaishan; Wen, Zhidan; Fang, Chong; Shang, Yingxin; Lv, Lili

2017-07-01

The spatial distributions of the fluorescence intensities Fmax for chromophoric dissolved organic matter (CDOM) components, the fluorescence indices (FI370 and FI310) and their correlations with water quality of 19 lakes in the Songhua River Basin (SHRB) across semiarid regions of Northeast China were examined with the data collected in September 2012 and 2015. The 19 lakes were divided into two groups according to EC (threshold value = 800 μS cm-1): fresh water (N = 13) and brackish water lakes (N = 6). The fluorescent characteristics of CDOM in the 19 lakes were investigated using excitation-emission matrix fluorescence spectroscopy (EEM) coupled with parallel factor (PARAFAC) and multivariate analysis. Two humic-like components (C1 and C3), one tryptophan-like component (C2), and one tyrosine-like component (C4) were identified by PARAFAC. The component C4 was not included in subsequent analyses due to the strong scatter in some colloidal water samples from brackish water lakes. The correlations between Fmax for the three EEM-PARAFAC extracted CDOM components C1-C3, the fluorescence indices (FI370 and FI310) and the water quality parameters (i.e., TN, TP, Chl-a, pH, EC, turbidity (Turb) and dissolved organic carbon (DOC)) were determined by redundancy analysis (RDA). The results of RDA analysis showed that spatial variation in land cover, pollution sources, and salinity/EC gradients in water quality affected Fmax for the fluorescent components C1-C3 and the fluorescence indices (FI370 and FI310). Further examination indicated that the CDOM fluorescent components and the fluorescence indices (FI370 and FI310) did not significantly differ (t-test, p > 0.05) in fresh water (N = 13) and brackish water lakes (N = 6). There was a difference in the distribution of the average Fmax for the CDOM fluorescent components between C1 to C3 from agricultural sources and urban wastewater sources in hypereutrophic brackish water lakes. The Fmax for humic-like components C1 and C3 spatially varied with land cover among the 19 lakes. Our results indicated that the spatial distributions of Fmax for CDOM fluorescent components and their correlations with water quality can be evaluated by EEM-PARAFAC and multivariate analysis among the 19 lakes across semiarid regions of Northeast China, which has potential implication for lakes with similar genesis.

Trimodal spectra for high discrimination of benign and malignant prostate tissue

NASA Astrophysics Data System (ADS)

Al Salhi, Mohamad; Masilamani, Vadivel; Trinka, Vijmasi; Rabah, Danny; Al Turki, Mohammed R.

2011-02-01

High false positives and over diagnosis is a major problem with management of prostate cancer. A non-invasive or a minimally invasive technique to accurately distinguish malignant prostate cancers from benign tumors will be extremely helpful to overcome this problem. In this paper, we had used three different fluorescence spectroscopy techniques viz., Fluorescence Emission Spectrum (FES), Stokes' Shift Spectrum (SSS) and Reflectance Spectrum (RS) to discriminate benign prostate tumor tissues (N=12) and malignant prostate cancer tissues (N=8). These fluorescence techniques were used to determine the relative concentration of naturally occurring biomolecules such as tryptophan, elastin, NADH and flavin which are found to be out of proportion in cancer tissues. Our studies show that combining all three techniques, benign and malignant prostate tissues could be classified with accuracy greater than 90%. This preliminary report is based on in vitro spectroscopy analysis. However, by employing fluorescence endoscopy techniques, this can be extended to in vivo analysis as well. This technique has the potential to identify malignant prostate tissues without surgery.

Determination of rice syrup adulterant concentration in honey using three-dimensional fluorescence spectra and multivariate calibrations

NASA Astrophysics Data System (ADS)

Chen, Quansheng; Qi, Shuai; Li, Huanhuan; Han, Xiaoyan; Ouyang, Qin; Zhao, Jiewen

2014-10-01

To rapidly and efficiently detect the presence of adulterants in honey, three-dimensional fluorescence spectroscopy (3DFS) technique was employed with the help of multivariate calibration. The data of 3D fluorescence spectra were compressed using characteristic extraction and the principal component analysis (PCA). Then, partial least squares (PLS) and back propagation neural network (BP-ANN) algorithms were used for modeling. The model was optimized by cross validation, and its performance was evaluated according to root mean square error of prediction (RMSEP) and correlation coefficient (R) in prediction set. The results showed that BP-ANN model was superior to PLS models, and the optimum prediction results of the mixed group (sunflower ± longan ± buckwheat ± rape) model were achieved as follow: RMSEP = 0.0235 and R = 0.9787 in the prediction set. The study demonstrated that the 3D fluorescence spectroscopy technique combined with multivariate calibration has high potential in rapid, nondestructive, and accurate quantitative analysis of honey adulteration.

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

PubMed Central

Kaščáková, Slávka; Maigre, Laure; Chevalier, Jacqueline; Réfrégiers, Matthieu; Pagès, Jean-Marie

2012-01-01

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack. PMID:22719907

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Comparative study of the fluorescence intensity of dental composites and human teeth submitted to artificial aging.

PubMed

Jablonski, Tatiana; Takahashi, Marcos Kenzo; Brum, Rafeal Torres; Rached, Rodrigo Nunes; Souza, Evelise M

2014-01-01

The aim of this study was to evaluate quantitatively the fluorescence of resin composites and human teeth, and to determine the stability of fluorescence after aging. Ten specimens were built using a 1 mm thick increment of dentin composite overlapped by a 0.5 mm thick increment of enamel composite. Ten sound human molars were sectioned and silicon carbide-polished to obtain enamel and dentin slabs 1.5 mm in thickness. Fluorescence measurements were carried out by a fluorescence spectrophotometer before and after thermocycling (2000 cycles, 5°C and 55°C). One-way analysis of variance (ANOVA) with repeated measures and Tukey's test were performed at a significance level of 5%. Most of the tested composites showed significant differences in fluorescence both before and after aging (P < 0.05). Opallis was the only composite whose fluorescence was similar to that of human teeth at both periods of evaluation (P > 0.05), and was the only composite that showed comparable results of fluorescence to the tooth structure before and after thermocycling. With the exception of Filtek Supreme, there were significant reductions in fluorescence intensity for all the tested composites (P < 0.05).

Elemental analysis using a handheld X-Ray fluorescence spectrometer

USGS Publications Warehouse

Groover, Krishangi D.; Izbicki, John

2016-06-24

The U.S. Geological Survey is collecting geologic samples from local stream channels, aquifer materials, and rock outcrops for studies of trace elements in the Mojave Desert, southern California. These samples are collected because geologic materials can release a variety of elements to the environment when exposed to water. The samples are to be analyzed with a handheld X-ray fluorescence (XRF) spectrometer to determine the concentrations of up to 27 elements, including chromium.

Laser-Excited Atomic Fluorescence and Ionization in a Graphite Furnace for the Determination of Metals and Nonmetals

NASA Astrophysics Data System (ADS)

Butcher, David James

1990-01-01

Here is reported novel instrumentation for atomic spectrometry that combined the use of a pulsed laser system as the light source and an electrothermal atomizer as the atom cell. The main goal of the research was to develop instrumentation that was more sensitive for elemental analysis than commercially available instruments and could be used to determine elements in real sample matrices. Laser excited atomic fluorescence spectrometry (LEAFS) in an electrothermal atomizer (ETA) was compared to ETA atomic absorption spectrometry (AAS) for the determination of thallium, manganese, and lead in food and agricultural standard reference materials (SRMs). Compared to ETA AAS, ETA LEAFS has a longer linear dynamic range (LDR) (5-7 orders of magnitude compared to 2-3 orders of magnitude) and higher sensitivity (10 ^{-16} to 10^{ -14} g as compared to 10^{ -13} to 10^{-11} g). Consequently, ETA LEAFS allows elemental analysis to be done over a wider range of concentrations with less dilution steps. Thallium was accurately determined in biological samples by ETA LEAFS at amounts five to one hundred times below the ETA AAS detection limit. ETA AAS and ETA LEAFS were compared for the determination of lead and manganese, and in general, the accuracies and precisions of ETA AAS were the same, with typical precisions between 3% and 6%. Fluorine was determined using laser excited molecular fluorescence spectrometry (LEMOFS) in an ETA. Molecular fluorescence from magnesium fluoride was collected, and the detection limit of 0.3 pg fluorine was two to six orders of magnitude more sensitive than other methods commonly used for the determination of fluorine. Significant interferences from ions were observed, but the sensitivity was high enough that fluorine could be determined in freeze dried urine SRMs by diluting the samples by a factor of one hundred to remove the interferences. Laser enhanced ionization (LEI) in an ETA was used for the determination of metals. For thallium, indium, and lithium, detection limits between 0.7 and 2 pg were obtained, with an LDR of 3.5 orders of magnitude. Sodium was shown to severely depress the indium LEI signal in an ETA.

Effects of iron on optical properties of dissolved organic matter.

PubMed

Poulin, Brett A; Ryan, Joseph N; Aiken, George R

2014-09-02

Iron is a source of interference in the spectroscopic analysis of dissolved organic matter (DOM); however, its effects on commonly employed ultraviolet and visible (UV-vis) light adsorption and fluorescence measurements are poorly defined. Here, we describe the effects of iron(II) and iron(III) on the UV-vis absorption and fluorescence of solutions containing two DOM fractions and two surface water samples. In each case, regardless of DOM composition, UV-vis absorption increased linearly with increasing iron(III). Correction factors were derived using iron(III) absorption coefficients determined at wavelengths commonly used to characterize DOM. Iron(III) addition increased specific UV absorbances (SUVA) and decreased the absorption ratios (E2:E3) and spectral slope ratios (SR) of DOM samples. Both iron(II) and iron(III) quenched DOM fluorescence at pH 6.7. The degree and region of fluorescence quenching varied with the iron:DOC concentration ratio, DOM composition, and pH. Regions of the fluorescence spectra associated with greater DOM conjugation were more susceptible to iron quenching, and DOM fluorescence indices were sensitive to the presence of both forms of iron. Analyses of the excitation-emission matrices using a 7- and 13-component parallel factor analysis (PARAFAC) model showed low PARAFAC sensitivity to iron addition.

Analysis of doxorubicin distribution in MCF-7 cells treated with drug-loaded nanoparticles by combination of two fluorescence-based techniques, confocal spectral imaging and capillary electrophoresis.

PubMed

Gautier, Juliette; Munnier, Emilie; Soucé, Martin; Chourpa, Igor; Douziech Eyrolles, Laurence

2015-05-01

The intracellular distribution of the antiancer drug doxorubicin (DOX) was followed qualitatively by fluorescence confocal spectral imaging (FCSI) and quantitatively by capillary electrophoresis (CE). FCSI permits the localization of the major fluorescent species in cell compartments, with spectral shifts indicating the polarity of the respective environment. However, distinction between drug and metabolites by FCSI is difficult due to their similar fluorochromes, and direct quantification of their fluorescence is complicated by quantum yield variation between different subcellular environments. On the other hand, capillary electrophoresis with fluorescence detection (CE-LIF) is a quantitative method capable of separating doxorubicin and its metabolites. In this paper, we propose a method for determining drug and metabolite concentration in enriched nuclear and cytosolic fractions of cancer cells by CE-LIF, and we compare these data with those of FCSI. Significant differences in the subcellular distribution of DOX are observed between the drug administered as a molecular solution or as a suspension of drug-loaded iron oxide nanoparticles coated with polyethylene glycol. Comparative analysis of the CE-LIF vs FCSI data may lead to a tentative calibration of this latter method in terms of DOX fluorescence quantum yields in the nucleus and more or less polar regions of the cytosol.

Effects of iron on optical properties of dissolved organic matter

USGS Publications Warehouse

Poulin, Brett; Ryan, Joseph N.; Aiken, George R.

2014-01-01

Iron is a source of interference in the spectroscopic analysis of dissolved organic matter (DOM); however, its effects on commonly employed ultraviolet and visible (UV–vis) light adsorption and fluorescence measurements are poorly defined. Here, we describe the effects of iron(II) and iron(III) on the UV–vis absorption and fluorescence of solutions containing two DOM fractions and two surface water samples. In each case, regardless of DOM composition, UV–vis absorption increased linearly with increasing iron(III). Correction factors were derived using iron(III) absorption coefficients determined at wavelengths commonly used to characterize DOM. Iron(III) addition increased specific UV absorbances (SUVA) and decreased the absorption ratios (E2:E3) and spectral slope ratios (SR) of DOM samples. Both iron(II) and iron(III) quenched DOM fluorescence at pH 6.7. The degree and region of fluorescence quenching varied with the iron:DOC concentration ratio, DOM composition, and pH. Regions of the fluorescence spectra associated with greater DOM conjugation were more susceptible to iron quenching, and DOM fluorescence indices were sensitive to the presence of both forms of iron. Analyses of the excitation–emission matrices using a 7- and 13-component parallel factor analysis (PARAFAC) model showed low PARAFAC sensitivity to iron addition.

Interaction of amino acid-functionalized silver nanoparticles and Candida albicans polymorphs: A deep-UV fluorescence imaging study.

PubMed

Dojčilović, Radovan; Pajović, Jelena D; Božanić, Dušan K; Bogdanović, Una; Vodnik, Vesna V; Dimitrijević-Branković, Suzana; Miljković, Miona G; Kaščaková, Slavka; Réfrégiers, Matthieu; Djoković, Vladimir

2017-07-01

The interaction of the tryptophan functionalized Ag nanoparticles and live Candida albicans cells was studied by synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging at the DISCO beamline of Synchrotron SOLEIL. DUV imaging showed that incubation of the fungus with functionalized nanoparticles results in significant increase in the fluorescence signal. The analysis of the images revealed that the interaction of the nanoparticles with (pseudo)hyphae polymorphs of the diploid fungus was less pronounced than in the case of yeast cells or budding spores. The changes in the intensity of the fluorescence signals of the cells after incubation were followed in [327-353nm] and [370-410nm] spectral ranges that correspond to the fluorescence of tryptophan in non-polar and polar environment, respectively. As a consequence of the environmental sensitivity of the silver-tryptophan fluorescent nanoprobe, we were able to determine the possible accumulation sites of the nanoparticles. The analysis of the intensity decay kinetics showed that the photobleaching effects were more pronounced in the case of the functionalized nanoparticle treated cells. The results of time-integrated emission in the mentioned spectral ranges suggested that the nanoparticles penetrate the cells, but that the majority of the nanoparticles attach to the cells' surfaces. Copyright © 2017 Elsevier B.V. All rights reserved.

Estuarial fingerprinting through multidimensional fluorescence and multivariate analysis.

PubMed

Hall, Gregory J; Clow, Kerin E; Kenny, Jonathan E

2005-10-01

As part of a strategy for preventing the introduction of aquatic nuisance species (ANS) to U.S. estuaries, ballast water exchange (BWE) regulations have been imposed. Enforcing these regulations requires a reliable method for determining the port of origin of water in the ballast tanks of ships entering U.S. waters. This study shows that a three-dimensional fluorescence fingerprinting technique, excitation emission matrix (EEM) spectroscopy, holds great promise as a ballast water analysis tool. In our technique, EEMs are analyzed by multivariate classification and curve resolution methods, such as N-way partial least squares Regression-discriminant analysis (NPLS-DA) and parallel factor analysis (PARAFAC). We demonstrate that classification techniques can be used to discriminate among sampling sites less than 10 miles apart, encompassing Boston Harbor and two tributaries in the Mystic River Watershed. To our knowledge, this work is the first to use multivariate analysis to classify water as to location of origin. Furthermore, it is shown that curve resolution can show seasonal features within the multidimensional fluorescence data sets, which correlate with difficulty in classification.

Pulp tissue in sex determination: A fluorescent microscopic study

PubMed Central

Nayar, Amit; Singh, Harkanwal Preet; Leekha, Swati

2014-01-01

Aims: To determine and compare the reliability of pulp tissue in determination of sex and to analyze whether caries have any effect on fluorescent body test. Materials and Methods: This study was carried on 50 maxillary and mandibular teeth (25 male teeth and 25 female teeth), which were indicated for extraction. The teeth are categorized into 5 groups, 10 each (5 from males and 5 from females) on the basis of caries progression. The pulp cells are stained with quinacrine hydrochloride and observed with fluorescent microscope for fluorescent body. Gender is determined by identification of Y chromosome fluorescence in dental pulp. Results: Fluorescent bodies were found to be more in sound teeth in males as the caries increase the mean percentage of fluorescent bodies observed decreases in males. We also observed the fluorescent spots in females, and the value of the spot increases in female as the caries progresses, thereby giving false positive results in females. Conclusion: Sex determination by fluorescent staining of the Y chromosome is a reliable technique in teeth with healthy pulps or caries with enamel or up to half way of dentin. Teeth with caries involving pulp cannot be used for sex determination. PMID:25125912

Fluorescence excitation and excited state intramolecular relaxation dynamics of jet-cooled methyl-2-hydroxy-3-naphthoate

NASA Astrophysics Data System (ADS)

McCarthy, Annemarie; Ruth, Albert A.

2013-11-01

Two distinct S0 → S1 fluorescence excitation spectra of methyl-2-hydroxy-3-napthoate (MHN23) have been obtained by monitoring fluorescence separately in the short (˜410 nm) and long (˜650 nm) wavelength emission bands. The short wavelength fluorescence is assigned to two MHN23 conformers which do not undergo excited state intramolecular proton transfer (ESIPT). Analysis of the 'long wavelength' fluorescence excitation spectrum, which arises from the proton transfer tautomer of MHN23 indicates an average lifetime of τ ⩾ 18 ± 2 fs for the initially excited states. Invoking the results of Catalan et al. [J. Phys. Chem. A, 1999, 103, 10921], who determined the N tautomer to decay predominantly via a fast non-radiative process, the limit of the rate of intramolecular excited proton transfer in MHN23 is calculated as, kpt ⩽ 1 × 1012 s-1.

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing.

PubMed

Sinchenko, Elena; Gibbs, W E Keith; Davis, Claire E; Stoddart, Paul R

2010-11-20

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.

Acridine-based fluorescence chemosensors for selective sensing of Fe3+ and Ni2+ ions

NASA Astrophysics Data System (ADS)

Wang, Chaoyu; Fu, Jiaxin; Yao, Kun; Xue, Kun; Xu, Kuoxi; Pang, Xiaobin

2018-06-01

Two novel acridine-based fluorescence chemosensors (L1 and L2) were prepared and their metal ions sensing properties were investigated. L1 (L2) exhibited an excellent selective fluorescence response toward Fe3+ (Ni2+) and the stoichiometry ratio of L1-Fe3+ and L2-Ni2+ were 1:1. The detection limits of L1 and L2 were calculated by the fluorescence titration to be 4.13 μM and 1.52 μM, respectively, which were below the maximum permissive level of Fe3+ and Ni2+ ions in drinking water set by the EPA. The possible mechanism of the fluorescence detection of Fe3+ and Ni2+ had been proposed according to the analysis of Job's plot, IR spectra and ESI-MS. The determination of Fe3+ and Ni2+ ions in living cells had been applied successfully.

Analysis of cholesterol trafficking with fluorescent probes

PubMed Central

Maxfield, Frederick R.; Wüstner, Daniel

2013-01-01

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly. PMID:22325611

Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oetting, W.S.; Lee, H.K.; Flanders, D.J.

The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) usingmore » primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5{prime} end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis. 15 refs., 2 figs., 4 tabs.« less

Understanding aquatic microbial processes using EEM's and in-situ fluorescence sensors

NASA Astrophysics Data System (ADS)

Fox, Bethany; Attridge, John; Rushworth, Cathy; Cox, Tim; Anesio, Alexandre; Reynolds, Darren

2015-04-01

The diverse origin of dissolved organic matter (DOM) in aquatic systems is well documented within the literature. Previous literature indicates that coloured dissolved organic matter (CDOM) is, in part, transformed by aquatic microbial processes, and that dissolved organic material derived from a microbial origin exhibits tryptophan-like fluorescence. However, this phenomenon is not fully understood and very little data is available within the current literature. The overall aim of our work is to reveal the microbial-CDOM interactions that give rise to the observed tryptophan-like fluorescence. The work reported here investigates the microbial processes that occur within freshwater aquatic samples, as defined by the biochemical oxygen demand (BOD) test, as a function of the T1 peak (λex/em 280/330-370 nm). A series of standard water samples were prepared using glucose, glutamic acid, BOD dilution water and a bacterial seed (Cole-Parmer BOD microbe capsules). Samples were spiked with CDOM (derived from an environmental water body) and subjected to time resolved BOD analysis and as excitation-emission fluorescence spectroscopy. All EEM spectral data was interrogated using parallel factor analysis (PARAFAC) in an attempt to determine the presence and dominance (relative intensities) of the CDOM-related and T1-related fluorophores within the samples. In-situ fluorescence sensors (Chelsea Technologies Group Ltd.) were also used to monitor the T1 fluorescence peak (UviLux Tryptophan) and the CDOM fluorescence peak (UviLux CDOM) during experiments. Tryptophan-like fluorescence was observed (albeit transient) in both spiked and un-spiked standard water samples. By furthering our understanding of aquatic organic matter fluorescence, its origin, transformation, fate and interaction with aquatic microbiological processes, we aim to inform the design of a new generation in-situ fluorescence sensor for the monitoring of aquatic ecosystem health.

Second-order advantage with excitation-emission photoinduced fluorimetry for the determination of the antiepileptic carbamazepine in environmental waters.

PubMed

Lozano, Valeria A; Escandar, Graciela M

2013-06-11

A photochemically induced fluorescence system combined with second-order chemometric analysis for the determination of the anticonvulsant carbamazepine (CBZ) is presented. CBZ is a widely used drug for the treatment of epilepsy and is included in the group of emerging contaminant present in the aquatic environment. CBZ is not fluorescent in solution but can be converted into a fluorescent compound through a photochemical reaction in a strong acid medium. The determination is carried out by measuring excitation-emission photoinduced fluorescence matrices of the products formed upon ultraviolet light irradiation in a laboratory-constructed reactor constituted by two simple 4 W germicidal tubes. Working conditions related to both the reaction medium and the photoreactor geometry are optimized by an experimental design. The developed approach enabled the determination of CBZ at trace levels without the necessity of applying separation steps, and in the presence of uncalibrated interferences which also display photoinduced fluorescence and may be potentially present in the investigated samples. Different second-order algorithms were tested and successful resolution was achieved using multivariate curve resolution-alternating least-squares (MCR-ALS). The study is employed for the discussion of the scopes and yields of each of the applied second-order chemometric tools. The quality of the proposed method is probed through the determination of the studied emerging pollutant in both environmental and drinking water samples. After a pre-concentration step on a C18 membrane using 50.0 mL of real water samples, a prediction relative error of 2% and limits of detection and quantification of 0.2 and 0.6 ng mL(-1) were respectively obtained. Copyright © 2013 Elsevier B.V. All rights reserved.

Determination of selenium in the environment and in biological material.

PubMed Central

Bem, E M

1981-01-01

This paper reviews the following problems, sampling, decomposition procedures and most important analytical methods used for selenium determination, e.g., neutron activation analysis, atomic absorption spectrometry, gas-liquid chromatography, spectrophotometry, fluorimetry, and x-ray fluorescence. This review covers the literature mainly from 1975 to 1977. PMID:7007035

Synchrotron X-Ray Microprobe In-Situ Analysis of Extraterrestrial Particles Collected in Aerogel on the MIR Space Station

NASA Technical Reports Server (NTRS)

Flynn, G. J.; Sutton, S. R.; Horz, F.

2000-01-01

Using in-situ x-ray fluorescence, we determined the Cr/Fe, Mn/Fe and Ni/Fe of a particle captured in aerogel on MIR are approximately chondritic, indicating an extraterrestrial origin. Impurity of the aerogel precluded determining the Cu and Zn.

Optimal fluorescence waveband determination for detecting defect cherry tomatoes using fluorescence excitation-emission matrix

USDA-ARS?s Scientific Manuscript database

A multi-spectral fluorescence imaging technique was used to detect defect cherry tomatoes. The fluorescence excitation and emission matrix was used to measure for defects, sound surface, and stem areas to determine the optimal fluorescence excitation and emission wavelengths for discrimination. Two-...

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor.

PubMed

Zhou, Zhixue; Du, Yan; Dong, Shaojun

2011-07-01

Double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) at low concentration of CuSO(4), and the formed Cu NPs have excellent fluorescence, whereas a single-strand DNA (ssDNA) template does not support Cu NPs' formation. This property of dsDNA-Cu NPs makes it suitable for DNA sensing. However, exploration of dsDNA-Cu NPs applied in biological analysis is still at an early stage. In this regard, we report herein for the first time a sensitive, cost-effective, and simple aptamer sensor (aptasensor) using dsDNA-Cu NPs as fluorescent probe. The design consists of a dsDNA with reporter DNA (here, aptamer) as template for the formation of Cu NPs, and the formed dsDNA-Cu NPs show high fluorescence. Using adenosine triphosphate (ATP) as a model analyte, the introduction of ATP triggers the structure switching of reporter DNA to form aptamer-ATP complex, causing the destruction of the double helix and thus no formation of the Cu NPs, resulting in low fluorescence. The preferable linear range (0.05-500 μM), sensitivity (LOD 28 nM), and simplicity for the detection of ATP indicate that dsDNA-Cu NPs may have great prospects in the field of biological analysis. We also use this novel fluorescent probe to determine ATP in 1% human serum and human adenocarcinoma HeLa cells. The dsDNA-Cu NPs probes provide recovery of 104-108% in 1% human serum and a prominent fluorescent signal is obtained in cellular ATP assay, revealing the practicality of using dsDNA-Cu NPs for the determination of ATP in real samples. Besides, this design is simply based on nucleic acid hybridization, so it can be generally applied to other aptamers for label-free detection of a broad range of analytes. Successful detection of cocaine with detection limit of 0.1 μM demonstrates its potential to be a general method.

Caracterisation of anthropogenic contribution to the coastal fluorescent organic matter

NASA Astrophysics Data System (ADS)

El Nahhal, Ibrahim; Nouhi, Ayoub; Mounier, Stéphane

2015-04-01

It is known that most of the coastal fluorescent organic matter is of a terrestrial origin (Parlanti, 2000; Tedetti, Guigue, & Goutx, 2010). However, the contribution of the anthropogenic organic matter to this pool is not well defined and evaluated. In this work the monitoring of little bay (Toulon Bay, France) was done in the way to determine the organic fluorescent response during a winter period. The sampling campaign consisted of different days during the month of December, 2014 ( 12th, 15th, 17th, 19th) on 21 different sampling sites for the fluorescence measurements (without any filtering of the samples) and the whole month of December for the bacterial and the turbidity measurements. Excitation Emission Matrices (EEMs) of fluorescence (from 200 to 400 nm and 220 to 420 nm excitation and emission range) were treated by parallel factor analysis (PARAFAC).The parafac analysis of the EEM datasets was conducted using PROGMEEF software in Matlab langage. On the same time that the turbidity and bacterial measurement (particularly the E.Coli concentration) were determined. The results gives in a short time range, information on the the contribution of the anthropogenic inputs to the coastal fluorescent organic matter. In addition, the effect of salinity on the photochemical degradation of the anthropogenic organic matter (especially those from wastewater treatment plants) will be studied to investigate their fate in the water end member by the way of laboratory experiments. Parlanti, E. (2000). Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs. Organic Geochemistry, 31(12), 1765-1781. doi:10.1016/S0146-6380(00)00124-8 Tedetti, M., Guigue, C., & Goutx, M. (2010). Utilization of a submersible UV fluorometer for monitoring anthropogenic inputs in the Mediterranean coastal waters. Marine Pollution Bulletin, 60(3), 350-62. doi:10.1016/j.marpolbul.2009.10.018

New fluorescent probes for detection and characterization of amyloid fibrils

NASA Astrophysics Data System (ADS)

Gorbenko, Galyna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Kalnina, Inta; Vasilev, Aleksey; Kaloyanova, Stefka; Deligeorgiev, Todor

2010-08-01

The applicability of the novel fluorescent probes, aminoderivative of benzanthrone ABM, squaraine dye SQ-1 and polymethine dye V2 to identification and structural analysis of amyloid fibrils has been evaluated using the lysozyme model system in which fibrillar aggregates have been formed in concentrated ethanol solution. The association constant, binding stoichiometry and molar fluorescence of the bound dye have been determined. ABM was found to surpass classical amyloid marker ThT in the sensitivity to the presence of fibrillar aggregates. Resonance energy transfer measurements involving ABM-SQ-1 and SQ-1-V2 donor-acceptor pairs yielded the limits for fractal-like dimension of lysozyme fibrils.

Interaction and energy transfer studies between bovine serum albumin and CdTe quantum dots conjugates: CdTe QDs as energy acceptor probes.

PubMed

Kotresh, M G; Inamdar, L S; Shivkumar, M A; Adarsh, K S; Jagatap, B N; Mulimani, B G; Advirao, G M; Inamdar, S R

2017-06-01

In this paper, a systematic investigation of the interaction of bovine serum albumin (BSA) with water-soluble CdTe quantum dots (QDs) of two different sizes capped with carboxylic thiols is presented based on steady-state and time-resolved fluorescence measurements. Efficient Förster resonance energy transfer (FRET) was observed to occur from BSA donor to CdTe acceptor as noted from reduction in the fluorescence of BSA and enhanced fluorescence from CdTe QDs. FRET parameters such as Förster distance, spectral overlap integral, FRET rate constant and efficiency were determined. The quenching of BSA fluorescence in aqueous solution observed in the presence of CdTe QDs infers that fluorescence resonance energy transfer is primarily responsible for the quenching phenomenon. Bimolecular quenching constant (k q ) determined at different temperatures and the time-resolved fluorescence data provide additional evidence for this. The binding stoichiometry and various thermodynamic parameters are evaluated by using the van 't Hoff equation. The analysis of the results suggests that the interaction between BSA and CdTe QDs is entropy driven and hydrophobic forces play a key role in the interaction. Binding of QDs significantly shortened the fluorescence lifetime of BSA which is one of the hallmarks of FRET. The effect of size of the QDs on the FRET parameters are discussed in the light of FRET parameters obtained. Copyright © 2016 John Wiley & Sons, Ltd.

In Situ Determination of Illegal Drugs in Oral Fluid by Portable Capillary Electrophoresis with Deep UV Excited Fluorescence Detection.

PubMed

Saar-Reismaa, Piret; Erme, Enn; Vaher, Merike; Kulp, Maria; Kaljurand, Mihkel; Mazina-Šinkar, Jekaterina

2018-05-15

The present study demonstrates the potential of a portable capillary electrophoresis (CE) instrument, coupled to deep UV fluorescence detector (FD) with a 230-255 nm excitation wavelength range, for the determination of the abuse of illegal drugs in oral fluids in situ. CE was introduced in this study due its exceptional power of separation and resolution, short analysis time, and ability for miniaturization for on-site assessment of different substances. The deep UV fluorescence detector was equipped with five interchangeable emission filters, in the emission wavelength range from 278 to 600 nm, and was successfully employed for determination of natively fluorescing illegal drugs, such as cocaine, cocaethylene, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxeamphetamine (MDA), 3,4-methylenedioxy- N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxy- N-methylamphetamine (PMMA), amphetamine (AMP), methamphetamine (METH), tetrahydrocannabinol (THC) and cannabidiol (CBD). The developed FD showed impressive sensitivity. The instrumental detection limit was 0.5 μg/L for MDMA. It also showed broad linearity, up to 50 mg/L for MDMA. The noise CV% was 1.1% for an empty capillary and 0.6% for a capillary filled with acetonitrile. The portable CE-FD with developed electrophoretic methodologies was successfully utilized for the determination of illegal abuse of drugs during "Weekend" 2016 and 2017 Music Festivals (Estonia). Moreover, CE-FD can be employed for detection of other natively fluorescing compounds in the proposed range (e.g., for different phenolic compounds, BTEX, naphthalene derivatives, and others), significantly widening the applicability of developed CE-FD instrument.

NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

PubMed

Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

2015-10-01

To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

FluoroSim: A Visual Problem-Solving Environment for Fluorescence Microscopy

PubMed Central

Quammen, Cory W.; Richardson, Alvin C.; Haase, Julian; Harrison, Benjamin D.; Taylor, Russell M.; Bloom, Kerry S.

2010-01-01

Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology. PMID:20431698

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2005-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Minamitani, Elizabeth Forsythe; Pusey, Marc L.

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Amiruddha

2005-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. Preliminary experiments show that the presence of the fluorescent probe does not affect the nucleation process or the quality of the X-ray data obtained.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth

2004-01-01

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Quaternized magnetic nanoparticles-fluorescent polymer system for detection and identification of bacteria.

PubMed

Wan, Yi; Sun, Yan; Qi, Peng; Wang, Peng; Zhang, Dun

2014-05-15

Nanomaterial-based 'chemical nose' sensor with sufficient sensing specificity is a useful analytical tool for the detection of toxicologically important substances in complicated biological systems. A sensor array containing three quaternized magnetic nanoparticles (q-MNPs)-fluorescent polymer systems has been designed to identify and quantify bacteria. The bacterial cell membranes disrupt the q-MNP-fluorescent polymer, generating unique fluorescence response array. The response intensity of the array is dependent on the level of displacement determined by the relative q-MNP-fluorescent polymer binding strength and bacteria cells-MNP interaction. These characteristic responses show a highly repeatable bacteria cells and can be differentiated by linear discriminant analysis (LDA). Based on the array response matrix from LDA, our approach has been used to measure bacteria with an accuracy of 87.5% for 10(7) cfu mL(-1) within 20 min. Combined with UV-vis measurement, the method can be successfully performed to identify and detect eight different pathogen samples with an accuracy of 96.8%. The measurement system has a potential for further applications and provides a facile and simple method for the rapid analysis of protein, DNA, and pathogens. Copyright © 2013 Elsevier B.V. All rights reserved.

X-ray fluorescence determination of Sn, Sb, Pb in lead-based bearing alloys using a solution technique

NASA Astrophysics Data System (ADS)

Tian, Lunfu; Wang, Lili; Gao, Wei; Weng, Xiaodong; Liu, Jianhui; Zou, Deshuang; Dai, Yichun; Huang, Shuke

2018-03-01

For the quantitative analysis of the principal elements in lead-antimony-tin alloys, directly X-ray fluorescence (XRF) method using solid metal disks introduces considerable errors due to the microstructure inhomogeneity. To solve this problem, an aqueous solution XRF method is proposed for determining major amounts of Sb, Sn, Pb in lead-based bearing alloys. The alloy samples were dissolved by a mixture of nitric acid and tartaric acid to eliminated the effects of microstructure of these alloys on the XRF analysis. Rh Compton scattering was used as internal standard for Sb and Sn, and Bi was added as internal standard for Pb, to correct for matrix effects, instrumental and operational variations. High-purity lead, antimony and tin were used to prepare synthetic standards. Using these standards, calibration curves were constructed for the three elements after optimizing the spectrometer parameters. The method has been successfully applied to the analysis of lead-based bearing alloys and is more rapid than classical titration methods normally used. The determination results are consistent with certified values or those obtained by titrations.

Quantitative analysis of essential oils of Thymus daenensis using laser-induced fluorescence and Raman spectroscopy.

PubMed

Khoshroo, H; Khadem, H; Bahreini, M; Tavassoli, S H; Hadian, J

2015-11-10

Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples.

[Determination of homocysteine level in plasma by high performance liquids chromatography with fluorescence detection].

PubMed

Da, Xu; Qian, Ling-Jia

2005-08-01

To establish a method for detection of plasma total homocysteine with HPLC. The chromatography analysis was carried out using a Symmetry Shield RP18. The mobile phase was sodium acetate (0.08 mol/L) and methanol (1%) and we utilized a HPLC system with fluorescence detection of plasma homocysteine derivatized from reaction with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). The average recoveries were 95.8 - 100.8% and the relative standard deviations were 1.2-2.0%. The results showed it to be a rapid and accurate method for the determination of homocysteine level in plasma.

Statistical Analysis of Bending Rigidity Coefficient Determined Using Fluorescence-Based Flicker-Noise Spectroscopy.

PubMed

Doskocz, Joanna; Drabik, Dominik; Chodaczek, Grzegorz; Przybyło, Magdalena; Langner, Marek

2018-06-01

Bending rigidity coefficient describes propensity of a lipid bilayer to deform. In order to measure the parameter experimentally using flickering noise spectroscopy, the microscopic imaging is required, which necessitates the application of giant unilamellar vesicles (GUV) lipid bilayer model. The major difficulty associated with the application of the model is the statistical character of GUV population with respect to their size and the homogeneity of lipid bilayer composition, if a mixture of lipids is used. In the paper, the bending rigidity coefficient was measured using the fluorescence-enhanced flicker-noise spectroscopy. In the paper, the bending rigidity coefficient was determined for large populations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. The quantity of obtained experimental data allows to perform statistical analysis aiming at the identification of the distribution, which is the most appropriate for the calculation of the value of the membrane bending rigidity coefficient. It has been demonstrated that the bending rigidity coefficient is characterized by an asymmetrical distribution, which is well approximated with the gamma distribution. Since there are no biophysical reasons for that we propose to use the difference between normal and gamma fits as a measure of the homogeneity of vesicle population. In addition, the effect of a fluorescent label and types of instrumental setups on determined values has been tested. Obtained results show that the value of the bending rigidity coefficient does not depend on the type of a fluorescent label nor on the type of microscope used.

Direct chemiluminescence of carbon dots induced by potassium ferricyanide and its analytical application

NASA Astrophysics Data System (ADS)

Amjadi, Mohammad; Manzoori, Jamshid L.; Hallaj, Tooba; Sorouraddin, Mohammad H.

2014-03-01

The chemiluminescence (CL) of water-soluble fluorescent carbon dots (C-dots) induced by direct chemical oxidation was investigated. C-dots were prepared by solvothermal method and characterized by fluorescence spectra and transmission electron microscopy. It was found that K3Fe(CN)6 could directly oxidize C-dots to produce a relatively intense CL emission. The mechanism of CL generation was investigated based on the fluorescence and CL emission spectra and the effect of radical scavengers on the CL intensity. The inhibitive effect of some metal ions and biologically important molecules on the CL intensity of the system was examined and the potential of the system for the determination of these species at trace levels was studied. In order to evaluate the capability of method to real sample analysis, it was applied to the determination of Cr(VI) and adrenaline in water and injection samples, respectively.

Application of X-Ray Fluorescence Analysis to Determine the Elemental Composition of Tissues from Different Ovarian Neoplasms

NASA Astrophysics Data System (ADS)

Motevich, I. G.; Strekal, N. D.; Papko, N. M.; Glebovich, M. I.; Shulha, A. V.; Maskevich, S. A.

2015-03-01

We present the results of x-ray fluorescence analysis of tissues from healthy ovaries and from ovaries with different pathologies: benign and borderline tumors, mucinous and endometrioid cancers, serous carcinomas. We determine the average copper, zinc, calcium, selenium, cadmium, lead, and mercury levels. We observed that in the benign ovarian tumors, we see a significant decrease in the cadmium, mercury, and lead levels compared with healthy tissues. In the borderline neoplasms, the copper level is reduced relative to zinc (Cu/Zn), cadmium, mercury, and lead, and also the zinc concentration is increased. In the ovarian carcinomas, we observed changes in the ratio of the chemical elements in the tumor tissues, depending on the histologic type. The results obtained can be used for differentiation, diagnosis, and adjustment of treatment for different ovarian neoplasms.

Determination of the anti-viral drug Ribavirin in dosage forms via micelle-enhanced spectrofluorimetric method.

PubMed

Alarfaj, Nawal A; El-Tohamy, Maha F

2013-01-01

A simple and sensitive spectrofluorimetric method was developed for the determination of Ribavirin in pharmaceutical formulations. The proposed method was based on the fluorescence spectral behaviour of Ribavirin in a sodium dodecyl sulfate (SDS) micellar system. In an aqueous acetate buffer solution of pH 4.0, the fluorescence intensity of Ribavirin was significantly enhanced by about 217% in the presence of SDS. Fluorescence intensity was measured at 396 nm after excitation at 270 nm for Ribavirin. The fluorescence-concentration plot was rectilinear over the range of 0.01-3.0 µg/mL for Ribavirin with a lower detection limit of 5.02 x 10(-3) µg/mL. The method was successfully applied to the analysis of the drug in its commercial capsules. Results were in good agreement with those obtained with the official method. The application of the proposed method was extended to stability studies of Ribavirin after exposure to different forced degradation conditions such as acidic, alkaline, photo and oxidative conditions according to ICH guidelines. Copyright © 2012 John Wiley & Sons, Ltd.

Micelle-enhanced spectrofluorimetric method for determination of cholesterol-reducing drug ezetimibe in dosage forms.

PubMed

Alarfaj, Nawal A; Aly, Fatma A

2012-01-01

A simple and sensitive spectrofluorimetric method was developed for the determination of ezetimibe in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of ezetimibe in sodium dodecyl sulfate (SDS) micellar system. In aqueous solution of acetate buffer pH 5.0, the fluorescence intensity of ezetimibe was greatly enhanced, 200% enhancement, in the presence of SDS. The fluorescence intensity of ezetimibe was measured at 380 nm after excitation at 268 nm. The fluorescence-concentration plot was rectilinear over the range of 0.03-3.0 μg/mL with lower detection limit of 3.08 × 10(-3) μg/mL. The method was successfully applied to the analysis of ezetimibe in its commercial tablets; the results were in good agreement with those obtained with the reported method. The application of the proposed method was extended to the stability studies of ezetimibe after exposure to different forced degradation conditions, such as acidic, alkaline, photo and oxidative conditions, according to ICH guidelines.

Determination of polycyclic aromatic hydrocarbons by four-way parallel factor analysis in presence of humic acid.

PubMed

Yang, Ruifang; Zhao, Nanjing; Xiao, Xue; Yu, Shaohui; Liu, Jianguo; Liu, Wenqing

2016-01-05

There is not effective method to solve the quenching effect of quencher in fluorescence spectra measurement and recognition of polycyclic aromatic hydrocarbons in aquatic environment. In this work, a four-way dataset combined with four-way parallel factor analysis is used to identify and quantify polycyclic aromatic hydrocarbons in the presence of humic acid, a fluorescent quencher and an ubiquitous substance in aquatic system, through modeling the quenching effect of humic acid by decomposing the four-way dataset into four loading matrices corresponding to relative concentration, excitation spectra, emission spectra and fluorescence quantum yield, respectively. It is found that Phenanthrene, pyrene, anthracene and fluorene can be recognized simultaneously with the similarities all above 0.980 between resolved spectra and reference spectra. Moreover, the concentrations of them ranging from 0 to 8μgL(-1) in the test samples prepared with river water could also be predicted successfully with recovery rate of each polycyclic aromatic hydrocarbon between 100% and 120%, which were higher than those of three-way PARAFAC. These results demonstrate that the combination of four-way dataset with four-way parallel factor analysis could be a promising method to recognize the fluorescence spectra of polycyclic aromatic hydrocarbons in the presence of fluorescent quencher from both qualitative and quantitative perspective. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of trace zinc in seawater by coupling solid phase extraction and fluorescence detection in the Lab-On-Valve format.

PubMed

Grand, Maxime M; Chocholouš, Petr; Růžička, Jarda; Solich, Petr; Measures, Christopher I

2016-06-07

By virtue of their compactness, long-term stability, minimal reagent consumption and robustness, miniaturized sequential injection instruments are well suited for automation of assays onboard research ships. However, in order to reach the sensitivity and limit of detection required for open-ocean determinations of trace elements, it is necessary to preconcentrate the analyte prior its derivatization and subsequent detection by fluorescence. In this work, a novel method for the determination of dissolved zinc (Zn) at subnanomolar levels in seawater is described. The proposed method combines, for the first time, automated matrix removal, extraction of the target element, and fluorescence detection within a miniaturized flow manifold, based on the Lab-On-Valve (LOV) concept. The key feature of the microfluidic manipulation of the sample is flow programming, designed to pass sample through a mini-column where the target analyte and other complexable cations are retained, while the seawater matrix is washed out. Next, zinc is eluted and merged with a Zn selective fluorescent probe (FluoZin-3) at the confluence point of the LOV central channel using two high-precision stepper motor driven pumps that are operated in concert. Finally, the thus formed Zn complex is transported to the LOV flow cell for selective fluorescence measurement. This work describes the characterization and optimization of the method including Solid Phase Extraction using the Toyopearl AF-Chelate-650M resin, and detailed assay protocol controlled by a commercially available software and instrument. The proposed method features a LOD of 0.02 nM, high precision (<3% at 0.1 and 2 nM Zn levels), an assay cycle of 13 min and a reagent consumption of 150 μL FluoZin-3 per sample, which makes the method highly suitable for oceanographic shipboard analysis. The accuracy of the method has been validated through the analysis of seawater reference standards and comparison with ICP-MS determinations on seawater samples collected in the upper 1300 m of the subtropical south Indian Ocean. This work confirms that integration of sample pretreatment with optical detection in the LOV format offers a widely applicable approach to trace analysis of seawater. Copyright © 2016. Published by Elsevier B.V.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

Nitrogen-doped carbon quantum dots as a fluorescence probe combined with magnetic solid-phase extraction purification for analysis of folic acid in human serum.

PubMed

Wang, Meng; Jiao, Yang; Cheng, Chunsheng; Hua, Jianhao; Yang, Yaling

2017-12-01

A novel and sensitive method based on nitrogen-doped carbon quantum dots as a fluorescence probe coupled with magnetic solid-phase extraction (MSPE) purification for analysis of folic acid (FA) in human serum samples has been established for the first time. In the developed system, magnetic nanoparticles coated with hexanoic acid (Fe 3 O 4 @C 6 ) were synthesized by a one-step chemical co-precipitation method with good magnetic properties and dispersibility for sample purification, and it is better to be separated from the sample. High fluorescence nitrogen-doped carbon quantum dots (N-CQDs), simply prepared using a one-step hydrothermal method with nitrilotriacetic acid, could be selectively quenched by FA. Based on this phenomenon, a fluorescence assay was proposed for specific determination of FA. Various operational experiment parameters have been studied and optimized in detail. Under the optimum experimental conditions, the detection limit of the proposed method for FA was evaluated to be 0.5 nM (S/N = 3), while the relative standard deviation (RSD) was 1.2% (n = 6). Finally, the proposed method was applied for determination of trace levels of FA from human serum samples and quantitative recoveries were achieved within the range of 95.7-103.5%. All of the results showed that the proposed method had significant application in further research. Graphical abstract Schematic of synthesis of N-CQDs and schematic of suggested mode for analysis of folic acid (FA).

A first evaluation of the analytical capabilities of the new X-ray fluorescence facility at International Atomic Energy Agency-Elettra Sincrotrone Trieste for multipurpose total reflection X-ray fluorescence analysis

NASA Astrophysics Data System (ADS)

Marguí, Eva; Hidalgo, Manuela; Migliori, Alessandro; Leani, Juan José; Queralt, Ignasi; Kallithrakas-Kontos, Nikolaos; Streli, Christina; Prost, Josef; Karydas, Andreas Germanos

2018-07-01

The aim of the work is to present a systematic evaluation of the analytical capabilities of the new X-ray fluorescence facility jointly operated between the International Atomic Energy Agency and the Elettra Sincrotrone Trieste for multipurpose total reflection X-ray fluorescence analysis. The analytical performance of the XRF beamline end-station (IAEAXspe) was systematically evaluated under TXRF excitation geometry by analyzing different types of aqueous (lake, waste and fresh water) and solid (soil, vegetal, biological) certified reference materials using an excitation energy of 13.0 keV (for the purpose of multielemental analysis). The results obtained for both types of samples in terms of limits of detection and accuracy were also compared with those obtained using laboratory X-ray tube based TXRF systems with different features (including Mo and W X-ray tube systems). Taking advantage of the possibility to work under high vacuum, the IAEAXspe set-up instrumental sensitivity was also determined using an excitation energy of 6.2 keV to explore the possibilities for light elements determination. A clear improvement of the element detection limits is achieved when comparing this facility to conventional X-ray tube based TXRF systems highlighting the benefits of using the monoenergetic synchrotron exciting radiation and the ultra-high vacuum chamber in comparison with conventional laboratory systems. The results of the present work are discussed in view of further exploitation of the facility for different environmental and biological related applications.

Efficacy of Sex Determination from Human Dental Pulp Tissue and its Reliability as a Tool in Forensic Dentistry.

PubMed

Khanna, Kaveri Surya

2015-01-01

Sex determination is one of the primary steps in forensics. Barr body can be used as a histological method for identification of sex as it is found to be specific to female somatic cells and rare in male cells. To demarcate human dental pulp as an important identification tool of sex in forensic odontology (FO) and to evaluate the time period till which sex can be determined from pulp tissue using three stains H and E, Feulgen, and acridine - orange under fluorescence so as. 90 pulp samples (45 males and 45 females) were subjected to Barr body analysis for determination of sex using light and fluorescent microscopy. Barr body was found to be positive for female samples and negative or rare in the male sample (<3%). Barr body from human dental pulp tissue can be used as a successful determinant of sex identification in FO.

Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

PubMed

Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

2018-05-07

Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

H2 Fluorescence in M dwarf Systems: A Stellar Origin

NASA Astrophysics Data System (ADS)

Kruczek, Nicholas; France, Kevin; Evonosky, William; Youngblood, Allison; Loyd, R. O. Parke

2017-01-01

Observations of Lyα-driven H2 fluorescence can be a useful tool for measuring the abundance of H2 in exoplanet atmospheres. This emission has been previously observed in M dwarfs with planetary systems but at too low of a signal to determine its origin. It may have been originating in the atmospheres of planets, but conditions within these systems also mean that the H2 could be residing on the stellar surface or in a circumstellar disk. We use observations from the ``Measurements of the Ultraviolet Spectral Characteristics of Low-mass Exoplanet Host Stars" (MUSCLES) Hubble Space Telescope (HST) Treasury Survey to study H2 fluorescence in M dwarfs with and without confirmed planets to determine the origin of the emission. The results are further supported by the direct imaging of a candidate M dwarf system using the HST-Advanced Camera for Surveys/Solar Blind Channel. We constrain the location of the fluorescing H2 through analysis of the line profiles and determine that the emission is originating on the star. We verify that this interpretation is consistent with 1D radiative transfer models that are optimized using the spectra of the MUSCLES stars and find that the H2 likely resides in starspots or a cool region of the lower chromosphere.

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of cinnarizine and nicergoline in pharmaceutical preparations.

PubMed

Walash, Mohamed I; Belal, Fathalla; El-Enany, Nahed; Abdelal, Amina

2008-01-01

A rapid, simple, and highly sensitive second-derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixtures of cinnarizine (CN) and nicergoline (NIC). The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 80 nm in aqueous methanol (50%, v/v). The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.025-1.5 and 0.25-5.5 microg/mL for CN and NIC, respectively, with lower detection limits of 0.58 and 0.82 ng/mL and quantitation limits of 1.93 and 2.73 ng/mL for CN and NIC, respectively. The proposed method was successfully applied for the determination of the studied compounds in synthetic mixtures and in commercial tablets. The results obtained were in good agreement with those obtained with reference methods. The high sensitivity attained by the proposed method allowed the determination of CN in real and spiked human plasma. The mean recovery in the case of spiked human plasma [number of trials (n) = 3] was 102.82 +/- 2.17%, while that in real human plasma (n = 3) was 105.25 +/- 2.05.

Development of a rapid, simple and sensitive HPLC-FLD method for determination of rhodamine B in chili-containing products.

PubMed

Qi, Ping; Lin, Zhihao; Li, Jiaxu; Wang, ChengLong; Meng, WeiWei; Hong, Hong; Zhang, Xuewu

2014-12-01

In this work, a simple, rapid and sensitive analytical method for the determination of rhodamine B in chili-containing foodstuffs is described. The dye is extracted from samples with methanol and analysed without further cleanup procedure by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD). The influence of matrix fluorescent compounds (capsaicin and dihydrocapsaicin) on the analysis was overcome by the optimisation of mobile-phase composition. The limit of determination (LOD) and limit of quantification (LOQ) were 3.7 and 10 μg/kg, respectively. Validation data show a good repeatability and within-lab reproducibility with relative standard deviations <10%. The overall recoveries are in the range of 98-103% in chili powder and in the range of 87-100% in chili oil depending on the concentration of rhodamine B in foodstuffs. This method is suitable for the routine analysis of rhodamine B due to its sensitivity, simplicity, reasonable time and cost. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid determination of nitrite in foods in acidic conditions by high-performance liquid chromatography with fluorescence detection.

PubMed

Wang, Xiao-Fang; Fan, Ji-Cai; Ren, Ren; Jin, Quan; Wang, Jing

2016-06-01

In this study, a simple, rapid, and sensitive method for the determination of nitrite (NO2 (-) ) in food samples by high-performance liquid chromatography with fluorescence detection in acidic conditions had been developed. The derivatization of the nitrite with 2,3-diaminonaphthalene was performed in acidic conditions to yield the highly fluorescent 2,3-naphthotriazole, which was directly analyzed by high-performance liquid chromatography with fluorescence detection without adjusting the solution to alkaline. The analysis column was reversed-phase C8 column. A constant flow rate of 1.0 mL/min was employed using water/acetonitrile as the mobile phase in isocratic mode (70:30, v/v). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. The standard calibration curves were linear for nitrite in different matrixes in the concentration range of 0-100 μg/L, and the correlation coefficients ranged from 0.9978 to 0.9998. The limits of detection and quantification were in the ranges of 0.012-0.060 and 0.040-0.20 mg/kg, respectively. The recoveries of nitrite from samples spiked at three different concentrations were 74.0-113.2%, and the relative standard deviations of the recovery results (n = 6) were 1.67-10.8%. The proposed method has good repeatability and is very sensitive and simple. It has been successfully used to determine nitrite in foods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells.

PubMed

Wüstner, Daniel; Lund, Frederik W; Röhrl, Clemens; Stangl, Herbert

2016-01-01

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Analysis Results for Lunar Soil Simulant Using a Portable X-Ray Fluorescence Analyzer

NASA Technical Reports Server (NTRS)

Boothe, R. E.

2006-01-01

Lunar soil will potentially be used for oxygen generation, water generation, and as filler for building blocks during habitation missions on the Moon. NASA s in situ fabrication and repair program is evaluating portable technologies that can assess the chemistry of lunar soil and lunar soil simulants. This Technical Memorandum summarizes the results of the JSC 1 lunar soil simulant analysis using the TRACeR III IV handheld x-ray fluorescence analyzer, manufactured by KeyMaster Technologies, Inc. The focus of the evaluation was to determine how well the current instrument configuration would detect and quantify the components of JSC-1.

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining wasmore » determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.« less

Multi-exon genotyping of SMN gene in spinal muscular atrophy by universal fluorescent PCR and capillary electrophoresis.

PubMed

Wang, Chun-Chi; Chang, Jan-Gowth; Chen, Yen-Ling; Jong, Yuh-Jyh; Wu, Shou-Mei

2010-07-01

In this study, we established the first method for simultaneous evaluation of nine exons in the survival motor neuron (SMN) genes for full-scale genotyping. This method was used not only to quantify the copy numbers of highly homogenous telomeric SMN (SMN1)/centromeric SMN genes in exons 7 and 8 but also to determine intragenic mutations in all nine exons for complete diagnosis of spinal muscular atrophy (SMA). Additionally, we utilized the "universal fluorescent PCR" for simultaneously fluorescent labeling of eleven gene fragments (nine exons in SMN and two internal standards). Such technique is very beneficial for multi-exon analysis due to only requirement of one universal fluorescent primer which could fluorescently amplify all gene fragments. Of all 262 detected individuals, three subjects possessing different ratios of SMN1/centromeric SMN in the two exons were determined as gene conversion, and we also detected three interesting intragenic mutations (c.1 -39A>G, c.22_23insA in exon 1, c.84C>T in exon 2a) which were associated with the SMA patients owning one copy of SMN1 including two mutations never reported previously. This high-resolved method provided better potential technique for genotyping and identifying SMA, carrier and normal controls in large population.

Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process.

PubMed

Douša, Michal; Doubský, Jan; Srbek, Jan

2016-07-01

An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

In situ microbial detection in Mojave Desert soil using native fluorescence.

PubMed

Smith, H D; Duncan, A G; Neary, P L; Lloyd, C R; Anderson, A J; Sims, R C; McKay, C P

2012-03-01

We report on the use of a portable instrument for microbial detection in the Mojave Desert soil and the potential for its use on Mars. The instrument is based on native fluorescence and employs four excitation wavelengths combined with four emission wavelengths. A soil dilution series in which known numbers of Bacillus subtilis spores were added to soil was used to determine the sensitivity of the instrument. We found that the fluorescence of the biological and organic components of the desert soil samples studied can be as strong as the fluorescence of the mineral component of these soils. Using the calibration derived from B. subtilis spores, we estimated that microbial content at our primary sampling site was 10(7) bacteria per gram of soil, a level confirmed by phospholipid fatty acid analysis. At a nearby site, but in a slightly different geological setting, we tested the instrument's ability to map out microbial concentrations in situ. Over a ∼50 m diameter circle, soil microbial concentrations determined with the B. subtilis calibration indicate that the concentrations of microorganisms detected varies from 10(4) to 10(7) cells per gram of soil. We conclude that fluorescence is a promising method for detecting soil microbes in noncontact applications in extreme environments on Earth and may have applications on future missions to Mars.

Synthesis, crystal structures, fluorescence and xanthine oxidase inhibitory activity of pyrazole-based 1,3,4-oxadiazole derivatives

NASA Astrophysics Data System (ADS)

Qi, De-Qiang; Yu, Chuan-Ming; You, Jin-Zong; Yang, Guang-Hui; Wang, Xue-Jie; Zhang, Yi-Ping

2015-11-01

A series of pyrazole-based 1,3,4-oxadiazole derivatives were rationally designed and synthesized in good yields by following a convenient route. All the newly synthesized molecules were fully characterized by IR, 1H NMR and elemental analysis. Eight compounds were structurally determined by single crystal X-ray diffraction analysis. The fluorescence properties of all the compounds were investigated in dimethyl sulfoxide media. In addition, these newly synthesized compounds were evaluated for in vitro inhibitory activity against commercial enzyme xanthine oxidase (XO) by measuring the formation of uric acid from xanthine. Among the compounds synthesized and tested, 3d and 3e were found to be moderate inhibitory activity against commercial XO with IC50 = 72.4 μM and 75.6 μM. The studies gave a new insight in further optimization of pyrazole-based 1,3,4-oxadiazole derivatives with excellent fluorescence properties and XO inhibitory activity.

Nuclear Resonance Fluorescence of U-235

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, Glen A.; Caggiano, Joseph A.; Hensley, Walter K.

Nuclear resonance fluorescence is a physical process that provides an isotopic-specific signature that could be used for the identification and characterization of materials. The technique involves the detection of prompt discrete-energy photons emitted from a sample which is exposed to photons in the MeV energy range. Potential applications of the technique range from detection of high explosives to characterization of special nuclear materials. One isotope of significant interest is 235U. Pacific Northwest National Laboratory and Passport Systems have collaborated to conduct measurements to search for a nuclear resonance fluorescence response of 235U below 3 MeV using a 200 g samplemore » of highly enriched uranium. Nine 235U resonances between 1650 and 2010 keV were identified in the preliminary analysis. Analysis of the measurement data to determine the integrated cross sections of the resonances is in progress.« less

Microanalysis of dental caries using laser-scanned fluorescence

NASA Astrophysics Data System (ADS)

Barron, Joseph R.; Paton, Barry E.; Zakariasen, Kenneth L.

1992-06-01

It is well known that enamel and dentin fluoresce when illuminated by short-wavelength optical radiation. Fluorescence emission from carious and non-carious regions of teeth have been studied using a new experimental scanning technique for fluorescence analysis of dental sections. Scanning in 2 dimensions will allow surface maps of dental caries to be created. These surface images are then enhanced using the conventional and newer image processing techniques. Carious regions can be readily identified and contour maps can be used to graphically display the degree of damage on both surfaces and transverse sections. Numerous studies have shown that carious fluorescence is significantly different than non-carious regions. The scanning laser fluorescence spectrometer focuses light from a 25 mW He-Cd laser at 442 nm through an objective lens onto a cross-section area as small as 3 micrometers in diameter. Microtome prepared dental samples 100 micrometers thick are laid flat onto an optical bench perpendicular to the incident beam. The sample is moved under computer control in X & Y with an absolute precision of 0.1 micrometers . The backscattered light is both spatial and wavelength filtered before being measured on a long wavelength sensitized photomultiplier tube. High precision analysis of dental samples allow detailed maps of carious regions to be determined. Successive images allow time studies of caries growth and even the potential for remineralization studies of decalcified regions.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis, fluorescence, TGA and crystal structure of thiazolyl-pyrazolines derived from chalcones

NASA Astrophysics Data System (ADS)

Suwunwong, T.; Chantrapromma, S.; Fun, H.-K.

2015-04-01

Thiazolyl-pyrazolines 3a-3d were synthesized in a three step procedure using chalcones as starting materials and characterized by FT-IR, UV-Vis, and 1H NMR techniques. The crystal structure of compound 3a was also determined by X-ray diffraction analysis. Compound 3a crystallized out in the orthorhombic P212121 space group with the unit cell dimensions: a = 5.2106(2) Å, b = 12.4341(5) Å, c = 33.3254(13) Å, α = β = γ = 90°, V = 2159.12(15) Å3, Z = 4, D cald = 1.372 M gm-3 and F(000) = 928. Fluorescence of 3a-3d were studied in solid state and acetonitrile solution. It was found that, these compounds exhibit the green fluorescence light (506-508 nm) in both solid and solution states. The pH stability on fluorescence property and the thermal gravimetric analysis of compound 3a were specifically carried out. It was revealed that 3a shows high thermal stability up to around 250°C and presenting high stability in various pH ranges in the acetonitrilewater matrix.

Structure-matched Phthalocyanine Ion Pair as a Red-emitting Fluorescent Optical Probe for the Analysis of Sodium Dodecylbenzenesulfonate with High Specificity and Sensitivity.

PubMed

Yu, Fei; Guo, Menglin; Deng, Yabin; Lu, Yin; Chen, Lin; Huang, Ping; Li, Donghui

2016-01-01

We have found that a positively charged cationic copper phthalocyanine, Alcian blue (Alcian blue 8GX), can efficiently quench the fluorescence of an oppositely charged red fluorescent phthalocyanine compound with a matched molecular structure, tetrasulfonated aluminum phthalocyanine (AlS4Pc), because of the formation of an ion pair complex (AlS4Pc-Alcian blue 8GX) that exhibits almost no fluorescence. An investigation was carried out on the fluorescence recovery of AlS4Pc-Alcian blue 8GX caused by a series of anionic surfactants containing a sulfonic group (sodium dodecylbenzenesulfonate (SDBS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate (SDS)). The results showed that SDBS exhibited a significant response, and the highest sensitivity among the surfactants. Due to its high efficiency of fluorescence quenching and the high level of fluorescence recovery, direct observes can even be performed by the naked eye. The results revealed that the Alcian blue 8GX-AlS4Pc ion-pair complex fluorescent probe only responded to SDBS in the low-concentration range. Based on the new founding, this study proposed a novel principle and method of fluorescence enhancement to specifically measure the concentration of SDBS, thereby achieving a highly sensitive and highly specific determination of SDBS. Under the optimal conditions, the fluorescence intensity (I(f)) of the system and the concentration of SDBS in the range of 1 × 10(-7) - 1 × 10(-5) mol/dm(3) exhibited a good linear relationship. This method is highly sensitive, and the operation is simple and rapid. It had been applied for the quantitative analysis of SDBS in environmental water, while achieving satisfactory results compared with those of the standard method. This study developed a new application of the fluorescent phthalocyanine compounds used as molecular probes in analytical sciences.

Custom blending of lamp phosphors

NASA Technical Reports Server (NTRS)

Klemm, R. E.

1978-01-01

Spectral output of fluorescent lamps can be precisely adjusted by using computer-assisted analysis for custom blending lamp phosphors. With technique, spectrum of main bank of lamps is measured and stored in computer memory along with emission characteristics of commonly available phosphors. Computer then calculates ratio of green and blue intensities for each phosphor according to manufacturer's specifications and plots them as coordinates on graph. Same ratios are calculated for measured spectrum. Once proper mix is determined, it is applied as coating to fluorescent tubing.

Ambient methods and apparatus for rapid laser trace constituent analysis

DOEpatents

Snyder, Stuart C.; Partin, Judy K.; Grandy, Jon D.; Jeffery, Charles L.

2002-01-01

A method and apparatus are disclosed for measuring trace amounts of constituents in samples by using laser induced breakdown spectroscopy and laser induced fluorescence under ambient conditions. The laser induced fluorescence is performed at a selected wavelength corresponding to an absorption state of a selected trace constituent. The intensity value of the emission decay signal which is generated by the trace constituent is compared to calibrated emission intensity decay values to determine the amount of trace constituent present.

LAMP2GO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Priye, Aashish

2017-06-08

The app is a unique image analysis software and acts as a fluorescence reader for multiplexed nucleic acid amplification reactions. If the reaction is positive, a bright fluorescent signal is emitted from the solution depending on the choice of fluorophore molecule. The app reads the Red, blue and green (RGB) channels of the emitted signal and transforms it to yield the chromaticity (x and y) and luminance of the signal. This new representation of signal is far superior to the RGB system in determining the signal color and intensity.

Alamar blue reagent interacts with cell-culture media giving different fluorescence over time: potential for false positives.

PubMed

Munshi, Soumyabrata; Twining, Robert C; Dahl, Russell

2014-01-01

The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence. We have used Opti-MEM, DMEM and 1:1 DMEM:Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test. Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p<0.05). We also show that the time-dependent change in RFU of 1:1 DMEM:Opti-MEM was more gradual compared to that of the other two media. These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability. Copyright © 2014 Elsevier Inc. All rights reserved.

Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry.

PubMed

Kurihara, Takamasa; Min, Jun Zhe; Hirata, Asuka; Toyo'oka, Toshimasa; Inagaki, Shinsuke

2009-05-01

Rapid, selective and sensitive determination of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N-linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N-linked oligosaccharides in various glycoproteins seems to be possible.

Development of a single-cell X-ray fluorescence flow cytometer

DOE PAGES

Crawford, Andrew M.; Kurecka, Patrick; Yim, Tsz Kwan; ...

2016-06-17

An X-ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X-ray beam. X-ray fluorescence was then used to determine the mass of metal in each cell. By making single-cell measurements, the population heterogeneity for metals in the µ M to m M concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approachmore » has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ~4 cells min –1. These data show evidence for surprisingly broad metal distributions. Lastly, details of the device design, data analysis and opportunities for further sensitivity improvement are described.« less

X-ray fluorescence analysis of K, Al and trace elements in chloroaluminate melts

NASA Astrophysics Data System (ADS)

Shibitko, A. O.; Abramov, A. V.; Denisov, E. I.; Lisienko, D. G.; Rebrin, O. I.; Bunkov, G. M.; Rychkov, V. N.

2017-09-01

Energy dispersive x-ray fluorescence spectrometry was applied to quantitative determination of K, Al, Cr, Fe and Ni in chloroaluminate melts. To implement the external standard calibration method, an unconventional way of samples preparation was suggested. A mixture of metal chlorides was melted in a quartz cell at 350-450 °C under a slightly excessive pressure of purified argon (99.999 %). The composition of the calibration samples (CSs) prepared was controlled by means of the inductively coupled plasma atomic emission spectrometry (ICP-AES). The optimal conditions for analytical lines excitation were determined, the analytes calibration curves were obtained. There was some influence of matrix effects in synthesized samples on the analytical signal of some elements. The CSs are to be stored in inert gas atmosphere. The precision, accuracy, and reproducibility factors of the quantitative chemical analysis were computed.

Development of capillary electrophoresis methods for quantitative determination of taurine in vehicle system and biological media.

PubMed

da Silva, Dayse L P; Rüttinger, Hans H; Mrestani, Yahia; Baum, Walter F; Neubert, Reinhard H H

2006-06-01

CE methods have been developed for the determination of taurine in pharmaceutical formulation (microemulsion) and in biological media such as sweat. The CE system with end-column pulsed amperometric detection has been found to be an interesting method in comparison with UV and fluorescence detection for its simplicity and rapidity. A gold-disk electrode of 100 mm diameter was used as the working electrode. The effects of a field decoupler at the end of the capillary, separation voltage, injection and pressure times were investigated. A detection limit of 4 x 10(-5) mol/L was reached using integrated pulsed amperometric detection, a method successfully applied to taurine analysis of the biological samples such as sweat. For taurine analysis of oil-in-water microemulsion, fluorescence detector was the favored method, the detection limit of which was 4 x 10(-11) mol/L.

Spectrofluorimetric analysis of famotidine in pharmaceutical preparations and biological fluids by derivatization with benzoin

NASA Astrophysics Data System (ADS)

Alamgir, Malik; Khuhawar, Muhammad Yar; Memon, Saima Q.; Hayat, Amir; Zounr, Rizwan Ali

2015-01-01

A sensitive and simple spectrofluorimetric method has been developed for the analysis of famotidine, from pharmaceutical preparations and biological fluids after derivatization with benzoin. The reaction was carried out in alkaline medium with measurement of fluorescence intensity at 446 nm with excitation wavelength at 286 nm. Linear calibration was obtained with 0.5-15 μg/ml with coefficient of determination (r2) 0.997. The factors affecting the fluorescence intensity were optimized. The pharmaceutical additives and amino acid did not interfere in the determination. The mean percentage recovery (n = 4) calculated by standard addition from pharmaceutical preparation was 94.8-98.2% with relative standard deviation (RSD) 1.56-3.34% and recovery from deproteinized spiked serum and urine of healthy volunteers was 98.6-98.9% and 98.0-98.4% with RSD 0.34-0.84% and 0.29-0.87% respectively.

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection.

PubMed

Jing, Peng; Kaneta, Takashi; Imasaka, Totaro

2002-08-01

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Determination of thorium by fluorescent x-ray spectrometry

USGS Publications Warehouse

Adler, I.; Axelrod, J.M.

1955-01-01

A fluorescent x-ray spectrographic method for the determination of thoria in rock samples uses thallium as an internal standard. Measurements are made with a two-channel spectrometer equipped with quartz (d = 1.817 A.) analyzing crystals. Particle-size effects are minimized by grinding the sample components with a mixture of silicon carbide and aluminum and then briquetting. Analyses of 17 samples showed that for the 16 samples containing over 0.7% thoria the average error, based on chemical results, is 4.7% and the maximum error, 9.5%. Because of limitations of instrumentation, 0.2% thoria is considered the lower limit of detection. An analysis can be made in about an hour.

Fluorescence microplate readers as an alternative to flow injection analysis for determination of wort beta-glucan

USDA-ARS?s Scientific Manuscript database

Wort beta-glucan concentration is a critical malting quality parameter used to identify and avoid potential brewhouse filtration problems. ASBC method Wort-18 is widely used in malt analysis laboratories and brewhouses to measure wort beta-glucan levels. However, the chemistry underlying the method...

Analysis of Dextromethorphan in Cough Drops and Syrups: A Medicinal Chemistry Laboratory

ERIC Educational Resources Information Center

Hamilton, Todd M.; Wiseman, Frank L., Jr.

2009-01-01

Fluorescence spectroscopy is used to determine the quantity of dextromethorphan hydrobromide (DM) in over-the-counter (OTC) cough drops and syrups. This experiment is appropriate for an undergraduate medicinal chemistry laboratory course when studying OTC medicines and active ingredients. Students prepare the cough drops and syrups for analysis,…

Molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin.

PubMed

Wang, Xiaoyan; Yu, Jialuo; Kang, Qi; Shen, Dazhong; Li, Jinhua; Chen, Lingxin

2016-03-15

A facile strategy was developed to prepare molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin (PC) based on fluorescence resonance energy transfer (FRET), via a sol-gel polymerization process using nitrobenzoxadiazole (NBD) as fluorescent signal source. The ratio of two fluorescence peak emission intensities of NBD and PC was utilized to determine the concentration of PC, which could effectively reduce the background interference and fluctuation of diverse conditions. As a result, this sensor obtained high sensitivity with a low detection limit of 0.14 nM within 6 min, and excellent recognition specificity for PC over its analogues with a high imprinting factor of 9.1. Furthermore, the sensor attained high recoveries in the range of 93.8-110.2% at three spiking levels of PC, with precisions below 4.7% in seawater and lake water samples. The developed sensor strategy demonstrated simplicity, reliability, rapidity, high selectivity and high sensitivity, proving to be a feasible way to develop high efficient fluorescence sensors and thus potentially applicable for ultratrace analysis of complicated matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation.

PubMed

Keller, Scott B; Dudley, Jonathan A; Binzel, Katherine; Jasensky, Joshua; de Pedro, Hector Michael; Frey, Eric W; Urayama, Paul

2008-10-15

Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.

Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

NASA Astrophysics Data System (ADS)

Lewis, William; Williams, Maura; Franco, Walfre

2017-02-01

The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells

PubMed Central

Filonov, Grigory S.

2016-01-01

RNA spatial dynamics play a crucial role in cell physiology and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This article focuses on imaging RNAs using an “RNA mimic of GFP”. This approach relies on a RNA aptamer, called dimeric Broccoli, which binds to and switches on the fluorescence of DFHBI, a small molecule mimicking the fluorophore in GFP. Dimeric Broccoli is tagged to heterologously expressed RNAs and upon DFHBI binding the fluorescent signal of dimeric Broccoli reports the transcript’s localization in cells. This protocol describes the process of validating the fluorescence of dimeric Broccoli-labeled transcripts in vitro and in cells, flow cytometry analysis to determine overall fluorescence levels in cells, and fluorescence imaging in bacterial and mammalian cells. Overall, the current protocol should be useful for researchers seeking to image high abundance RNAs, such as transcribed off the T7 promoter in bacteria or off Pol III-dependent promoters in mammalian cells. PMID:26995352

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters.

PubMed

Hosseini, Morteza; Mehrabi, Fatemeh; Ganjali, Mohammad Reza; Norouzi, Parviz

2016-11-01

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer-templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA-AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as-prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA-AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

New insight in quantitative analysis of vascular permeability during immune reaction (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kalchenko, Vyacheslav; Molodij, Guillaume; Kuznetsov, Yuri; Smolyakov, Yuri; Israeli, David; Meglinski, Igor; Harmelin, Alon

2016-03-01

The use of fluorescence imaging of vascular permeability becomes a golden standard for assessing the inflammation process during experimental immune response in vivo. The use of the optical fluorescence imaging provides a very useful and simple tool to reach this purpose. The motivation comes from the necessity of a robust and simple quantification and data presentation of inflammation based on a vascular permeability. Changes of the fluorescent intensity, as a function of time is a widely accepted method to assess the vascular permeability during inflammation related to the immune response. In the present study we propose to bring a new dimension by applying a more sophisticated approach to the analysis of vascular reaction by using a quantitative analysis based on methods derived from astronomical observations, in particular by using a space-time Fourier filtering analysis followed by a polynomial orthogonal modes decomposition. We demonstrate that temporal evolution of the fluorescent intensity observed at certain pixels correlates quantitatively to the blood flow circulation at normal conditions. The approach allows to determine the regions of permeability and monitor both the fast kinetics related to the contrast material distribution in the circulatory system and slow kinetics associated with extravasation of the contrast material. Thus, we introduce a simple and convenient method for fast quantitative visualization of the leakage related to the inflammatory (immune) reaction in vivo.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

A step towards standardization: A method for end-point titer determination by fluorescence index of an automated microscope. End-point titer determination by fluorescence index.

PubMed

Carbone, Teresa; Gilio, Michele; Padula, Maria Carmela; Tramontano, Giuseppina; D'Angelo, Salvatore; Pafundi, Vito

2018-05-01

Indirect Immunofluorescence (IIF) is widely considered the Gold Standard for Antinuclear Antibody (ANA) screening. However, the high inter-reader variability remains the major disadvantage associated with ANA testing and the main reason for the increasing demand of the computer-aided immunofluorescence microscope. Previous studies proposed the quantification of the fluorescence intensity as an alternative for the classical end-point titer evaluation. However, the different distribution of bright/dark light linked to the nature of the self-antigen and its location in the cells result in different mean fluorescence intensities. The aim of the present study was to correlate Fluorescence Index (F.I.) with end-point titers for each well-defined ANA pattern. Routine serum samples were screened for ANA testing on HEp-2000 cells using Immuno Concepts Image Navigator System, and positive samples were serially diluted to assign the end-point titer. A comparison between F.I. and end-point titers related to 10 different staining patterns was made. According to our analysis, good technical performance of F.I. (97% sensitivity and 94% specificity) was found. A significant correlation between quantitative reading of F.I. and end-point titer groups was observed using Spearman's test and regression analysis. A conversion scale of F.I. in end-point titers for each recognized ANA-pattern was obtained. The Image Navigator offers the opportunity to improve worldwide harmonization of ANA test results. In particular, digital F.I. allows quantifying ANA titers by using just one sample dilution. It could represent a valuable support for the routine laboratory and an effective tool to reduce inter- and intra-laboratory variability. Copyright © 2018. Published by Elsevier B.V.

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour - Determination by high-performance thin-layer chromatography-fluorescence detection.

PubMed

Oellig, Claudia

2017-07-21

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography-fluorescence detection (HPTLC-FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC-FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26μg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Ionic calcium determination in skim milk with molecular probes and front-face fluorescence spectroscopy: simple linear regression.

PubMed

Gangidi, R R; Metzger, L E

2006-11-01

The purpose of this study was to determine if the ionic calcium content of skim milk could be determined using molecular probes and front-face fluorescence spectroscopy. Current methods for determining ionic calcium are not sensitive, overestimate ionic calcium, or require complex procedures. Molecular probes designed specifically for measuring ionic calcium could potentially be used to determine the ionic calcium content of skim milk. The goal of the current study was to develop foundation methods for future studies to determine ionic calcium directly in skim milk and other dairy products with molecular probes and fluorescence spectroscopy. In this study, the effect of pH on calcium-sensitive fluorescent probe (Rhod-5N and Fluo-5N) performance using various concentrations of skim milk was determined. The pH of diluted skim milk (1.9 to 8.9% skim milk), was adjusted to either 6.2 or 7.0, after which the samples were analyzed with fluorescent probes (1 microM) and front-face fluorescence spectroscopy. The ionic calcium content of each sample was also determined using a calcium ion-selective electrode. The results demonstrated that the ionic calcium content of each sample was highly correlated (R2 > 0.989) with the fluorescence intensities of the probe-calcium adduct using simple linear regression. Higher than suggested ionic calcium contents of 1,207 and 1,973 microM were determined with the probes (Fluo-5N and Rhod-5N) in diluted skim milk with pH 7.0 and 6.2, respectively. The fluorescence intensity of the probe-calcium adduct decreased with a decrease in pH for the same ionic calcium concentration. This study demonstrates that Fluo-5N and Rhod-5N can be used to determine the ionic-calcium content of diluted milk with front-face fluorescence spectroscopy. Furthermore, these probes may also have the potential to determine the ionic calcium content of undiluted skim milk.

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

PubMed

Rich, Ryan M; Stankowska, Dorota L; Maliwal, Badri P; Sørensen, Thomas Just; Laursen, Bo W; Krishnamoorthy, Raghu R; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

2013-02-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

Selective detection of Co2+ by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and biological activities

NASA Astrophysics Data System (ADS)

Mahajan, Prasad G.; Dige, Nilam C.; Desai, Netaji K.; Patil, Shivajirao R.; Kondalkar, Vijay V.; Hong, Seong-Karp; Lee, Ki Hwan

2018-06-01

Nowadays scientist over the world are engaging to put forth improved methods to detect metal ion in an aqueous medium based on fluorescence studies. A simple, selective and sensitive method was proposed for detection of Co2+ ion using fluorescent organic nanoparticles. We synthesized a fluorescent small molecule viz. 4,4‧-{benzene-1,4-diylbis-[(Z)methylylidenenitrilo]}dibenzoic acid (BMBA) to explore its suitability as sensor for Co2+ ion and biocompatibility in form of nanoparticles. Fluorescence nanoparticles (BMBANPs) prepared by simple reprecipitation method. Aggregation induced enhanced emission of BMBANPs exhibits the narrower particle size of 68 nm and sphere shape morphology. The selective fluorescence quenching was observed by addition of Co2+ and does not affected by presence of other coexisting ion solutions. The photo-physical properties, viz. UV-absorption, fluorescence emission, and lifetime measurements are in support of ligand-metal interaction followed by static fluorescence quenching phenomenon in emission of BMBANPs. Finally, we develop a simple analytical method for selective and sensitive determination of Co2+ ion in environmental samples. The cell culture E. coli, Bacillus sps., and M. tuberculosis H37RV strain in the vicinity of BMBANPs indicates virtuous anti-bacterial and anti-tuberculosis activity which is of additional novel application shown by prepared nanoparticles.

Rapid enantiomeric separation and simultaneous determination of phenethylamines by ultra high performance liquid chromatography with fluorescence and mass spectrometric detection: application to the analysis of illicit drugs distributed in the Japanese market and biological samples.

PubMed

Inagaki, Shinsuke; Hirashima, Haruo; Taniguchi, Sayuri; Higashi, Tatsuya; Min, Jun Zhe; Kikura-Hanajiri, Ruri; Goda, Yukihiro; Toyo'oka, Toshimasa

2012-12-01

A rapid enantiomeric separation and simultaneous determination method based on ultra high performance liquid chromatography (UHPLC) was developed for phenethylamine-type abused drugs using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(-)-DBD-Py-NCS) as the chiral fluorescent derivatization reagent. The derivatives were rapidly enantiomerically separated by reversed-phase UHPLC using a column of 2.3-µm octadecylsilica (ODS) particles by isocratic elution with water-methanol or water-acetonitrile systems as the mobile phase. The proposed method was applied to the analysis of products containing illicit drugs distributed in the Japanese market. Among the products, 1-(3,4-methylenedioxyphenyl)butan-2-amine (BDB) and 1-(2-methoxy4,5-methylenedioxyphenyl)propan-2-amine (MMDA-2) were detected in racemic form. Furthermore, the method was successfully applied to the analysis of hair specimens from rats that were continuously dosed with diphenyl(pyrrolidin-2-yl)methanol (D2PM). Using UHPLC-fluorescence (FL) detection, (R)- and (S)-D2PM from hair specimens were enantiomerically separated and detected with high sensitivity. The detection limits of (R)- and (S)-D2PM were 0.12 and 0.21 ng/mg hair, respectively (signal-to-noise ratio (S/N) = 3). Copyright © 2012 John Wiley & Sons, Ltd.

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

PubMed Central

2012-01-01

Background Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport. PMID:23148417

Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.

PubMed

Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

2011-01-01

Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

Clinicopathological characteristics of Xp11.2 translocation renal cell carcinoma in adolescents and adults: Diagnosis using immunostaining of transcription factor E3 and fluorescence in situ hybridization analysis.

PubMed

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tonooka, Akiko; Hasegawa, Tadashi; Tsukamoto, Taiji

2016-02-01

To determine the rate and clinicopathological features of Xp11.2 translocation carcinoma using immunostaining of transcription factor E3 and fluorescence in situ hybridization analysis. We evaluated 638 patients with renal cell carcinoma treated at Sapporo Medical University Hospital, Sapporo, Japan, from 1990 to 2009 by reviewing all hematoxylin-eosin-stained sections and carrying out immunostaining of transcription factor E3 for all cases. Fluorescence in situ hybridization analysis was carried out for patients with positive immunostaining or with findings suspicious for Xp11.2 translocation carcinoma on hematoxylin-eosin-stained sections. In this analysis, we set a cut-off level for split signals of at least 10% of nuclei. Of the 631 patients, 20 (3.2%) were positive for immunostaining. Finally, five patients were diagnosed with Xp11.2 translocation carcinoma (0.8%). Four of these patients were female and aged less than 50 years, and three cases were diagnosed as stage IV with multiple regional lymph nodal or visceral metastases. The positive predictive value of immunostaining was 25%. Patients with Xp11 translocation renal cell carcinoma tend to be younger, more frequently female and diagnosed at a more advanced stage. Immunostaining followed by fluorescence in situ hybridization analysis is an accurate and cost-effective approach for diagnosis of Xp11 translocation renal cell carcinoma. © 2015 The Japanese Urological Association.

Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots

NASA Astrophysics Data System (ADS)

Bocsi, Jozsef; Mittag, Anja; Varga, Viktor S.; Molnar, Bela; Tulassay, Zsolt; Sack, Ulrich; Lenz, Dominik; Tarnok, Attila

2006-02-01

Scanning Fluorescence Microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al. Cytometry 2004, 61A:1). The ratio of CD4+/CD8+ cells is an important in immune diagnostics in immunodeficiency and HIV. Therefor a four-color staining protocol (DNA, CD3, CD4 and CD8) for automated SFM analysis of lymphocytes was developed. EDTA uncoagulated blood was stained with organic and inorganic (Quantum dots) fluorochromes in different combinations. Aliquots of samples were measured by Flow Cytometry (FCM) and SFM. By SFM specimens were scanned and digitized using four fluorescence filter sets. Automated cell detection (based on Hoechst 33342 fluorescence), CD3, CD4 and CD8 detection were performed, CD4/CD8 ratio was calculated. Fluorescence signals were well separable on SFM and FCM. Passing and Bablok regression of all CD4/CD8 ratios obtained by FCM and SFM (F(X)=0.0577+0.9378x) are in the 95% confidence interval. Cusum test did not show significant deviation from linearity (P>0.10). This comparison indicates that there is no systemic bias between the two different methods. In SFM analyses the inorganic Quantum dot staining was very stable in PBS in contrast to the organic fluorescent dyes, but bleached shortly after mounting with antioxidant and free radical scavenger mounting media. This shows the difficulty of combinations of organic dyes and Quantum dots. Slide based multi-fluorescence labeling system and automated SFM are applicable tools for the CD4/CD8 ratio determination in peripheral blood samples. Quantum Dots are stable inorganic fluorescence labels that may be used as reliable high resolution dyes for cell labeling.

Determination of mycotoxins produced by Fusarium isolates from banana fruits by capillary gas chromatography and high-performance liquid chromatography.

PubMed

Jiménez, M; Mateo, R

1997-08-22

A method of analysis for trichothecenes (nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, T-2 tetraol, T-2 and HT-2 toxins), zearalenone and zearalenols, and another method for determination of fumonisin B1 are described and applied to cultures of Fusarium isolated from bananas. Both methods were adapted from different techniques of extraction, clean-up and determination of these mycotoxins. The first method involves extraction with methanol-1% aqueous sodium chloride, clean-up of extracts by partition with hexane and dichloromethane, additional solid reversed-phase clean-up and analysis of two eluates by both high-performance liquid chromatography with ultraviolet detection and capillary gas chromatography. The method for fumonisin B1 implies extraction with aqueous methanol, concentration, clean-up with water and methanol on Amberlite XAD-2 column, formation of a fluorescent 4-fluoro-7-nitrobenzofurazan derivative and analysis by high-performance liquid chromatography with fluorescence detection. Both procedures give good limits of detection and recoveries, and are considered suitable for the detection and quantification of the studied toxins in corn and rice cultures of Fusarium spp. isolated from banana fruits.

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

Targeting human c-Myc promoter duplex DNA with actinomycin D by use of multi-way analysis of quantum-dot-mediated fluorescence resonance energy transfer.

PubMed

Gholami, Somayeh; Kompany-Zareh, Mohsen

2013-07-01

Actinomycin D (Act D), an oncogenic c-Myc promoter binder, interferes with the action of RNA polymerase. There is great demand for high-throughput technology able to monitor the activity of DNA-binding drugs. To this end, binding of 7-aminoactinomycin D (7AAD) to the duplex c-Myc promoter was investigated by use of 2D-photoluminescence emission (2D-PLE), and the resulting data were subjected to analysis by use of convenient and powerful multi-way approaches. Fluorescence measurements were performed by use of the quantum dot (QD)-conjugated c-Myc promoter. Intercalation of 7AAD within duplex base pairs resulted in efficient energy transfer from drug to QD via fluorescence resonance energy transfer (FRET). Multi-way analysis of the three-way data array obtained from titration experiments was performed by use of restricted Tucker3 and hard trilinear decomposition (HTD). These techniques enable analysis of high-dimensional and complex data from nanobiological systems which include several spectrally overlapped structures. It was almost impossible to obtain robust and meaningful information about the FRET process for such high overlap data by use of classical analysis. The soft approach had the important advantage over univariate classical methods of enabling us to investigate the source of variance in the fluorescence signal of the DNA-drug complex. It was established that hard trilinear decomposition analysis of FRET-measured data overcomes the problem of rank deficiency, enabling calculation of concentration profiles and pure spectra for all species, including non-fluorophores. The hard modeling approach was also used for determination of equilibrium constants for the hybridization and intercalation equilibria, using nonlinear fit data analysis. The intercalation constant 3.6 × 10(6) mol(-1) L and hybridization stability 1.0 × 10(8) mol(-1) L obtained were in good agreement with values reported in the literature. The analytical concentration of the QD-labeled DNA was determined by use of nonlinear fitting, without using external standard calibration samples. This study was a successful application of multi-way chemometric methods to investigation of nano-biotechnological systems where several overlapped species coexist in solution.

[Analysis of fluorescence spectrum of petroleum-polluted water].

PubMed

Huang, Miao-Fen; Song, Qing-Jun; Xing, Xu-Feng; Jian, Wei-Jun; Liu, Yuan; Zhao, Zu-Long

2014-09-01

In four ratio experiments, natural waters, sampled from the mountain reservoir and the sea water around Dalian city, were mixed with the sewage from petroleum refinery and petroleum exploitation plants. The fluorescence spectra of water samples containing only chromophoric dissolved organic matters(CDOM), samples containing only petroleum, and samples containing a mixture of petroleum and CDOM were analyzed, respectively. The purpose of this analysis is to provide a basis for determining the contribution of petroleum substances and CDOM to the total absorption coefficient of the petroleum-contaminated water by using fluorescence technique. The results showed that firstly, CDOM in seawater had three main fluorescence peaks at Ex: 225-230 nm/Em: 320-330 nm, Ex: 280 nm/Em: 340 nm and Ex: 225-240 nm/Em: 430-470 nm, respectively, and these may arise from the oceanic chlorophyll. CDOM in natural reservoir water had two main fluorescence peaks at EX: 240- 260 nm/Em: 420-450 nm and Ex: 310~350 nm/Em: 420--440 nm, respectively, and these may arise from the terrestrial sources; secondly, the water samples containing only petroleum extracted with n-hexane had one to three fluorescence spectral peaksat Ex: 220-240 nm/Em: 320-340 nm, Ex: 270-290 nm/Em: 310-340 nm and Ex: 220-235 nm/Em: 280-310 nm, respectively, caused by their hydrocarbon component; finally, the water samples containing both petroleum and CDOM showed a very strong fluorescence peak at Ex: 230-250 nm/Em: 320-370 nm, caused by the combined effect of CDOM and petroleum hydrocarbons.

Development and Validation of highly Sensitive Stability Indicating Spectrofluorimetric Method for Determination of Amlodipine in Pharmaceutical Preparations and Human Plasma.

PubMed

Mohamed, Abdel-Maaboud I; Omar, Mahmoud A; Hammad, Mohamed A; Mohamed, Abobakr A

2016-11-01

A highly sensitive and simple spectrofluorimetric method was developed for the determination of Amlodipine besylate (AML) in its pharmaceutical formulations and spiked human plasma. The proposed method is based on the investigation of the fluorescence spectral behaviour of AML in Tween-80 micellar system. In aqueous solution, the fluorescence intensity of AML was greatly enhanced (160 %) in the presence of Tween-80. The fluorescence intensity was measured at 427 nm after excitation at 385 nm. The fluorescence-concentration plot was rectilinear over the concentration range 0.1-4.0 μg/ml, with lower detection limit of 0.03 μg/ml. The suggested method was successfully applied for the analysis of AML in its commercial tablets alone or in combination with either Atorvastatin or Valsartan. The application of the proposed method was extended to the assay of AML in spiked human plasma and stability studies of AML after exposure to different forced degradation conditions, such as acidic, alkaline, photo- and oxidative conditions, according to ICH guidelines. The results were statistically compared to those obtained by comparison methods and were found to be in good agreement.

A simple, reversible, colorimetric and water-soluble fluorescent chemosensor for the naked-eye detection of Cu2+ in ~100% aqueous media and application to real samples.

PubMed

Sun, Tao; Niu, Qingfen; Li, Tianduo; Guo, Zongrang; Liu, Haixia

2018-01-05

A simple, reversible, colorimetric and water-soluble fluorescent chemosensor ADA for the naked-eye detection of Cu 2+ was developed. Sensor ADA showed high selectivity and sensitivity toward Cu 2+ in ~100% aqueous media over wide pH range. Sensor ADA exhibited a red-shift in the absorption spectra from 466 to 480nm that is accompanied by significant color change from light yellow to yellowish brown instantaneously. The Cu 2+ recognition is based on the chelation-enhanced fluorescence quenching (CHEQ) effect of the paramagnetic nature. The lowest detection limit is determined to be 15.8nM, which is much lower than the allowable level of Cu 2+ in drinking water set by U.S. Environmental Protection Agency (~20μM) and the World Health Organization (~30μM). The 1:1 binding process was confirmed by fluorescence measurements, IR analysis and DFT studies. Moreover, sensor ADA was successfully applied for determination of trace level of Cu 2+ with 4 reuse cycles in various water samples, which affords promising potential in ion-detection field. Copyright © 2017 Elsevier B.V. All rights reserved.

A simple, reversible, colorimetric and water-soluble fluorescent chemosensor for the naked-eye detection of Cu2 + in 100% aqueous media and application to real samples

NASA Astrophysics Data System (ADS)

Sun, Tao; Niu, Qingfen; Li, Tianduo; Guo, Zongrang; Liu, Haixia

2018-01-01

A simple, reversible, colorimetric and water-soluble fluorescent chemosensor ADA for the naked-eye detection of Cu2 + was developed. Sensor ADA showed high selectivity and sensitivity toward Cu2 + in 100% aqueous media over wide pH range. Sensor ADA exhibited a red-shift in the absorption spectra from 466 to 480 nm that is accompanied by significant color change from light yellow to yellowish brown instantaneously. The Cu2 + recognition is based on the chelation-enhanced fluorescence quenching (CHEQ) effect of the paramagnetic nature. The lowest detection limit is determined to be 15.8 nM, which is much lower than the allowable level of Cu2 + in drinking water set by U.S. Environmental Protection Agency ( 20 μM) and the World Health Organization ( 30 μM). The 1:1 binding process was confirmed by fluorescence measurements, IR analysis and DFT studies. Moreover, sensor ADA was successfully applied for determination of trace level of Cu2 + with 4 reuse cycles in various water samples, which affords promising potential in ion-detection field.

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

NASA Astrophysics Data System (ADS)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies. Electronic supplementary information (ESI) available: Absorbance and fluorescence spectra of quantum dot nanoprobes, electrophoresis analysis, and experimental setup for fluorescence imaging with dual channels. See DOI: 10.1039/c3nr03291d

The Analysis of Fluorescence Decay by a Method of Moments

PubMed Central

Isenberg, Irvin; Dyson, Robert D.

1969-01-01

The fluorescence decay of the excited state of most biopolymers, and biopolymer conjugates and complexes, is not, in general, a simple exponential. The method of moments is used to establish a means of analyzing such multi-exponential decays. The method is tested by the use of computer simulated data, assuming that the limiting error is determined by noise generated by a pseudorandom number generator. Multi-exponential systems with relatively closely spaced decay constants may be successfully analyzed. The analyses show the requirements, in terms of precision, that data must meet. The results may be used both as an aid in the design of equipment and in the analysis of data subsequently obtained. PMID:5353139

Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

NASA Astrophysics Data System (ADS)

Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

2011-07-01

Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

2016-12-01

Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.

Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

2011-07-01

Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Huanjun; Cho, Hyo-Min; Molloi, Sabee, E-mail: symolloi@uci.edu

Purpose: To investigate the feasibility of characterizing a Si strip photon-counting detector using x-ray fluorescence. Methods: X-ray fluorescence was generated by using a pencil beam from a tungsten anode x-ray tube with 2 mm Al filtration. Spectra were acquired at 90° from the primary beam direction with an energy-resolved photon-counting detector based on an edge illuminated Si strip detector. The distances from the source to target and the target to detector were approximately 19 and 11 cm, respectively. Four different materials, containing silver (Ag), iodine (I), barium (Ba), and gadolinium (Gd), were placed in small plastic containers with a diametermore » of approximately 0.7 cm for x-ray fluorescence measurements. Linear regression analysis was performed to derive the gain and offset values for the correlation between the measured fluorescence peak center and the known fluorescence energies. The energy resolutions and charge-sharing fractions were also obtained from analytical fittings of the recorded fluorescence spectra. An analytical model, which employed four parameters that can be determined from the fluorescence calibration, was used to estimate the detector response function. Results: Strong fluorescence signals of all four target materials were recorded with the investigated geometry for the Si strip detector. The average gain and offset of all pixels for detector energy calibration were determined to be 6.95 mV/keV and −66.33 mV, respectively. The detector’s energy resolution remained at approximately 2.7 keV for low energies, and increased slightly at 45 keV. The average charge-sharing fraction was estimated to be 36% within the investigated energy range of 20–45 keV. The simulated detector output based on the proposed response function agreed well with the experimental measurement. Conclusions: The performance of a spectral imaging system using energy-resolved photon-counting detectors is very dependent on the energy calibration of the detector. The proposed x-ray fluorescence technique offers an accurate and efficient way to calibrate the energy response of a photon-counting detector.« less

Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens.

PubMed

Sohn, Miryeong; Himmelsbach, David S; Barton, Franklin E; Fedorka-Cray, Paula J

2009-11-01

This study deals with the rapid detection and differentiation of Escherichia coli, Salmonella, and Campylobacter, which are the most commonly identified commensal and pathogenic bacteria in foods, using fluorescence spectroscopy and multivariate analysis. Each bacterial sample cultured under controlled conditions was diluted in physiologic saline for analysis. Fluorescence spectra were collected over a range of 200-700 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. The synchronous scan technique was employed to find the optimum excitation (lambda(ex)) and emission (lambda(em)) wavelengths for individual bacteria with the wavelength interval (Deltalambda) being varied from 10 to 200 nm. The synchronous spectra and two-dimensional plots showed two maximum lambda(ex) values at 225 nm and 280 nm and one maximum lambda(em) at 335-345 nm (lambda(em) = lambda(ex) + Deltalambda), which correspond to the lambda(ex) = 225 nm, Deltalambda = 110-120 nm, and lambda(ex) = 280 nm, Deltalambda = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the fluorescence spectra resulted in successful classification of the bacteria by their genus as well as determining their concentration. The detection limit was approximately 10(3)-10(4) cells/mL for each bacterial sample. These results demonstrated that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to classify bacterial pathogens in liquids. The methodology is rapid (>10 min), inexpensive, and requires minimal sample preparation compared to standard analytical methods for bacterial detection.

Using fluorescence-parallel factor analysis for assessing disinfection by-product formation and natural organic matter removal efficiency in secondary treated synthetic drinking waters.

PubMed

Watson, Kalinda; Farré, Maria José; Leusch, Frederic D L; Knight, Nicole

2018-05-28

Parallel factor (PARAFAC) analysis of fluorescence excitation-emission matrices (EEMs) was used to investigate the organic matter and DBP formation characteristics of untreated, primary treated (enhanced coagulation; EC) and secondary treated synthetic waters prepared using a Suwannee River natural organic matter (SR-NOM) isolate. The organic matter was characterised by four different fluorescence components; two humic acid-like (C1 and C2) and two protein-like (C3 and C4). Secondary treatment methods tested, following EC treatment, were; powdered activated carbon (PAC), granular activated carbon (GAC), 0.1% silver-impregnated activated carbon (SIAC), and MIEX® resin. Secondary treatments were more effective at removing natural organic matter (NOM) and fluorescent DBP-precursor components than EC alone. The formation of a suite of 17 DBPs including chlorinated, brominated and iodinated trihalomethanes (THMs), dihaloacetonitriles (DHANs), chloropropanones (CPs), chloral hydrate (CH) and trichloronitromethane (TCNM) was determined after chlorinating water sampled before and after each treatment step. Regression analysis was used to investigate the relationship between peak component fluorescence intensity (F MAX ), DBP concentration and speciation, and more commonly used aggregate parameters such as DOC, UV 254 and SUVA 254 . PARAFAC component 1 (C1) was in general a better predictor of DBP formation than other aggregate parameters, and was well correlated (R ≥ 0.80) with all detected DBPs except dibromochloromethane (DBCM) and dibromoacetonitrile (DBAN). These results indicate that the fluorescence-PARAFAC approach could provide a robust analytical tool for predicting DBP formation, and for evaluating the removal of NOM fractions relevant to DBP formation during water treatment. Copyright © 2018. Published by Elsevier B.V.

Counter tube window and X-ray fluorescence analyzer study

NASA Technical Reports Server (NTRS)

Hertel, R.; Holm, M.

1973-01-01

A study was performed to determine the best design tube window and X-ray fluorescence analyzer for quantitative analysis of Venusian dust and condensates. The principal objective of the project was to develop the best counter tube window geometry for the sensing element of the instrument. This included formulation of a mathematical model of the window and optimization of its parameters. The proposed detector and instrument has several important features. The instrument will perform a near real-time analysis of dust in the Venusian atmosphere, and is capable of measuring dust layers less than 1 micron thick. In addition, wide dynamic measurement range will be provided to compensate for extreme variations in count rates. An integral pulse-height analyzer and memory accumulate data and read out spectra for detail computer analysis on the ground.

A method of lead determination in human teeth by energy dispersive X-ray fluorescence (EDXRF).

PubMed

Sargentini-Maier, M L; Frank, R M; Leroy, M J; Turlot, J C

1988-12-01

A systematic sampling procedure was combined with a method of energy dispersive X-ray fluorescence (EDXRF) to study lead content and its variations in human teeth. On serial ground sections made on unembedded permanent teeth of inhabitants of Strasbourg with a special diamond rotating disk, 2 series of 500 microns large punch biopsies were made systematically in 5 directions from the tooth surface to the inner pulpal dentine with a micro-punching unit. In addition, pooled fragments of enamel and dentine were made for each tooth. On each punched fragment or pooled sample, lead content was determined after dissolution in ultrapure nitric acid, on a 4 microns thick polypropylene film, and irradiation with a Siemens EDXRF prototype with direct sample excitation by a high power X-ray tube with a molybdenum anode. Fluorescence was detected by a Si(Li) detector and calcium was used as an internal standard. This technique allowed a rapid, automatic, multielementary and non-destructive analysis of microsamples with good detection limits.

Calorimetric and spectroscopic studies of the interaction between zidovudine and human serum albumin

NASA Astrophysics Data System (ADS)

Pîrnău, Adrian; Mic, Mihaela; Neamţu, Silvia; Floare, Călin G.; Bogdan, Mircea

2018-02-01

A quantitative analysis of the interaction between zidovudine (AZT) and human serum albumin (HSA) was achieved using Isothermal titration calorimetry (ITC) in combination with fluorescence and 1H NMR spectroscopy. ITC directly measure the heat during a biomolecular binding event and gave us thermodynamic parameters and the characteristic association constant. By fluorescence quenching, the binding parameters of AZT-HSA interaction was determined and location to binding site I of HSA was confirmed. Via T1 NMR selective relaxation time measurements the drug-protein binding extent was evaluated as dissociation constants Kd and the involvement of azido moiety of zidovudine in molecular complex formation was put in evidence. All three methods indicated a very weak binding interaction. The association constant determined by ITC (3.58 × 102 M- 1) is supported by fluorescence quenching data (2.74 × 102 M- 1). The thermodynamic signature indicates that at least hydrophobic and electrostatic type interactions played a main role in the binding process.

Fluorescence-based multi-parameter approach to characterize dynamics of organic carbon, faecal bacteria and particles at alpine karst springs.

PubMed

Frank, Simon; Goeppert, Nadine; Goldscheider, Nico

2018-02-15

Karst springs, especially in alpine regions, are important for drinking water supply but also vulnerable to contamination, especially after rainfall events. This high variability of water quality requires rapid quantification of contamination parameters. Here, we used a fluorescence-based multi-parameter approach to characterize the dynamics of organic carbon, faecal bacteria, and particles at three alpine karst springs. We used excitation emission matrices (EEMs) to identify fluorescent dissolved organic material (FDOM). At the first system, peak A fluorescence and total organic carbon (TOC) were strongly correlated (Spearman's r s of 0.949), indicating that a large part of the organic matter is related to humic-like substances. Protein-like fluorescence and cultivation-based determination of coliform bacteria also had a significant correlation with r s =0.734, indicating that protein-like fluorescence is directly related to faecal pollution. At the second system, which has two spring outlets, the absolute values of all measured water-quality parameters were lower; there was a significant correlation between TOC and humic-like fluorescence (r s =0.588-0.689) but coliform bacteria and protein-like fluorescence at these two springs were not correlated. Additionally, there was a strong correlation (r s =0.571-0.647) between small particle fractions (1.0 and 2.0μm), a secondary turbidity peak and bacteria. At one of these springs, discharge was constant despite the reaction of all other parameters to the rainfall event. Our results demonstrated that i) all three springs showed fast and marked responses of all investigated water-quality parameters after rain events; ii) a constant discharge does not necessarily mean constant water quality; iii) at high contamination levels, protein-like fluorescence is a good indicator of bacterial contamination, while at low contamination levels no correlation between protein-like fluorescence and bacterial values was detected; and iv) a combination of fluorescence measurements and particle-size analysis is a promising approach for a rapid assessment of organic contamination, especially relative to time-consuming conventional bacterial determination methods. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel quantitative analysis method of three-dimensional fluorescence spectra for vegetable oils contents in edible blend oil

NASA Astrophysics Data System (ADS)

Xu, Jing; Wang, Yu-Tian; Liu, Xiao-Fei

2015-04-01

Edible blend oil is a mixture of vegetable oils. Eligible blend oil can meet the daily need of two essential fatty acids for human to achieve the balanced nutrition. Each vegetable oil has its different composition, so vegetable oils contents in edible blend oil determine nutritional components in blend oil. A high-precision quantitative analysis method to detect the vegetable oils contents in blend oil is necessary to ensure balanced nutrition for human being. Three-dimensional fluorescence technique is high selectivity, high sensitivity, and high-efficiency. Efficiency extraction and full use of information in tree-dimensional fluorescence spectra will improve the accuracy of the measurement. A novel quantitative analysis is proposed based on Quasi-Monte-Carlo integral to improve the measurement sensitivity and reduce the random error. Partial least squares method is used to solve nonlinear equations to avoid the effect of multicollinearity. The recovery rates of blend oil mixed by peanut oil, soybean oil and sunflower are calculated to verify the accuracy of the method, which are increased, compared the linear method used commonly for component concentration measurement.

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655

Determination of low molecular weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

NASA Technical Reports Server (NTRS)

Fahey, Robert C.; Newton, Gerald L.

1988-01-01

Methods are described for the preparation and high-performance liquid chromatography (HPLC) analysis of monobromobimane derivatives of low molecular weight thiols in extracts of biological samples. Typical problems encountered in the development and application of these methods are discussed. Analysis of mung bean extract is used as an example.

Palus Somni - Anomalies in the correlation of Al/Si X-ray fluorescence intensity ratios and broad-spectrum visible albedos. [lunar surface mineralogy

NASA Technical Reports Server (NTRS)

Clark, P. E.; Andre, C. G.; Adler, I.; Weidner, J.; Podwysocki, M.

1976-01-01

The positive correlation between Al/Si X-ray fluorescence intensity ratios determined during the Apollo 15 lunar mission and a broad-spectrum visible albedo of the moon is quantitatively established. Linear regression analysis performed on 246 1 degree geographic cells of X-ray fluorescence intensity and visible albedo data points produced a statistically significant correlation coefficient of .78. Three distinct distributions of data were identified as (1) within one standard deviation of the regression line, (2) greater than one standard deviation below the line, and (3) greater than one standard deviation above the line. The latter two distributions of data were found to occupy distinct geographic areas in the Palus Somni region.

Fluorescence from polystyrene - Photochemical processes in polymeric systems, 7

NASA Technical Reports Server (NTRS)

Gupta, M. C.; Gupta, A.

1983-01-01

Results are presented for measurements of the fluorescence spectra of polystyrene in dilute solution and in pure solid films. It is determined that a major potential source of experimental error is the concurrent photooxidative degradation in air which may obscure fluorescence emission from monomeric sites in solid films at 25 C. The fluorescence spectra of oriented films are evaluated in terms of the monomer to excimer fluorescence intensity ratio and the excimer 'red shift'. The monomer to excimer fluorescence intensity ratio is determined to be significantly higher in fluid solution than in solid film.

Fluorescence guided evaluation of photodynamic therapy as acne treatment

NASA Astrophysics Data System (ADS)

Ericson, Marica B.; Horfelt, Camilla; Cheng, Elaine; Larsson, Frida; Larko, Olle; Wennberg, Ann-Marie

2005-08-01

Photodynamic therapy (PDT) is an attractive alternative treatment for patients with acne because of its efficiency and few side effects. Propionibacterium acnes (P.acnes) are bacteria present in the skin, which produce endogenous porphyrins that act as photosensitisers. In addition, application of aminolaevulinic acid or its methyl ester (mALA) results in increased accumulation of porphyrins in the pilosebaceous units. This makes it possible to treat acne with PDT. This initial study investigates the possibility of fluorescence imaging as assessment tool in adjunct to PDT of patients with acne. Twenty-four patients with acne on the cheeks have been treated with PDT with and without mALA. Fluorescence images have been obtained before and after treatment. The clinical acne score was assessed as base line before PDT, and at every follow up visit. Additionally the amount of P.acnes was determined. The clinical evaluation showed a general improvement of acne, even though no difference between treatment with and without mALA was observed. By performing texture analysis and multivariate data analsysis on the fluorescence images, the extracted texture features were found to correlate with the corresponding clinical assessment (67%) and amount of P.acnes (72%). The analysis showed that features describing the highly fluorescent pores could be related to the clinical assessment. This result suggests that fluorescence imaging can be used as an objective assessment of acne, but further improvement of the technique is possible, for example by including colour images.

CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe- ions in aqueous solution.

PubMed

Wu, Chuan-Liu; Zhao, Yi-Bing

2007-06-01

Water-soluble cadmium sulfide (CdS) quantum dots (QDs) capped by mercaptoacetic acid were synthesized by aqueous-phase arrested precipitation, and characterized by transmission electron microscopy, spectrofluorometry, and UV-Vis spectrophotometry. The prepared luminescent water-soluble CdS QDs were evaluated as fluorescence probes for the detection of highly reactive hydrogen selenide ions (HSe(-) ions). The quenching of the fluorescence emission of CdS QDs with the addition of HSe(-) ions is due to the elimination of the S(2-) vacancies which are luminescence centers. Quantitative analysis based on chemical interaction between HSe(-) ions and the surface of CdS QDs is very simple, easy to develop, and has demonstrated very high sensitivity and selectivity features. The effect of foreign ions (common anions and biologically relevant cations) on the fluorescence of the CdS QDs was examined to evaluate the selectivity. Only Cu(2+) and S(2-) ions exhibit significant effects on the fluorescence of CdS QDs. With the developed method, we are able to determine the concentration of HSe(-) ions in the range from 0.10 to 4.80 micromol L(-1), and the limit of detection is 0.087 micromol L(-1). The proposed method was successfully applied to monitor the obtained HSe(-) ions from the reaction of glutathione with selenite. To the best of our knowledge, this is the first report on fluorescence analysis of HSe(-) ions in aqueous solution.

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro.

PubMed

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-03-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence-labeled polystyrene particles and to study the respective method́s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

PubMed

Zhang, Xuzhu; Poniewierski, Andrzej; Jelińska, Aldona; Zagożdżon, Anna; Wisniewska, Agnieszka; Hou, Sen; Hołyst, Robert

2016-10-04

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C 12 E 8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

Authentication of the botanical and geographical origin of honey by front-face fluorescence spectroscopy.

PubMed

Ruoff, Kaspar; Luginbühl, Werner; Künzli, Raphael; Bogdanov, Stefan; Bosset, Jacques Olivier; von der Ohe, Katharina; von der Ohe, Werner; Amado, Renato

2006-09-06

Front-face fluorescence spectroscopy, directly applied on honey samples, was used for the authentication of 11 unifloral and polyfloral honey types (n = 371 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis. Excitation spectra (220-400 nm) were recorded with the emission measured at 420 nm. In addition, emission spectra were recorded between 290 and 500 nm (excitation at 270 nm) as well as between 330 and 550 nm (excitation at 310 nm). A total of four different spectral data sets were considered for data analysis. Chemometric evaluation of the spectra included principal component analysis and linear discriminant analysis; the error rates of the discriminant models were calculated by using Bayes' theorem. They ranged from <0.1% (polyfloral and chestnut honeys) to 9.9% (fir honeydew honey) by using single spectral data sets and from <0.1% (metcalfa honeydew, polyfloral, and chestnut honeys) to 7.5% (lime honey) by combining two data sets. This study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey and may also be useful for the determination of the geographical origin within the same unifloral honey type.

Studying aerosol light scattering based on aspect ratio distribution observed by fluorescence microscope.

PubMed

Li, Li; Zheng, Xu; Li, Zhengqiang; Li, Zhanhua; Dubovik, Oleg; Chen, Xingfeng; Wendisch, Manfred

2017-08-07

Particle shape is crucial to the properties of light scattered by atmospheric aerosol particles. A method of fluorescence microscopy direct observation was introduced to determine the aspect ratio distribution of aerosol particles. The result is comparable with that of the electron microscopic analysis. The measured aspect ratio distribution has been successfully applied in modeling light scattering and further in simulation of polarization measurements of the sun/sky radiometer. These efforts are expected to improve shape retrieval from skylight polarization by using directly measured aspect ratio distribution.

Efficacy of Sex Determination from Human Dental Pulp Tissue and its Reliability as a Tool in Forensic Dentistry

PubMed Central

Khanna, Kaveri Surya

2015-01-01

Background: Sex determination is one of the primary steps in forensics. Barr body can be used as a histological method for identification of sex as it is found to be specific to female somatic cells and rare in male cells. To demarcate human dental pulp as an important identification tool of sex in forensic odontology (FO) and to evaluate the time period till which sex can be determined from pulp tissue using three stains H and E, Feulgen, and acridine - orange under fluorescence so as. Materials and Methods: 90 pulp samples (45 males and 45 females) were subjected to Barr body analysis for determination of sex using light and fluorescent microscopy. Results: Barr body was found to be positive for female samples and negative or rare in the male sample (<3%). Conclusion: Barr body from human dental pulp tissue can be used as a successful determinant of sex identification in FO. PMID:26668474

[Determination of cyclamate in complex matrix using HPLC after column derivatization with 4-fluoro-7-nitrobenzofurazan].

PubMed

Hauck, M; Köbler, H

1990-01-01

A method for the analysis of cyclamate in complex foodstuffs has been developed. This method is applicable in strongly coloured and protein-rich foodstuffs. The quantitative determination depends on oxidation of cyclamate to cyclohexylamine and derivatisation with 4-fluoro-7-nitrobenzofuran (NBD-F). The derivatives are analysed by HPLC on a C18: reversed-phase column, their minimal stability being 12 h. There are two possible methods of detection: (a) absorbance at 485 nm and (b) fluorescence with excitation at 485 nm and emission at 530 nm. The detection limit of cyclamate is 5 mg/kg foodstuff, with fluorescence detection 0.4 mg/kg. The recoveries are in the range of 88% to 104%.

Temporal binning of time-correlated single photon counting data improves exponential decay fits and imaging speed

PubMed Central

Walsh, Alex J.; Sharick, Joe T.; Skala, Melissa C.; Beier, Hope T.

2016-01-01

Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (<10%) required to reduce bias towards short lifetimes. Here, simulated fluorescence lifetime decays are analyzed by SPCImage and SLIM Curve software to determine the limiting lifetime parameters and photon requirements of fluorescence lifetime decays that can be accurately fit. Data analysis techniques to improve fitting accuracy for low photon count data were evaluated. Temporal binning of the decays from 256 time bins to 42 time bins significantly (p<0.0001) improved fit accuracy in SPCImage and enabled accurate fits with low photon counts (as low as 700 photons/decay), a 6-fold reduction in required photons and therefore improvement in imaging speed. Additionally, reducing the number of free parameters in the fitting algorithm by fixing the lifetimes to known values significantly reduced the lifetime component error from 27.3% to 3.2% in SPCImage (p<0.0001) and from 50.6% to 4.2% in SLIM Curve (p<0.0001). Analysis of nicotinamide adenine dinucleotide–lactate dehydrogenase (NADH-LDH) solutions confirmed temporal binning of TCSPC data and a reduced number of free parameters improves exponential decay fit accuracy in SPCImage. Altogether, temporal binning (in SPCImage) and reduced free parameters are data analysis techniques that enable accurate lifetime estimation from low photon count data and enable TCSPC imaging speeds up to 6x and 300x faster, respectively, than traditional TCSPC analysis. PMID:27446663

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

PubMed Central

Sieracki, M E; Reichenbach, S E; Webb, K L

1989-01-01

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge. Images PMID:2516431

Functionalized vertical GaN micro pillar arrays with high signal-to-background ratio for detection and analysis of proteins secreted from breast tumor cells.

PubMed

Choi, Mun-Ki; Kim, Gil-Sung; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Won-Yong; Umar, Ahmad; Lee, Sang-Kwon

2017-11-02

The detection of cancer biomarkers has recently attracted significant attention as a means of determining the correct course of treatment with targeted therapeutics. However, because the concentration of these biomarkers in blood is usually relatively low, highly sensitive biosensors for fluorescence imaging and precise detection are needed. In this study, we have successfully developed vertical GaN micropillar (MP) based biosensors for fluorescence sensing and quantitative measurement of CA15-3 antigens. The highly ordered vertical GaN MP arrays result in the successful immobilization of CA15-3 antigens on each feature of the arrays, thereby allowing the detection of an individual fluorescence signal from the top surface of the arrays owing to the high regularity of fluorophore-tagged MP spots and relatively low background signal. Therefore, our fluorescence-labeled and CA15-3 functionalized vertical GaN-MP-based biosensor is suitable for the selective quantitative analysis of secreted CA15-3 antigens from MCF-7 cell lines, and helps in the early diagnosis and prognosis of serious diseases as well as the monitoring of the therapeutic response of breast cancer patients.

Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.

PubMed

Kitamura, Akira; Nagata, Kazuhiro; Kinjo, Masataka

2015-03-16

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

Discrimination of geographical origin and detection of adulteration of kudzu root by fluorescence spectroscopy coupled with multi-way pattern recognition

NASA Astrophysics Data System (ADS)

Hu, Leqian; Ma, Shuai; Yin, Chunling

2018-03-01

In this work, fluorescence spectroscopy combined with multi-way pattern recognition techniques were developed for determining the geographical origin of kudzu root and detection and quantification of adulterants in kudzu root. Excitation-emission (EEM) spectra were obtained for 150 pure kudzu root samples of different geographical origins and 150 fake kudzu roots with different adulteration proportions by recording emission from 330 to 570 nm with excitation in the range of 320-480 nm, respectively. Multi-way principal components analysis (M-PCA) and multilinear partial least squares discriminant analysis (N-PLS-DA) methods were used to decompose the excitation-emission matrices datasets. 150 pure kudzu root samples could be differentiated exactly from each other according to their geographical origins by M-PCA and N-PLS-DA models. For the adulteration kudzu root samples, N-PLS-DA got better and more reliable classification result comparing with the M-PCA model. The results obtained in this study indicated that EEM spectroscopy coupling with multi-way pattern recognition could be used as an easy, rapid and novel tool to distinguish the geographical origin of kudzu root and detect adulterated kudzu root. Besides, this method was also suitable for determining the geographic origin and detection the adulteration of the other foodstuffs which can produce fluorescence.

Second derivative synchronous fluorimetric method for simultaneous determination of harman and norharman in coffee samples

NASA Astrophysics Data System (ADS)

Wabaidur, Saikh Mohammad; Lee, Sang Hak; Alothman, Zeid Abdullah; Siddiqui, Masoom Raza; Alam, Seikh Mafiz

2013-06-01

The simultaneous determination of harman and norharman using second derivative synchronous fluorescence method has been developed based on their natural fluorescence. Due to their similar molecular structures, it is difficult to determine them simultaneously in the mixture using conventional fluorimetry. Overlapping of fluorescence spectra was resolved by using a constant second derivative synchronous fluorimetry. The derivative synchronous spectrum, maintaining a constant difference of Δλ = 150 nm between emission and excitation for both the compounds, has been selected for the analysis. The range of application is between 0.182 and 18.2 μg/mL (correlation coefficient, R = 0.9982) for harman and between 0.504 and 16.8 μg/mL (correlation coefficient, R = 0.9962) for norharman. The recovery ranges of 98.5-101.1% for harman and 97.5-99.1% for norharman from their synthetic mixture was reported. The detection limits are 0.016 μg/mL and 0.038 μg/mL for harman and norharman, respectively. Similarly, the quantitation limit of the two compounds was found to be 0.049 and 0.109 μg/mL, respectively. The method was applied to the simultaneous determination of both compounds in coffee samples with satisfactory results.

Second derivative synchronous fluorimetric method for simultaneous determination of harman and norharman in coffee samples.

PubMed

Wabaidur, Saikh Mohammad; Lee, Sang Hak; Alothman, Zeid Abdullah; Siddiqui, Masoom Raza; Alam, Seikh Mafiz

2013-06-01

The simultaneous determination of harman and norharman using second derivative synchronous fluorescence method has been developed based on their natural fluorescence. Due to their similar molecular structures, it is difficult to determine them simultaneously in the mixture using conventional fluorimetry. Overlapping of fluorescence spectra was resolved by using a constant second derivative synchronous fluorimetry. The derivative synchronous spectrum, maintaining a constant difference of Δλ=150 nm between emission and excitation for both the compounds, has been selected for the analysis. The range of application is between 0.182 and 18.2 μg/mL (correlation coefficient, R=0.9982) for harman and between 0.504 and 16.8 μg/mL (correlation coefficient, R=0.9962) for norharman. The recovery ranges of 98.5-101.1% for harman and 97.5-99.1% for norharman from their synthetic mixture was reported. The detection limits are 0.016 μg/mL and 0.038 μg/mL for harman and norharman, respectively. Similarly, the quantitation limit of the two compounds was found to be 0.049 and 0.109 μg/mL, respectively. The method was applied to the simultaneous determination of both compounds in coffee samples with satisfactory results. Copyright © 2013 Elsevier B.V. All rights reserved.

Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Kimura, Hitoshi; Ogura, Yusuke; Tanida, Jun

2018-06-01

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.

[Determination of chromphoric dissolved organic matter in water from different sources].

PubMed

Liu, Xian-ping; Li, Lei; Dai, Jin-feng; Wang, Xiao-ru; Lee, Frank S C

2007-10-01

Chromophoric dissolved organic matter (CDOM) represents the fraction of the dissolved organic pool which absorbs light in the visible as well as UV ranges. It could affect the color of the waters. It is necessary to study it during in research on ecosystem, remote sensing of the water color and the cycle of carbon in waters. CDOM can fluoresce when excited, so fluorescence spectrum has been used to study its origin, distribution, and change. In the present article the fluorescence spectrophotometer was used to study the relation between the fluorescence intensity, spectrum area and the concentration of CDOM. When the concentration of CDOM is low (less than 75 mg x L(-1)), there is a better linear relationship (r2 > 0.98) between the fluorescence intensity, the spectrum area and the concentration of CDOM. Meanwhile good linear relations were found between the fluorescence intensity and spectrum area, which showed the same changeable trend of the fluorescence intensity and spectrum area with the concentration change of CDOM. A method was established to quantify the concentration of CDOM in water from different source using the linear relationship between the spectrum area and the concentration. It suits the complicated constituent analysis of CDOM and could really and accurately show the concentration of CDOM in natural water.

Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

PubMed

Zerjatke, Thomas; Gak, Igor A; Kirova, Dilyana; Fuhrmann, Markus; Daniel, Katrin; Gonciarz, Magdalena; Müller, Doris; Glauche, Ingmar; Mansfeld, Jörg

2017-05-30

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Comparison of light-induced and laser-induced fluorescence methods for the detection and quantification of enamel demineralization

NASA Astrophysics Data System (ADS)

Ando, Masatoshi; Analoui, Mostafa; Schemehorn, Bruce R.; Stookey, George K.

1999-05-01

The Quantitative Laser-Induced Fluorescence (QLF) technique has been sued for diagnosis of early caries in permanent teeth (PT). The objective of this study was to determine the caries quantification ability of QLF in deciduous teeth (DT). Sixty sound teeth, thirty DT and thirty PT, were used. All teeth were cleaned to remove debris and equally divided into three groups. Lesions were created in small windows (0.8x2.0 mm2) on buccal or labial surface for 48, 72, and 96 hr. Lesion images were made with a 488 nm argon laser (QLF I) and then with a 370 +/- 80 nm violet-blue light (QLF II). Both images were analyzed to determine the mean percent change in fluorescence radiance (ΔF). A center section from the lesions was taken for analysis with microradiography. The lesion depth and loss of mineral content were determined. The correlations between ΔF and lesion depth as well as ΔZ in DT were 0.76 and 0.84 with QLF I, 0.81 and 0.88 with QLF II, respectively. It can be concluded the ability of QLF to quantify white-spots in DT is better than in PT.

Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

PubMed

Sahore, Vishal; Sonker, Mukul; Nielsen, Anna V; Knob, Radim; Kumar, Suresh; Woolley, Adam T

2018-01-01

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (μCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a μCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined μCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and μCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

Protein Multiplexed Immunoassay Analysis with R.

PubMed

Breen, Edmond J

2017-01-01

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex ® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex ® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex ® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays.

Electroadsorption-assisted direct determination of trace arsenic without interference using transmission X-ray fluorescence spectroscopy.

PubMed

Jiang, Tian-Jia; Guo, Zheng; Liu, Jin-Huai; Huang, Xing-Jiu

2015-08-18

An analytical technique based on electroadsorption and transmission X-ray fluorescence (XRF) for the quantitative determination of arsenic in aqueous solution with ppb-level limits of detection (LOD) is proposed. The approach uses electroadsorption to enhance the sensitivity and LOD of the arsenic XRF response. Amine-functionalized carbonaceous microspheres (NH2-CMSs) are found to be the ideal materials for both the quantitative adsorption of arsenic and XRF analysis due to the basic amine sites on the surface and their noninterference in the XRF spectrum. In electroadsorptive X-ray fluorescence (EA-XRF), arsenic is preconcentrated by a conventional three-electrode system with a positive electricity field around the adsorbents. Then, the quantification of arsenic on the adsorbents is achieved using XRF. The electroadsorption preconcentration can realize the fast transfer of arsenic from the solution to the adsorbents and improve the LOD of conventional XRF compared with directly determining arsenic solution by XRF alone. The sensitivity of 0.09 cnt ppb(-1) is obtained without the interferences from coexisted metal ions in the determination of arsenic, and the LOD is found to be 7 ppb, which is lower than the arsenic guideline value of 10 ppb given by the World Health Organization (WHO). These results demonstrated that XRF coupled with electroadsorption was able to determine trace arsenic in real water sample.

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

PubMed

Fujikawa, Takahisa; Hirose, Tetsuro; Fujii, Hideaki; Oe, Shoshiro; Yasuchika, Kentaro; Azuma, Hisaya; Yamaoka, Yoshio

2003-08-01

Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Imaging of size-dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles.

PubMed

Awaad, Aziz; Nakamura, Michihiro; Ishimura, Kazunori

2012-07-01

We investigated size-dependent uptake of fluorescent thiol-organosilica particles by Peyer's patches (PPs). We performed an oral single-particle administration (95, 130, 200, 340, 695 and 1050 nm) and a simultaneous dual-particle administration using 2 kinds of particles. Histological imaging and quantitative analysis revealed that particles taken up by the PP subepithelial dome were size dependent, and there was an optimal size range for higher uptake. Quantitative analysis of simultaneous dual-particle administration revealed that the percentage of fluorescence areas for 95, 130, 200, 340, 695 and 1050 nm with respect to 110 nm area was 124.0, 89.1, 73.8, 20.2, 9.2 and 0.5%, respectively. Additionally, imaging using fluorescent thiol-organosilica particles could detect 2 novel pathways through mouse PP epithelium: the transcellular pathway and the paracellular pathway. The uptake of nanoparticles based on an optimal size range and 2 novel pathways could indicate a new approach for vaccine delivery and nanomedicine development. Studying various sizes of fluorescent organosilica particles and their uptake in Peyer's patches, this team of authors determined the optimal size range of administration. Two novel pathways through mouse Peyer's patch epithelium were detected, i.e., the transcellular pathway and the paracellular pathway. This observation may have important applications in future vaccine delivery and nano-drug delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

PubMed Central

Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

2016-01-01

Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

PubMed Central

Rich, Ryan M.; Stankowska, Dorota L.; Maliwal, Badri P.; Sørensen, Thomas Just; Laursen, Bo W.; Krishnamoorthy, Raghu R.; Gryczynski, Zygmunt; Borejdo, Julian

2013-01-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background. PMID:23254457

Simple transmission Raman measurements using a single multivariate model for analysis of pharmaceutical samples contained in capsules of different colors.

PubMed

Lee, Yeojin; Kim, Jaejin; Lee, Sanguk; Woo, Young-Ah; Chung, Hoeil

2012-01-30

Direct transmission Raman measurements for analysis of pharmaceuticals in capsules are advantageous since they can be used to determine active pharmaceutical ingredient (API) concentrations in a non-destructive manner and with much less fluorescence background interference from the capsules themselves compared to conventional back-scattering measurements. If a single calibration model such as developed from spectra simply collected in glass vials could be used to determine API concentrations of samples contained in capsules of different colors rather than constructing individual models for each capsule color, the utility of transmission measurements would be further enhanced. To evaluate the feasibility, transmission Raman spectra of binary mixtures of ambroxol and lactose were collected in a glass vial and a partial least squares (PLS) model for the determination of ambroxol concentration was developed. Then, the model was directly applied to determine ambroxol concentrations of samples contained in capsules of 4 different colors (blue, green, white and yellow). Although the prediction performance was slightly degraded when the samples were placed in blue or green capsules, due to the presence of weak fluorescence, accurate determination of ambroxol was generally achieved in all cases. The prediction accuracy was also investigated when the thickness of the capsule was varied. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of energy response for photon-counting detectors using x-ray fluorescence

PubMed Central

Ding, Huanjun; Cho, Hyo-Min; Barber, William C.; Iwanczyk, Jan S.; Molloi, Sabee

2014-01-01

Purpose: To investigate the feasibility of characterizing a Si strip photon-counting detector using x-ray fluorescence. Methods: X-ray fluorescence was generated by using a pencil beam from a tungsten anode x-ray tube with 2 mm Al filtration. Spectra were acquired at 90° from the primary beam direction with an energy-resolved photon-counting detector based on an edge illuminated Si strip detector. The distances from the source to target and the target to detector were approximately 19 and 11 cm, respectively. Four different materials, containing silver (Ag), iodine (I), barium (Ba), and gadolinium (Gd), were placed in small plastic containers with a diameter of approximately 0.7 cm for x-ray fluorescence measurements. Linear regression analysis was performed to derive the gain and offset values for the correlation between the measured fluorescence peak center and the known fluorescence energies. The energy resolutions and charge-sharing fractions were also obtained from analytical fittings of the recorded fluorescence spectra. An analytical model, which employed four parameters that can be determined from the fluorescence calibration, was used to estimate the detector response function. Results: Strong fluorescence signals of all four target materials were recorded with the investigated geometry for the Si strip detector. The average gain and offset of all pixels for detector energy calibration were determined to be 6.95 mV/keV and −66.33 mV, respectively. The detector’s energy resolution remained at approximately 2.7 keV for low energies, and increased slightly at 45 keV. The average charge-sharing fraction was estimated to be 36% within the investigated energy range of 20–45 keV. The simulated detector output based on the proposed response function agreed well with the experimental measurement. Conclusions: The performance of a spectral imaging system using energy-resolved photon-counting detectors is very dependent on the energy calibration of the detector. The proposed x-ray fluorescence technique offers an accurate and efficient way to calibrate the energy response of a photon-counting detector. PMID:25471962

Variation in the Humification Degree of Dissolved Organic Matter from Cattle Manure during Composting as Analyzed by Ultraviolet-Visible and Fluorescence Spectroscopy.

PubMed

Chen, Yukun; Jiang, Zhao; Zhang, Xiuyuan; Cao, Bo; Yang, Fan; Wang, Ziyi; Zhang, Ying

2017-11-01

This study investigated the degree of humification of dissolved organic matter (DOM) during different periods of cattle manure composting using ultraviolet-visible (UV-vis) and fluorescence spectroscopy (emission, synchronous scan, and excitation-emission matrix) and determined which method is more suitable for analysis of the humification degree of DOM. Two composting piles were prepared by mixing manure and corn straw. One pile (Pile A [PA]) contained inoculated exogenous composite agents at a ratio of 2% (v/v), and a pile without the addition of inoculants (PNA) served as the control treatment. The results showed that ultraviolet integrated absorption intensities in the range of 226 to 400 nm and 260 to 280 nm and specific ultraviolet absorbances at 254 and 280 nm of both PA and PNA gradually increased with composting time. Based on the fluorescence regional integration analysis and parallel factor analysis, the humic-like substances became the main components of the DOM after composting. Our study demonstrated that the humification degree of DOM was enhanced during composting and that the inoculation composite agent was beneficial for the humification of DOM at the mesophilic and thermophilic phases of the composting process. Moreover, the results of correlation analysis and principal component analysis demonstrated that the fluorescence spectral parameters evaluated the humification degree of DOM during the whole cattle manure composting process better than the UV-vis spectral parameters. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Small-Volume Injections: Evaluation of Volume Administration Deviation From Intended Injection Volumes.

PubMed

Muffly, Matthew K; Chen, Michael I; Claure, Rebecca E; Drover, David R; Efron, Bradley; Fitch, William L; Hammer, Gregory B

2017-10-01

In the perioperative period, anesthesiologists and postanesthesia care unit (PACU) nurses routinely prepare and administer small-volume IV injections, yet the accuracy of delivered medication volumes in this setting has not been described. In this ex vivo study, we sought to characterize the degree to which small-volume injections (≤0.5 mL) deviated from the intended injection volumes among a group of pediatric anesthesiologists and pediatric postanesthesia care unit (PACU) nurses. We hypothesized that as the intended injection volumes decreased, the deviation from those intended injection volumes would increase. Ten attending pediatric anesthesiologists and 10 pediatric PACU nurses each performed a series of 10 injections into a simulated patient IV setup. Practitioners used separate 1-mL tuberculin syringes with removable 18-gauge needles (Becton-Dickinson & Company, Franklin Lakes, NJ) to aspirate 5 different volumes (0.025, 0.05, 0.1, 0.25, and 0.5 mL) of 0.25 mM Lucifer Yellow (LY) fluorescent dye constituted in saline (Sigma Aldrich, St. Louis, MO) from a rubber-stoppered vial. Each participant then injected the specified volume of LY fluorescent dye via a 3-way stopcock into IV tubing with free-flowing 0.9% sodium chloride (10 mL/min). The injected volume of LY fluorescent dye and 0.9% sodium chloride then drained into a collection vial for laboratory analysis. Microplate fluorescence wavelength detection (Infinite M1000; Tecan, Mannedorf, Switzerland) was used to measure the fluorescence of the collected fluid. Administered injection volumes were calculated based on the fluorescence of the collected fluid using a calibration curve of known LY volumes and associated fluorescence.To determine whether deviation of the administered volumes from the intended injection volumes increased at lower injection volumes, we compared the proportional injection volume error (loge [administered volume/intended volume]) for each of the 5 injection volumes using a linear regression model. Analysis of variance was used to determine whether the absolute log proportional error differed by the intended injection volume. Interindividual and intraindividual deviation from the intended injection volume was also characterized. As the intended injection volumes decreased, the absolute log proportional injection volume error increased (analysis of variance, P < .0018). The exploratory analysis revealed no significant difference in the standard deviations of the log proportional errors for injection volumes between physicians and pediatric PACU nurses; however, the difference in absolute bias was significantly higher for nurses with a 2-sided significance of P = .03. Clinically significant dose variation occurs when injecting volumes ≤0.5 mL. Administering small volumes of medications may result in unintended medication administration errors.

Tracking of fluorescence nanoparticles with nanometre resolution in a biological system: assessing local viscosity and microrheology.

PubMed

Marki, Alex; Ermilov, Eugeny; Zakrzewicz, Andreas; Koller, Akos; Secomb, Timothy W; Pries, Axel R

2014-04-01

The aim of the study was to establish a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometre precision in a standard fluorescence microscope using a point spread function (PSF) approach, and to evaluate validity and precision for different analysis methods and optical conditions with particular application to microcirculatory flow dynamics and cell biology. Images of fluorescent particles were obtained with a standard fluorescence microscope equipped with a piezo positioner for the objective. Whole pattern (WP) comparison with a PSF recorded for the specific set-up and measurement of the outermost ring radius (ORR) were used for analysis. Images of fluorescent particles were recorded over a large range (about 7μm) of vertical positions, with and without distortion by overlapping particles as well as in the presence of cultured endothelial cells. For a vertical range of 6.5μm the standard deviation (SD) from the predicted value, indicating validity, was 9.3/8.7 nm (WP/ORR) in the vertical and 8.2/11.7 nm in the horizontal direction. The precision, determined by repeated measurements, was 5.1/3.8 nm in the vertical and 2.9/3.7 nm in the horizontal direction. WP was more robust with respect to underexposure or overlapping images. On the surface of cultured endothelial cells, a layer with 2.5 times increased viscosity and a thickness of about 0.8μm was detected. With a validity in the range of 10 nm and a precision down to about 3-5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle localizations, including the assessment of endothelial cell microenvironment.

Analysis of elemental concentration censored distributions in breast malignant and breast benign neoplasm tissues

NASA Astrophysics Data System (ADS)

Kubala-Kukuś, A.; Banaś, D.; Braziewicz, J.; Góźdź, S.; Majewska, U.; Pajek, M.

2007-07-01

The total reflection X-ray fluorescence method was applied to study the trace element concentrations in human breast malignant and breast benign neoplasm tissues taken from the women who were patients of Holycross Cancer Centre in Kielce (Poland). These investigations were mainly focused on the development of new possibilities of cancer diagnosis and therapy monitoring. This systematic comparative study was based on relatively large (˜ 100) population studied, namely 26 samples of breast malignant and 68 samples of breast benign neoplasm tissues. The concentrations, being in the range from a few ppb to 0.1%, were determined for thirteen elements (from P to Pb). The results were carefully analysed to investigate the concentration distribution of trace elements in the studied samples. The measurements of concentration of trace elements by total reflection X-ray fluorescence were limited, however, by the detection limit of the method. It was observed that for more than 50% of elements determined, the concentrations were not measured in all samples. These incomplete measurements were treated within the statistical concept called left-random censoring and for the estimation of the mean value and median of censored concentration distributions, the Kaplan-Meier estimator was used. For comparison of concentrations in two populations, the log-rank test was applied, which allows to compare the censored total reflection X-ray fluorescence data. Found statistically significant differences are discussed in more details. It is noted that described data analysis procedures should be the standard tool to analyze the censored concentrations of trace elements analysed by X-ray fluorescence methods.

Establishment of integrated protocols for automated high throughput kinetic chlorophyll fluorescence analyses.

PubMed

Tschiersch, Henning; Junker, Astrid; Meyer, Rhonda C; Altmann, Thomas

2017-01-01

Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (Φ PSII ). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of Φ PSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

Ratiometric fluorescent detection of chromium(VI) in real samples based on dual emissive carbon dots.

PubMed

Ma, Yunxia; Chen, Yonglei; Liu, Juanjuan; Han, Yangxia; Ma, Sudai; Chen, Xingguo

2018-08-01

As we know, hexavalent chromium (Cr(VI)) was usually used as an additive to improve the color fastness during the printing and dyeing process, and thus posing tremendous threat to our health and living quality. In this work, the dual emissive carbon dots (DECDs) were synthesized through hydrothermal treatment of m-aminophenol and oxalic acid. The obtained DECDs not only exhibited dual emission fluorescence peaks (430 nm, 510 nm) under the single excitation wavelength of 380 nm, but also possessed good water solubility and excellent fluorescence stability. A ratiometric fluorescent method for the determination of Cr(VI) was developed using the DECDs as a probe. Under the optimal conditions, a linear range was obtained from 2 to 300 μM with a limit of detection of 0.4 μM. Furthermore, the proposed ratiometric fluorescent method was applied to the analysis of Cr(VI) in textile, steel, industrial wastewater and chromium residue samples with satisfactory recoveries (88.4-106.8%). Copyright © 2018 Elsevier B.V. All rights reserved.

The Effects of Heteroatoms Si and S on Tuning the Optical Properties of Rhodamine- and Fluorescein-Based Fluorescence Probes: A Theoretical Analysis.

PubMed

Zhou, Panwang; Ning, Cai; Alsaedi, Ahmed; Han, Keli

2016-10-05

The effects of the incorporated heteroatoms Si and S on tuning the optical properties of rhodamine- and fluorescein-based fluorescence probes is investigated using DFT and time-dependent DFT with four different functionals. As previously proposed, the large redshift (90 nm) produced by a Si atom in both the absorption and emission spectra can be attributed to the σ*-π* conjugation between the σ* orbital of the Si atom and the π* orbital of the adjacent carbon atoms. However, the presence of a Si atom does not alter the fluorescence quenching mechanism of the nonfluorescent forms of the investigated compounds. For the first time, these theoretical results indicate that the n orbital of the S atom plays an important role in determining the optical properties of the nonfluorescent form of rhodamine-based fluorescence probes. It alters the fluorescence quenching mechanism by lowering the energy of the dark nπ* state, which is due to breakage of the C10-S52 bond upon photoexcitation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Green synthesis of carbon dots originated from Lycii Fructus for effective fluorescent sensing of ferric ion and multicolor cell imaging.

PubMed

Sun, Xiaohan; He, Jiang; Yang, Shenghong; Zheng, Mingda; Wang, Yingying; Ma, Shuang; Zheng, Haipeng

2017-10-01

Green, economical and effective method was developed for synthesis of fluorescent carbon dots (CDs), using one-pot hydrothermal treatment of Lycii Fructus. Optical and structural properties of the CDs have been extensively studied by UV-visible and fluorescence spectroscopic, x-ray diffraction (XRD) techniques, transmission electron microscopy (TEM) and high resolution TEM (HRTEM). Surface functionality and composition of CDs has been illustrated by Fourier transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS) spectra and elemental analysis. The fabricated CDs possess stable fluorescent properties. The fluorescent quantum yield of the CDs can reach 17.2%. The prepared CDs emitted a broad fluorescence between 415 and 545nm and their fluorescence was tuned by changing excitation wavelength. Meanwhile, the fluorescence intensity of the CDs could be significantly quenched by Fe 3+ (turn-off). The CDs exhibit captivating sensitivity and selectivity toward Fe 3+ with a linear range from 0 to 30μM and a detection limit of 21nM. The prepared CDs were successfully applied to the determination of Fe 3+ in the urine samples, the water samples from the from the Yellow River and living HeLa (Henrietta Lacks) cells. Moreover, the low-toxicity and excellent biocompatibility of the CDs were evaluated through MTT assay on HeLa cells. The CDs were also employed as fluorescent probes for multicolor imaging of HeLa cells successfully. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous quantitative analysis of arsenic, bismuth, selenium, and tellurium in soil samples using multi-channel hydride-generation atomic fluorescence spectrometry.

PubMed

Wang, Fang; Zhang, Gai

2011-03-01

The basic principles and the application of hydride-generation multi-channel atomic fluorescence spectrometry (HG-MC-AFS) in soil analysis are described. It is generally understood that only one or two elements can be simultaneously detected by commonly used one- or two-channel HG-AFS. In this work, a new sample-sensitive and effective method for the analysis of arsenic, bismuth, tellurium, and selenium in soil samples by simultaneous detection using HG-MC-AFS was developed. The method detection limits for arsenic, bismuth, tellurium, and selenium are 0.19 μg/g, 0.10 μg/g, 0.11 μg/g, and 0.08 μg/g, respectively. This method was successfully applied to the simultaneous determination of arsenic, bismuth, tellurium, and selenium in soil samples.

Colorimetric and fluorometric determination of uric acid based on the use of nitrogen-doped carbon quantum dots and silver triangular nanoprisms.

PubMed

Wang, Yanying; Yang, Yan; Liu, Wei; Ding, Fang; Zhao, Qingbiao; Zou, Ping; Wang, Xianxiang; Rao, Hanbing

2018-05-04

A dual-read detection system is described for non-enzymatic and non-aggregation based analysis of uric acid (UA). Silver triangular nanoprisms (AgTNPs) were used as colorimetric probes, while the reduction in the fluorescence of nitrogen-doped carbon quantum dots (N-CQDs) served as the fluorometric readout. The absorption band of the AgTNPs overlaps the emission band of N-CQDs (with a peak at 440 nm). Therefore, fluorescence is reduced owing to an inner filter effect. The AgTNPs are etched if exposed to H 2 O 2 , and round nanodiscs are formed. In the presence of UA, etching of the AgTNPs is suppressed because the facets of the AgTNPs are coated with UA. The absorbance, best measured at 683 nm, increases with the concentration of the pre-added UA. The colorimetric assay works in the 0.1-45 μM UA concentration range, and the fluorometric assay between 1 and 42 μM of UA. The respective detection limits are 50 and 200 nM, respectively. The probe can be used for direct visualization of UA. The method was successfully applied to the determination of UA in urine samples. Graphical abstract The fluorescence of nitrogen-doped carbon quantum dots (N-CQDs) is quenched by AgTNPs (silver triangular nanoprisms). As the AgTNPs are etched by H 2 O 2 , fluorescence recovers in the system after H 2 O 2 is added, and also undergoes a color change. Uric acid (UA) protects the AgTNPs from etching because the facets of the AgTNPs are coated with UA. The fluorescence of N-CQDs decreases. Thus, a dual-read probe is developed for determination of UA.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

PubMed

Kramer, S; Blaschke, G

2001-02-10

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

Comparison of the effect of UV laser radiation and of a radiomimetic substance on chromatin

NASA Astrophysics Data System (ADS)

Radulescu, Irina; Radu, Liliana; Serbanescu, Ruxandra; Nelea, V. D.; Martin, C.; Mihailescu, Ion N.

1998-07-01

The damages of the complex of deoxyribonucleic acid (DNA) and proteins from chromatin, produced by the UV laser radiation and/or by treatment with a radiomimetic substance, bleomycin, were compared. The laser radiation and bleomycin effects on chromatin structure were determined by the static and dynamic fluorimetry of chromatin complexes with the DNA specific ligand-- proflavine and by the analysis of tryptophan chromatin intrinsic fluorescence. Time resolved spectroscopy is a sensitive technique which allows to determine the excited state lifetimes of chromatin--proflavine complexes. Also, the percentage contributions to the fluorescence of proflavine, bound and unbound to chromatin DNA, were evaluated. The damages produced by the UV laser radiation on chromatin are similar with those of radiomimetic substance action and consists in DNA and proteins destruction. The DNA damage degree has been determined. The obtained results may constitute some indications in the laser utilization in radiochimiotherapy.

Determination of minor and trace elements concentration in kidney stones using elemental analysis techniques

NASA Astrophysics Data System (ADS)

Srivastava, Anjali

The determination of accurate material composition of a kidney stone is crucial for understanding the formation of the kidney stone as well as for preventive therapeutic strategies. Radiations probing instrumental activation analysis techniques are excellent tools for identification of involved materials present in the kidney stone. The X-ray fluorescence (XRF) and neutron activation analysis (NAA) experiments were performed and different kidney stones were analyzed. The interactions of X-ray photons and neutrons with matter are complementary in nature, resulting in distinctly different materials detection. This is the first approach to utilize combined X-ray fluorescence and neutron activation analysis for a comprehensive analysis of the kideny stones. Presently, experimental studies in conjunction with analytical techniques were used to determine the exact composition of the kidney stone. The use of open source program Python Multi-Channel Analyzer was utilized to unfold the XRF spectrum. A new type of experimental set-up was developed and utilized for XRF and NAA analysis of the kidney stone. To verify the experimental results with analytical calculation, several sets of kidney stones were analyzed using XRF and NAA technique. The elements which were identified from XRF technique are Br, Cu, Ga, Ge, Mo, Nb, Ni, Rb, Se, Sr, Y, Zr. And, by using Neutron Activation Analysis (NAA) are Au, Br, Ca, Er, Hg, I, K, Na, Pm, Sb, Sc, Sm, Tb, Yb, Zn. This thesis presents a new approach for exact detection of accurate material composition of kidney stone materials using XRF and NAA instrumental activation analysis techniques.

Determination of adenosine disodium triphosphate (ATP) using oxytetracycline-Eu 3+ as a fluorescence probe by spectrofluorimetry

NASA Astrophysics Data System (ADS)

Hou, Faju; Miao, Yanhong; Jiang, Chongqiu

2005-10-01

A new spectrofluorimetric method was developed for determination of adenosine disodium triphosphate (ATP). We studied the interactions between oxytetracycline (OTC)-Eu 3+ complex and adenosine disodium triphosphate (ATP) by using UV-vis absorption and fluorescence spectra. Using oxytetracycline (OTC)-Eu 3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the OTC-Eu 3+ complex at λ = 612 nm and the enhanced fluorescence intensity of Eu 3+ ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP are 8.00 × 10 -8-1.50 × 10 -6 mol L -1 with detection limits of 2.67 × 10 -9 mol L -1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in samples. The mechanism of fluorescence enhancement between oxytetracycline (OTC)-Eu 3+ complex and ATP was also studied.

Analysis of the photophysical properties of zearalenone using density functional theory

USDA-ARS?s Scientific Manuscript database

The intrinsic photophysical properties of the resorcylic acid moiety of zearalenone offer a convenient label free method to determine zearalenone levels in contaminated agricultural products. Density functional theory and steady-state fluorescence methods were applied to investigate the role of stru...

Rapid analysis of 3,4-methylenedioxymethamphetamine: a comparison of nonaqueous capillary electrophoresis/fluorescence detection with GC/MS.

PubMed

Fang, Ching; Chung, Yu-Lin; Liu, Ju-Tsung; Lin, Cheng-Huang

2002-02-18

Because of the increasing use of 3,4-methylenedioxymethamphetamine (3,4-MDMA), a rapid and sensitive analytical technique is required for its detection and determination. Using nonaqueous capillary electrophoresis/fluorescence spectroscopy (NACE/FS) detection, it is possible to determine this drug at the level 0.5 ppm without any pre-treatment in less than 5 min. After liquid-liquid extraction, the sample can be condensed and a detection limit of 3,4-MDMA in urine of 50 ppb (S/N = 3) can be achieved. The precision of the method was evaluated by measuring the repeatability and intermediate precision of migration time and the corrected peak height by comparison with a 3,4-MDMA-D5 internal standard. With the conventional GC/MS method, it is necessary to derivatize the 3,4-MDMA before injection and the GC migration time also is in excess of 20 min. Therefore, NACE/FS represents a good complementary method to GC/MS for use in forensic analysis.

Spectrofluorimetric analysis of famotidine in pharmaceutical preparations and biological fluids by derivatization with benzoin.

PubMed

Alamgir, Malik; Khuhawar, Muhammad Yar; Memon, Saima Q; Hayat, Amir; Zounr, Rizwan Ali

2015-01-05

A sensitive and simple spectrofluorimetric method has been developed for the analysis of famotidine, from pharmaceutical preparations and biological fluids after derivatization with benzoin. The reaction was carried out in alkaline medium with measurement of fluorescence intensity at 446 nm with excitation wavelength at 286 nm. Linear calibration was obtained with 0.5-15 μg/ml with coefficient of determination (r(2)) 0.997. The factors affecting the fluorescence intensity were optimized. The pharmaceutical additives and amino acid did not interfere in the determination. The mean percentage recovery (n=4) calculated by standard addition from pharmaceutical preparation was 94.8-98.2% with relative standard deviation (RSD) 1.56-3.34% and recovery from deproteinized spiked serum and urine of healthy volunteers was 98.6-98.9% and 98.0-98.4% with RSD 0.34-0.84% and 0.29-0.87% respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectral study and protein labeling of inclusion complex between dye and calixarene sulfonate.

PubMed

Fei, Xuening; Zhang, Yong; Zhu, Sen; Liu, Lijuan; Yu, Lu

2013-05-01

The host-guest inclusion complex of calix[6]arene sulfonate (SCA6) with thiazole orange (TO) formed in aqueous solution was studied. Absorption and fluorescence techniques were used for the analysis of this inclusion complex. The addition of calixarene sulfonate leads to a decrease in both absorption and fluorescence intensity of the dye, indicating that the inclusion complex was formed. Simultaneously, the inclusion phenomenon of another cyanine dye, Cy3, with calixarene sulfonate was investigated. The stability constant of the two complexes was determined, and the results were compared. The water solubility of TO dye was increased in the presence of calixarene sulfonate, and further protein labeling experiments suggested that this TO-SCA6 complex can act as a fluorescent probe for labeling of biomolecules.

Spectroscopic analysis of autofluorescence distribution in digestive organ for unstained metabolism-based tumor detection

NASA Astrophysics Data System (ADS)

Arimoto, Hidenobu; Iwata, Atsushi; Kagawa, Keiichiro; Sanomura, Yoji; Yoshida, Shigeto; Kawahito, Shoji; Tanaka, Shinji

2017-02-01

Auto fluorescence distribution of coenzymes NADH and FAD is investigated for the unstained tumor detection using an [?] originally designed confocal spectroscope. The tumor region in digestive organ can be determined by evaluating the redox index which is defined as the raio of NADH and FAD concentration. However, the redox index is largely influenced by the presence of collagen in the submucosal layer because its auto fluorescence spectrum overlaps considerably with that of NADH. Therefore, it is necessary to know in advance the distribution of NADH, FAD, and collagen in the mucosal layer. The purpose of our study is to investigate the vertical distribution of the redox index in tissue using depth-sensitive auto fluorescence spectroscopy. The experimental procedure and the results are presented.

Evaluation of a Portable Microchip Electrophoresis Fluorescence Detection System for the Analysis of Amino Acid Neurotransmitters in Brain Dialysis Samples

PubMed Central

OBORNY, Nathan J.; COSTA, Elton E. Melo; SUNTORNSUK, Leena; ABREU, Fabiane C.; LUNTE, Susan M.

2016-01-01

A portable fluorescence detection system for use with microchip electrophoresis was developed and compared to a benchtop system. Using this system, six neuroactive amines commonly found in brain dialysate—arginine, citrulline, taurine, histamine, glutamate, and aspartate—were derivatized offline with naphthalene-2,3-dicarboxaldehyde/cyanide, separated electrophoretically, and detected by fluorescence. Limits of detection for the analytes of interest were 50nM – 250nM for the benchtop system and 250 nM – 1.3 μM for the portable system, both of which were adequate for analyte determination in brain microdialysis samples. The portable system was then demonstrated for the detection of the same six amines in a rat brain microdialysis sample. PMID:26753703

Tunicamycin Enhances Neuroinvasion and Pathogenicity in Mice with Venezuelan Equine Encephalitis Virus

DTIC Science & Technology

2003-01-01

H. Analytical Tests 35 1. Virus Titrations 35 2. RNA Analysis 37 3. Determination of TNF-a and Total Nitrite Levels 37...fluorescent microscope. H. Analytical Tests 1. Virus Titrations For determination of virus titers, brain samples were homogenized in Eppendorf tubes...tissues were mounted on silane-coated slides (Sigma Diagnostics , St. Louis, MO) and labeled for VEE virus antigen by immunohistochemistry. Additional

Determination of emamectin residues in the tissues of Atlantic salmon (Salmo salar L.) using HPLC with fluorescence detection.

PubMed

Kim-Kang, H; Crouch, L S; Bova, A; Robinson, R A; Wu, J

2001-11-01

An accurate, reliable, and reproducible assay for the determination of residual concentrations of emamectin B(1a) in muscle, skin, and intact muscle/skin in natural proportions from Atlantic salmon treated with SCH 58854 (emamectin benzoate) is described. The determinative method was developed and validated using fortified control tissues at five levels over a range of 50-800 ng/g as well as tissues containing incurred levels in the same range. Incurred tissues were obtained from a metabolism study of [(3)H]emamectin benzoate in Atlantic salmon. The assay employs processing of a tissue ethyl acetate extract on a propylsulfonic acid solid phase extraction cartridge, followed by derivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. Following separation using reversed phase HPLC, the amount of derivatized emamectin B(1a) is determined by fluorescence detection. The theoretical limits of detection were determined from the analysis of control tissue matrices to be 2.6, 3.3, and 3.8 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. Likewise, the theoretical limits of quantitation (LOQ) were determined to be 6.9, 8.1, and 9.5 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. The lowest fortification level used for method validation was 50 ng/g, which served as the effective LOQ for the method. The overall percent recoveries (+/-% CV) were 94.4 +/- 6.89% (n = 25) for muscle, 88.4 +/- 5.35% (n = 25) for skin, and 88.0 +/- 3.73% for intact muscle/skin (n = 25). Accuracy, precision, linearity, selectivity, and ruggedness were demonstrated. The structure of the final fluorescent derivative of emamectin B(1a) free base was identified by ESI(+)/LC-MS. The frozen storage stability of [(3)H]emamectin B(1a) in tissues with incurred residues was demonstrated for approximately 15 months by radiometric analysis and for an additional approximately 13 months by fluorometric analysis for a total of approximately 28 months.

Utility of gold nanoparticles in luminescence determination of trovafloxacin: comparison of chemiluminescence and fluorescence detection.

PubMed

Alarfaj, Nawal A; El-Tohamy, Maha F

2015-12-01

Two novel sensitive sequential injection chemiluminescence analysis and fluorescence methods for trovafloxacin mesylate detection have been developed. The methods were based on the enhancement effect of gold nanoparticles on luminol-ferricyanide-trovafloxacin and europium(III)-trovafloxacin complex systems. The optimum conditions for both detection methods were investigated. The chemiluminescence signal was emitted due to the enhanced effect of gold nanoparticles on the reaction of luminol-ferricyanide-trovafloxacin in an alkaline medium. The response was linear over a concentration range of 1.0 × 10(-9) to 1.0 × 10(-2) mol/L (%RSD = 1.3), (n = 9, r = 0.9991) with a detection limit of 1.7 × 10(-10) mol/L (S/N = 3). The weak fluorescence intensity signal of the oxidation complex of europium(III)-trovafloxacin was strongly enhanced by gold nanoparticles and detected at λex = 330 and λem = 540 nm. Fluorescence detection enabled the determination of trovafloxacin mesylate over a linear range of 1.0 × 10(-8) to 1.0 × 10(-3) mol/L (%RSD = 1.2), (n = 6, r = 0.9993) with a detection limit of 3.3 × 10(-9) mol/L. The proposed methods were successfully applied to the determination of the studied drug in its bulk form and in pharmaceutical preparations. The results were treated statistically and compared with those obtained from other reported methods. Copyright © 2015 John Wiley & Sons, Ltd.

Validated spectrofluorimetric methods for the determination of apixaban and tirofiban hydrochloride in pharmaceutical formulations.

PubMed

El-Bagary, Ramzia I; Elkady, Ehab F; Farid, Naira A; Youssef, Nadia F

2017-03-05

Apixaban and Tirofiban Hydrochloride are low molecular weight anticoagulants. The two drugs exhibit native fluorescence that allow the development of simple and valid spectrofluorimetric methods for the determination of Apixaban at λ ex/λ em=284/450nm and tirofiban HCl at λ ex/λ em=227/300nm in aqueous media. Different experimental parameters affecting fluorescence intensities were carefully studied and optimized. The fluorescence intensity-concentration plots were linear over the ranges of 0.2-6μgml -1 for apixaban and 0.2-5μgml -1 for tirofiban HCl. The limits of detection were 0.017 and 0.019μgml -1 and quantification limits were 0.057 and 0.066μgml -1 for apixaban and tirofiban HCl, respectively. The fluorescence quantum yield of apixaban and tirofiban were calculated with values of 0.43 and 0.49. Method validation was evaluated for linearity, specificity, accuracy, precision and robustness as per ICH guidelines. The proposed spectrofluorimetric methods were successfully applied for the determination of apixaban in Eliquis tablets and tirofiban HCl in Aggrastat intravenous infusion. Tolerance ratio was tested to study the effect of foreign interferences from dosage forms excipients. Using Student's t and F tests, revealed no statistically difference between the developed spectrofluorimetric methods and the comparison methods regarding the accuracy and precision, so can be contributed to the analysis of apixaban and tirofiban HCl in QC laboratories as an alternative method. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe.

PubMed

Xu, Yujuan; Zhang, Peng; Wang, Zhen; Lv, Shaoping; Ding, Caifeng

2018-02-27

Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity. Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

PubMed Central

Valades Cruz, Cesar Augusto; Shaban, Haitham Ahmed; Kress, Alla; Bertaux, Nicolas; Monneret, Serge; Mavrakis, Manos; Savatier, Julien; Brasselet, Sophie

2016-01-01

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells. PMID:26831082

Correlation of tryptophan fluorescence intensity decay parameters with sup 1 H NMR-determined rotamer conformations: (tryptophan sup 2 )oxytocin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ross, J.B.A.; Schwartz, G.P.; Laws, W.R.

1992-02-18

While the fluorescence decay kinetics of tyrosine model compounds can be explained in terms of heterogeneity derived from the three ground-state {chi}{sup 1} rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from {chi}{sup 2} rotations. In this paper, the time-resolved and steady-state fluorescence properties of (tryptophan{sup 2})oxytocin at pH 3 are presented and compared with {sup 1}H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength-independent decay constants, only three exponential terms aremore » required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the {sup 1}H NMR-determined {chi}{sup 1} rotamer populations of the indole side chain. {sup 15}N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the {chi}{sup 1} rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two {chi}{sup 2} populations.« less

Comparison of fluorescence-based methods to determine nanoparticle uptake by phagocytes and non-phagocytic cells in vitro

PubMed Central

Claudia, Meindl; Kristin, Öhlinger; Jennifer, Ober; Eva, Roblegg; Eleonore, Fröhlich

2017-01-01

At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence–labeled polystyrene particles and to study the respective method’s suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non- or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and non-phagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements. PMID:28065592

Chromophoric and fluorescent dissolved organic matter in and above the oxygen minimum zone off Peru

NASA Astrophysics Data System (ADS)

Loginova, A. N.; Thomsen, S.; Engel, A.

2016-11-01

As a result of nutrient upwelling, the Peruvian coastal system is one of the most productive regions in the ocean. Sluggish ventilation of intermediate waters, characteristic for the Eastern Tropical South Pacific (ETSP) and microbial degradation of a high organic matter load promotes deoxygenation at depth. Dissolved organic matter (DOM) plays a key role in microbial respiration and carbon cycling, but little is known on DOM distribution and cycling in the ETSP. DOM optical properties give important insights on DOM sources, structure and biogeochemical reactivity. Here, we present data and a conceptual view on distribution and cycling of chromophoric (CDOM) and fluorescent (FDOM) DOM in and above the oxygen minimum zone (OMZ) off Peru. Five fluorescent components were identified during PARAFAC analysis. Highest intensities of CDOM and of the amino acid-like fluorescent component (C3) occurred above the OMZ and coincided with maximum chl a concentrations, suggesting phytoplankton productivity as major source. High intensities of a marine humic-like fluorescent component (C1), observed in subsurface waters, indicated in situ microbial reworking of DOM. FDOM release from inner shelf sediment was determined by seawater analysis and continuous glider sensor measurement and included a humic-like component (C2) with a signature typical for terrestrially derived humic acids. Upwelling supplied humic-like substances to the euphotic zone. Photo-reactions were likely involved in the production of a humic-like fluorescent component (C5). Our data show that variable biological and physical processes need to be considered for understanding DOM cycling in a highly dynamic coastal upwelling system like the ETSP off Peru.

Validation of fluorescent-labeled microspheres for measurement of relative blood flow in severely injured lungs

NASA Technical Reports Server (NTRS)

Hubler, M.; Souders, J. E.; Shade, E. D.; Hlastala, M. P.; Polissar, N. L.; Glenny, R. W.

1999-01-01

The aim of the study was to validate a nonradioactive method for relative blood flow measurements in severely injured lungs that avoids labor-intensive tissue processing. The use of fluorescent-labeled microspheres was compared with the standard radiolabeled-microsphere method. In seven sheep, lung injury was established by using oleic acid. Five pairs of radio- and fluorescent-labeled microspheres were injected before and after established lung injury. Across all animals, 175 pieces were selected randomly. The radioactivity of each piece was determined by using a scintillation counter. The fluorescent dye was extracted from each piece with a solvent without digestion or filtering. The fluorescence was determined with an automated fluorescent spectrophotometer. Perfusion was calculated for each piece from both the radioactivity and fluorescence and volume normalized. Correlations between flow determined by the two methods were in the range from 0.987 +/- 0.007 (SD) to 0.991 +/- 0.002 (SD) after 9 days of soaking. Thus the fluorescent microsphere technique is a valuable tool for investigating regional perfusion in severely injured lungs and can replace radioactivity.

Fluorescent optical position sensor

DOEpatents

Weiss, Jonathan D.

2005-11-15

A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

PubMed

Patra, Digambara

2010-01-15

A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

NASA Astrophysics Data System (ADS)

Plank, David M.; Sussman, Mark A.

2005-06-01

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Fourth-derivative synchronous spectrofluorimetry and HPLC with fluorescence detection as two analytical techniques for the simultaneous determination of itopride and domperidone.

PubMed

Ibrahim, Fawzia; Nasr, Jenny Jeehan

2016-02-01

Two simple, rapid and sensitive methods, namely, fourth-derivative synchronous spectrofluorimetry (method I) and HPLC with fluorescence detection (method II) were developed for the simultaneous analysis of a binary mixture of itopride HCl (ITP) and domperidone (DOM) without prior separation. The first method was based on measuring the fourth derivative of the synchronous fluorescence spectra of the two drugs at Δλ = 40 nm in methanol. The different experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully optimized. Chromatographic separation was performed in < 6.0 min using a RP C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) with fluorescence detection at 344 nm after excitation at 285 nm. A mobile phase composed of a mixture of 0.02 M phosphate buffer with acetonitrile in a ratio of 55 : 45, pH 4.5, was used at a flow rate of 1 mL/min. Linearity ranges were found to be 0.1-2 µg/mL for ITP in both methods, whereas those for DOM were found to be 0.08-2 and 0.05-1.5 µg/mL in methods I and II, respectively. The proposed methods were successfully applied for the determination of the studied drugs in synthetic mixtures and laboratory-prepared tablets. Copyright © 2015 John Wiley & Sons, Ltd.

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis.

PubMed

Welder, Frank; Paul, Beverly; Nakazumi, Hiroyuki; Yagi, Shigeyuki; Colyer, Christa L

2003-08-05

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.

Evaluation of slide based cytometry (SBC) for concentration measurements of fluorescent dyes in solution

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Marecka, Monika; Müller, Hans-Willy; Bocsi, József; Tárnok, Attila

2009-02-01

Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The novel slide-based imaging system is suitable for detection of fluorescence differences over a broad range of concentrations. This approach may lead to novel assays for measuring concentration differences in cell free solutions and cell cultures e.g. in secretion assays.

The Statistical Value of Raw Fluorescence Signal in Luminex xMAP Based Multiplex Immunoassays

PubMed Central

Breen, Edmond J.; Tan, Woei; Khan, Alamgir

2016-01-01

Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required. PMID:27243383

Simultaneous determination of micellar structure and drag reduction in a surfactant solution flow using the fluorescence probe method

NASA Astrophysics Data System (ADS)

Wakimoto, Tatsuro; Araga, Koichi; Katoh, Kenji

2018-03-01

As widely known, the addition of a specific type of surfactant to water reduces drag in a pipe flow. This effect is considered to be a result of the suppression of turbulent transition caused by the ordered structure of rod-like micelles that is referred to as a shear-induced structure (SIS). However, it is typically difficult to determine the SIS since it is necessary to noninvasively detect the SIS with several hundred nanometers in the actual moving flow. In this study, we used the fluorescence probe method to locally determine the SIS in a pipe flow. When hydrophobic fluorescence molecules are added to the surfactant solution, the fluorescence molecules are trapped in micelles. Thus, fluorescence intensity varies based on the change in the micellar structure. We verified the applicability of the fluorescence probe method to the SIS detection and determined the relationship between the micellar structure and the drag reduction in the pipe flow by simultaneously measuring the fluorescence intensity and pipe friction factor. The experimental result demonstrates that the SIS formation in the near-wall region is closely correlated with the drag reduction and suggests that the near-wall SIS suppresses the turbulent transition.

Fluorescence of crop residue: postmortem analysis of crop conditions

NASA Astrophysics Data System (ADS)

McMurtrey, James E., III; Kim, Moon S.; Daughtry, Craig S. T.; Corp, Lawrence A.; Chappelle, Emmett W.

1997-07-01

Fluorescence of crop residues at the end of the growing season may provide an indicator of the past crop's vegetative condition. Different levels of nitrogen (N) fertilization were applied to field grown corn and wheat at Beltsville, Maryland. The N fertilizer treatments produce a range of physiological conditions, pigment concentrations, biomass levels, and grain yields that resulted in varying growth and stress conditions in the living crops. After normal harvesting procedures the crop residues remained. The fluorescence spectral characteristics of the plant residues from crops grown under different levels of N nutrition were analyzed. The blue-green fluorescence response of in-vitro residue biomass of the N treated field corn had different magnitudes. A blue-green- yellow algorithm, (460/525)*600 nm, gave the best separations between prior corn growth conditions at different N fertilization levels. Relationships between total dry biomass, the grain yield, and fluorescence properties in the 400 - 670 nm region of the spectrum were found in both corn and wheat residues. The wheat residue was analyzed to evaluate the constituents responsible for fluorescence. A ratio of the blue-green, 450/550 nm, images gave the best separation among wheat residues at different N fertilization levels. Fluorescence of extracts from wheat residues showed inverse fluorescence intensities as a function of N treatments compared to that of the intact wheat residue or ground residue samples. The extracts also had an additional fluorescence emission peak in the red, 670 nm. Single band fluorescence intensity in corn and wheat residues is due mostly to the quantity of the material on the soil surface. Ratios of fluorescence bands varied as a result of the growth conditions created by the N treatments and are thought to be indicative of the varying concentrations of the plant residues fluorescing constituents. Estimates of the amount and cost effectiveness of N fertilizers to satisfy optimal plant growth condition for specific areas of the field for the next growing season may be useful indicators for crop management. Analysis of plant constituent qualities and quantities of dead crop materials during the harvesting practice or after harvest could be useful indicators of the previous crop's conditions. These measures could be used as a tool in determining precision farming management practices for site specific areas in a field.

Photoinduced azidohydroperoxidation of myrtenyl hydroperoxide with semiconductor particles and lucigenin as PET-catalysts.

PubMed

Griesbeck, Axel G; Reckenthäler, Melissa; Uhlig, Johannes

2010-06-01

The allylic hydroperoxide 2 (myrtenyl hydroperoxide), available from singlet oxygen photooxygenation of beta-pinene (1), is converted into the azido bis-hydroperoxide 3 by an electron-transfer induced azidyl radical formation and trapping of the initial tertiary carbon radical by triplet oxygen. The azido bis-hydroperoxide 3 is reduced to the azido 1,2-diol 4 or the amino diol 5, respectively. Beside classical fluorescent PET sensitizers such as rhodamines, also nanosized semiconductor particles as well as lucigenin were applied as catalysts. The electron transfer rate of azide oxidation was determined for lucigenin by fluorescence quenching analysis.

Energy Dispersive X-Ray Fluorescent Analysis of Soil in the Vicinity of Industrial Areas and Heavy Metal Pollution Assessment

NASA Astrophysics Data System (ADS)

Singh, V.; Joshi, G. C.; Bisht, D.

2017-05-01

The soil of two agricultural sites near an industrial area was investigated for heavy metal pollution using energy dispersive X-ray fluorescence (EDXRF). The concentration values for 17 elements were determined in the soil samples including eight heavy metal elements, i.e., Fe, Ni, As, Pb, Mn, Cr, Cu, and Zn. The soil near a pulp and paper mill was found to be highly polluted by the heavy metals. The concentration data obtained by EDXRF were further examined by calculating the pollution index and Nemerow integrated pollution index.

Determination of stoichiometry and concentration of trace elements in thin BaxSr1-xTiO3 perovskite layers.

PubMed

Becker, J S; Boulyga, S F

2001-07-01

This paper describes an analytical procedure for determining the stoichiometry of BaxSr1-xTiO3 perovskite layers using inductively coupled plasma mass spectrometry (ICP-MS). The analytical results of mass spectrometry measurements are compared to those of X-ray fluorescence analysis (XRF). The performance and the limits of solid-state mass spectrometry analytical methods for the surface analysis of thin BaxSr1-xTiO3 perovskite layers sputtered neutral mass spectrometry (SNMS)--are investigated and discussed.

INDIVIDUAL VARIATION IN THE FREQUENCY OF SPERM ANEUPLOIDY IN HUMANS

EPA Science Inventory

To examine interindividual differences in sperm chromosome aneuploidy, repeated semen specimens were obtained from a group of ten healthy men, aged 20-21 at the start of the study, and analyzed by multi-color fluorescence in situ hybridization (FISH) analysis to determine the fre...

Hydroxypropyl-beta-cyclodextrin enhanced determination for the Vitamin B12 by fluorescence quenching method.

PubMed

Sun, Jing; Zhu, Xiashi; Wu, Ming

2007-05-01

A novel fluorescence quenching method for the determination of Vitamin B12(VB12) had been developed. It was based on that the fluorescence intensity of erythrosine sodium(ES) could be enhanced by Hydroxypropyl-beta-cyclodextrin(HP-beta-CD) due to the formation of inclusion complex (HP-beta-CD-ES), while the fluorescence intensity of HP-beta-CD-ES was diminished after adding VB12 into the system, and there was a linear relationship between the fluorescence quenching value of the system (DeltaF) and the concentration of VB12 (c). The mechanism of the determination of VB12 was discussed. The results showed that under the optimal conditions, the linear range of calibration curve for the determination of VB12 was 0.0 approximately 2.1 x 10(-5) mol/L, and the detection limit was 1.8 x 10(-7) mol/ L. It could be satisfactorily applied to the determination of VB12 in injections.

Antibiotics mediated facile one-pot synthesis of gold nanoclusters as fluorescent sensor for ferric ions.

PubMed

Yu, Mengqun; Zhu, Zheguo; Wang, Hong; Li, Linyao; Fu, Fei; Song, Yang; Song, Erqun

2017-05-15

In this paper, the cheap, easily obtained small antibiotic molecule of vancomycin was employed as reducer/stabilizer for facile one-pot synthesis of water exhibited a bluish fluorescence emission at 410nm within a short synthesis time about 50min. Based on the strong fluorescence quenching due to electron transfer mechanism by the introduction of ferric ions(Fe 3+ ), the Van-AuNCs were interestingly designed for sensitive and selective detecting Fe 3+ with a limit of 1.4μmol L -1 in the linear range of 2-100μmol L -1 within 20min. The Van-AuNCs based method was successfully applied to determine Fe 3+ in tap water, lake water, river water and sea water samples with the quantitative spike recoveries from 97.50-111.14% with low relative standard deviations ranging from 0.49-1.87%, indicating the potential application of this Van-AuNCs based fluorescent sensor for environmental sample analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescent chemosensor for pyridine based on N-doped carbon dots.

PubMed

Campos, B B; Abellán, C; Zougagh, M; Jimenez-Jimenez, J; Rodríguez-Castellón, E; Esteves da Silva, J C G; Ríos, A; Algarra, M

2015-11-15

Fluorescent carbon dots (CDs) and its nitrogen doped (N-CDs) nanoparticles have been synthesized from lactose as precursor using a bottom-up hydrothermal methodology. The synthesized nanoparticles have been characterized by elemental analysis, FTIR, Raman, TEM, DLS, XPS, and steady-state and life-time fluorescence. The synthesized carbon nanoparticles, CDs and N-CDs, have a size at about 7.7±2.4 and 50±15nm, respectively, and quantum yields of 8% (CDs) and 11% (N-CDs). These techniques demonstrated the effectiveness of the synthesis procedure and the functionalization of the CDs surface with amine and amide groups in the presence of NH3 in aqueous media. The effect of excitation wavelength and pH on the luminescent properties was studied. Under the optimal conditions, the nitrogen doped nanoparticles can be used as pyridine sensor in aqueous media because they show an enhancement of its fluorescence with a good linear relationship. The analytical method is simple, reproducible and very sensitive for pyridine determination. Copyright © 2015 Elsevier Inc. All rights reserved.

Photoswitchable red fluorescent protein with a large Stokes shift

PubMed Central

Piatkevich, Kiryl D.; English, Brian P.; Malashkevich, Vladimir N.; Xiao, Hui; Almo, Steven C.; Singer, Robert H.; Verkhusha, Vladislav V.

2014-01-01

SUMMARY Subclass of fluorescent proteins, large Stokes shift fluorescent proteins, is characterized by their increased spread between the excitation and emission maxima. Here we report a photoswitchable variant of a red fluorescent protein with a large Stokes shift, PSLSSmKate, which initially exhibits excitation/emission at 445/622 nm, but irradiation with violet light photoswitches PSLSSmKate into a common red form with excitation/emission at 573/621 nm. We characterize spectral, photophysical and biochemical properties of PSLSSmKate in vitro and in mammalian cells, and determine its crystal structure in the large Stokes shift form. Mass-spectrometry, mutagenesis and spectroscopic analysis of PSLSSmKate allow us to propose molecular mechanisms for the large Stokes shift, pH dependence and light-induced chromophore transformation. We demonstrate applicability of PSLSSmKate to superresolution PALM microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects. PMID:25242289

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Simple determination of aflatoxins in rice by ultra-high performance liquid chromatography coupled to chemical post-column derivatization and fluorescence detection.

PubMed

Huertas-Pérez, José Fernando; Arroyo-Manzanares, Natalia; Hitzler, Dominik; Castro-Guerrero, Francisco Germán; Gámiz-Gracia, Laura; García-Campaña, Ana M

2018-04-15

A fast and simple analytical method was developed and characterized for the determination of aflatoxins (B 1 , B 2 , G 1 and G 2 ) in rice. The procedure is based on a simple solid-liquid extraction without further clean-up, and analysis by ultra-high performance liquid chromatography coupled with fluorescence detection. Fluorescence emission of aflatoxins B 1 and G 1 was enhanced by post-column chemical derivatization using pyridinium bromide perbromide. The analytical method was satisfactorily characterized in white and brown rice. Under optimum conditions, external calibration in solvent could be used for quantification purposes and limits of quantification were below the maximum contents established by the European Union regulation for these contaminants/commodity group combination (0.07-0.14 µg/kg for white rice and 0.20-0.28 µg/kg for brown rice). Recovery studies carried out at three different concentration levels (0.5, 2 and 5 µg/kg) showed values in the range of 84.5-105.3%, and RSDs ≤ 5%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.

PubMed

Jin, Byung-Ju; Ko, Eun-A; Namkung, Wan; Verkman, A S

2013-10-07

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

PubMed Central

Tang, Jianyong; Akerboom, Jasper; Vaziri, Alipasha; Looger, Loren L.; Shank, Charles V.

2010-01-01

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution. PMID:20472826

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

PubMed Central

Chatelier, R C; Ashcroft, R G; Lloyd, C J; Nice, E C; Whitehead, R H; Sawyer, W H; Burgess, A W

1986-01-01

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry. PMID:3015587

Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

NASA Astrophysics Data System (ADS)

Smith, Clint; Edwards, Jarrod; Fisher, Andmorgan

2010-04-01

Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.

PubMed

Brooks, Morgan M; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy; Cammarata, Patrick R

2013-01-01

Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O₂) followed by exposure to atmospheric oxygen (approximately 21% O₂). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Inhibition of GSK-3β activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3β activity by the GSK-3β inhibitor SB216763 provides positive protection against mitochondrial depolarization.

Usage of K-cluster and factor analysis for grouping and evaluation the quality of olive oil in accordance with physico-chemical parameters

NASA Astrophysics Data System (ADS)

Milev, M.; Nikolova, Kr.; Ivanova, Ir.; Dobreva, M.

2015-11-01

25 olive oils were studied- different in origin and ways of extraction, in accordance with 17 physico-chemical parameters as follows: color parameters - a and b, light, fluorescence peaks, pigments - chlorophyll and β-carotene, fatty-acid content. The goals of the current study were: Conducting correlation analysis to find the inner relation between the studied indices; By applying factor analysis with the help of the method of Principal Components (PCA), to reduce the great number of variables into a few factors, which are of main importance for distinguishing the different types of olive oil;Using K-means cluster to compare and group the tested types olive oils based on their similarity. The inner relation between the studied indices was found by applying correlation analysis. A factor analysis using PCA was applied on the basis of the found correlation matrix. Thus the number of the studied indices was reduced to 4 factors, which explained 79.3% from the entire variation. The first one unified the color parameters, β-carotene and the related with oxidative products fluorescence peak - about 520 nm. The second one was determined mainly by the chlorophyll content and related to it fluorescence peak - about 670 nm. The third and the fourth factors were determined by the fatty-acid content of the samples. The third one unified the fatty-acids, which give us the opportunity to distinguish olive oil from the other plant oils - oleic, linoleic and stearin acids. The fourth factor included fatty-acids with relatively much lower content in the studied samples. It is enquired the number of clusters to be determined preliminary in order to apply the K-Cluster analysis. The variant K = 3 was worked out because the types of the olive oil were three. The first cluster unified all salad and pomace olive oils, the second unified the samples of extra virgin oilstaken as controls from producers, which were bought from the trade network. The third cluster unified samples from pomace and extra virgin oils, which distinguish one from another in accordance with their parameters from the natural olive oils, because of presence of plant oils impurities.

A convenient method for determination of quizalofop-p-ethyl based on the fluorescence quenching of eosin Y in the presence of Pd(II)

NASA Astrophysics Data System (ADS)

Wu, Huan; Zhao, Yanmei; Tan, Xuanping; Zeng, Xiaoqing; Guo, Yuan; Yang, Jidong

2017-03-01

A convenient fluorescence quenching method for determination of Quizalofop-p-ethyl(Qpe) was proposed in this paper. Eosin Y(EY) is a red dye with strong green fluorescence (λex/λem = 519/540 nm). The interaction between EY, Pd(II) and Qpe was investigated by fluorescence spectroscopy, resonance Rayleigh scattering(RRS) and UV-Vis absorption. Based on changes in spectrum, Pd(II) associated with Qpe giving a positively charged chelate firstly, then reacted with EY through electrostatic and hydrophobic interaction formed ternary chelate could be demonstrated. Under optimum conditions, the fluorescence intensity of EY could be quenched by Qpe in the presence of Pd(II) and the RRS intensity had a remarkable enhancement, which was directly proportional to the Qpe concentration within a certain concentration range, respectively. Based on the fluorescence quenching of EY-Pd(II) system by Qpe, a novel, convenient and specific method for Qpe determination was developed. To our knowledge, this is the first fluorescence method for determination of Qpe was reported. The detection limit for Qpe was 20.3 ng/mL and the quantitative determination range was 0.04-1.0 μg/mL. The method was highly sensitive and had larger detection range compared to other methods. The influence of coexisting substances was investigated with good anti-interference ability. The new analytical method has been applied to determine of Qpe in real samples with satisfactory results.

A sensitive flow-batch system for on board determination of ultra-trace ammonium in seawater: Method development and shipboard application.

PubMed

Zhu, Yong; Yuan, Dongxing; Huang, Yongming; Ma, Jian; Feng, Sichao

2013-09-10

Combining fluorescence detection with flow analysis and solid phase extraction (SPE), a highly sensitive and automatic flow system for measurement of ultra-trace ammonium in open ocean water was established. Determination was based on fluorescence detection of a typical product of o-phthaldialdehyde and ammonium. In this study, the fluorescence reaction product could be efficiently extracted onto an SPE cartridge (HLB, hydrophilic-lipophilic balance). The extracted fluorescence compounds were rapidly eluted with ethanol and directed into a flow cell for fluorescence detection. Compared with the common used fluorescence method, the proposed one offered the benefits of improved sensitivity, reduced reagent consumption, negligible salinity effect and lower cost. Experimental parameters were optimized using a univariate experimental design. Calibration curves, ranging from 1.67 to 300nM, were obtained with different reaction times. The recoveries were between 89.5 and 96.5%, and the detection limits in land-based and shipboard laboratories were 0.7 and 1.2nM, respectively. The relative standard deviation was 3.5% (n=5) for an aged seawater sample spiked with 20nM ammonium. Compared with the analytical results obtained using the indophenol blue method coupled to a long-path liquid waveguide capillary cell, the proposed method showed good agreement. The method had been applied on board during a South China Sea cruise in August 2012. A vertical profile of ammonium in the South East Asia Time-Series (SEATS, 18° N, 116° E) station was produced. The distribution of ammonium in the surface seawater of the Qiongdong upwelling in South China Sea is also presented. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of new analytical methods for the determination of caffeine content in aqueous solution of green coffee beans.

PubMed

Weldegebreal, Blen; Redi-Abshiro, Mesfin; Chandravanshi, Bhagwan Singh

2017-12-05

This study was conducted to develop fast and cost effective methods for the determination of caffeine in green coffee beans. In the present work direct determination of caffeine in aqueous solution of green coffee bean was performed using FT-IR-ATR and fluorescence spectrophotometry. Caffeine was also directly determined in dimethylformamide solution using NIR spectroscopy with univariate calibration technique. The percentage of caffeine for the same sample of green coffee beans was determined using the three newly developed methods. The caffeine content of the green coffee beans was found to be 1.52 ± 0.09 (% w/w) using FT-IR-ATR, 1.50 ± 0.14 (% w/w) using NIR and 1.50 ± 0.05 (% w/w) using fluorescence spectroscopy. The means of the three methods were compared by applying one way analysis of variance and at p = 0.05 significance level the means were not significantly different. The percentage of caffeine in the same sample of green coffee bean was also determined by using the literature reported UV/Vis spectrophotometric method for comparison and found to be 1.40 ± 0.02 (% w/w). New simple, rapid and inexpensive methods were developed for direct determination of caffeine content in aqueous solution of green coffee beans using FT-IR-ATR and fluorescence spectrophotometries. NIR spectrophotometry can also be used as alternative choice of caffeine determination using reduced amount of organic solvent (dimethylformamide) and univariate calibration technique. These analytical methods may therefore, be recommended for the rapid, simple, safe and cost effective determination of caffeine in green coffee beans.

Simultaneous determination of estrogens (ethinylestradiol and norgestimate) concentrations in human and bovine serum albumin by use of fluorescence spectroscopy and multivariate regression analysis.

PubMed

Hordge, LaQuana N; McDaniel, Kiara L; Jones, Derick D; Fakayode, Sayo O

2016-05-15

The endocrine disruption property of estrogens necessitates the immediate need for effective monitoring and development of analytical protocols for their analyses in biological and human specimens. This study explores the first combined utility of a steady-state fluorescence spectroscopy and multivariate partial-least-square (PLS) regression analysis for the simultaneous determination of two estrogens (17α-ethinylestradiol (EE) and norgestimate (NOR)) concentrations in bovine serum albumin (BSA) and human serum albumin (HSA) samples. The influence of EE and NOR concentrations and temperature on the emission spectra of EE-HSA EE-BSA, NOR-HSA, and NOR-BSA complexes was also investigated. The binding of EE with HSA and BSA resulted in increase in emission characteristics of HSA and BSA and a significant blue spectra shift. In contrast, the interaction of NOR with HSA and BSA quenched the emission characteristics of HSA and BSA. The observed emission spectral shifts preclude the effective use of traditional univariate regression analysis of fluorescent data for the determination of EE and NOR concentrations in HSA and BSA samples. Multivariate partial-least-squares (PLS) regression analysis was utilized to correlate the changes in emission spectra with EE and NOR concentrations in HSA and BSA samples. The figures-of-merit of the developed PLS regression models were excellent, with limits of detection as low as 1.6×10(-8) M for EE and 2.4×10(-7) M for NOR and good linearity (R(2)>0.994985). The PLS models correctly predicted EE and NOR concentrations in independent validation HSA and BSA samples with a root-mean-square-percent-relative-error (RMS%RE) of less than 6.0% at physiological condition. On the contrary, the use of univariate regression resulted in poor predictions of EE and NOR in HSA and BSA samples, with RMS%RE larger than 40% at physiological conditions. High accuracy, low sensitivity, simplicity, low-cost with no prior analyte extraction or separation required makes this method promising, compelling, and attractive alternative for the rapid determination of estrogen concentrations in biomedical and biological specimens, pharmaceuticals, or environmental samples. Published by Elsevier B.V.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

PubMed

Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

2015-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

Click strategies for single-molecule protein fluorescence.

PubMed

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

NASA Astrophysics Data System (ADS)

Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

2001-05-01

In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

ALA-induced PpIX spectroscopy for brain tumor image-guided surgery

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Leblond, Frederic; Kim, Anthony; Harris, Brent T.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2011-03-01

Maximizing the extent of brain tumor resection correlates with improved survival and quality of life outcomes in patients. Optimal surgical resection requires accurate discrimination between normal and abnormal, cancerous tissue. We present our recent experience using quantitative optical spectroscopy in 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence-guided resection. Exogenous administration of ALA leads to preferential accumulation in tumor tissue of the fluorescent compound, PpIX, which can be used for in vivo surgical guidance. Using the state of the art approach with a fluorescence surgical microscope, we have been able to visualize a subset of brain tumors, but the sensitivity and accuracy of fluorescence detection for tumor tissue with this system are low. To take full advantage of the biological selectivity of PpIX accumulation in brain tumors, we used a quantitative optical spectroscopy system for in vivo measurements of PpIX tissue concentrations. We have shown that, using our quantitative approach for determination of biomarker concentrations, ALA-induced PpIX fluorescence-guidance can achieve accuracies of greater than 90% for most tumor histologies. Here we show multivariate analysis of fluorescence and diffuse reflectance signals in brain tumors with comparable diagnostic performance to our previously reported quantitative approach. These results are promising, since they show that technological improvements in current fluorescence-guided surgical technologies and more biologically relevant approaches are required to take full advantage of fluorescent biomarkers, achieve better tumor identification, increase extent of resection, and subsequently, lead to improve survival and quality of life in patients.

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Multiple Velocity Profile Measurements in Hypersonic Flows using Sequentially-Imaged Fluorescence Tagging

NASA Technical Reports Server (NTRS)

Bathel, Brett F.; Danehy, Paul M.; Inmian, Jennifer A.; Jones, Stephen B.; Ivey, Christopher B.; Goyne, Christopher P.

2010-01-01

Nitric-oxide planar laser-induced fluorescence (NO PLIF) was used to perform velocity measurements in hypersonic flows by generating multiple tagged lines which fluoresce as they convect downstream. For each laser pulse, a single interline, progressive scan intensified CCD camera was used to obtain separate images of the initial undelayed and delayed NO molecules that had been tagged by the laser. The CCD configuration allowed for sub-microsecond acquisition of both images, resulting in sub-microsecond temporal resolution as well as sub-mm spatial resolution (0.5-mm x 0.7-mm). Determination of axial velocity was made by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. Quantification of systematic errors, the contribution of gating/exposure duration errors, and influence of collision rate on fluorescence to temporal uncertainty were made. Quantification of the spatial uncertainty depended upon the analysis technique and signal-to-noise of the acquired profiles. This investigation focused on two hypersonic flow experiments: (1) a reaction control system (RCS) jet on an Orion Crew Exploration Vehicle (CEV) wind tunnel model and (2) a 10-degree half-angle wedge containing a 2-mm tall, 4-mm wide cylindrical boundary layer trip. The experiments were performed at the NASA Langley Research Center's 31-inch Mach 10 wind tunnel.

Fluorescence Determination of Tryptophan Side-Chain Accessibility and Dynamics in Triple-Helical Collagen-Like Peptides

PubMed Central

Simon-Lukasik, Kristine V.; Persikov, Anton V.; Brodsky, Barbara; Ramshaw, John A. M.; Laws, William R.; Alexander Ross, J. B.; Ludescher, Richard D.

2003-01-01

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)n. About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5–9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter (∼3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels. PMID:12524302

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2004-09-01

Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression of the GFP marker and cell- surface endothelial...express the green fluorescent protein (GFP) and clonal MSC populations can be isolated and phenotypically and genotypically analyzed by flow cytometry ...monoclonal populations of these GFP+ murine MSCs and conducted flow cytometry analysis to determine their phenotype. Specifically, we determined if

Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

NASA Astrophysics Data System (ADS)

Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2017-03-01

Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

Analysis of laser fluorosensor systems for remote algae detection and quantification

NASA Technical Reports Server (NTRS)

Browell, E. V.

1977-01-01

The development and performance of single- and multiple-wavelength laser fluorosensor systems for use in the remote detection and quantification of algae are discussed. The appropriate equation for the fluorescence power received by a laser fluorosensor system is derived in detail. Experimental development of a single wavelength system and a four wavelength system, which selectively excites the algae contained in the four primary algal color groups, is reviewed, and test results are presented. A comprehensive error analysis is reported which evaluates the uncertainty in the remote determination of the chlorophyll a concentration contained in algae by single- and multiple-wavelength laser fluorosensor systems. Results of the error analysis indicate that the remote quantification of chlorophyll a by a laser fluorosensor system requires optimum excitation wavelength(s), remote measurement of marine attenuation coefficients, and supplemental instrumentation to reduce uncertainties in the algal fluorescence cross sections.

[Three dimensional fluorescent characteristics of soil dissolved organic matter (DOM) in Jiaozhou Bay coastal wetlands, China].

PubMed

Zi, Yuan Yuan; Kong, Fan Long; Xi, Min; Li, Yue; Yang, Ling

2016-12-01

In order to elucidate the structure characteristics of soil dissolved organic matter (DOM) and analyze the sources in Jiaozhou Bay coastal wetlands, four typical types of wetlands around Jiaozhou Bay were chosen, including Spartina anglica wetland, the barren wetland, Suaeda glauca wetland and Phragmites australis wetland. The soil samples were collected in January 2014. The contents of soil DOM were determined and the spectral analysis was made by three-dimensional fluorescent technology. The results showed that the contents of soil dissolved organic carbon (DOC) in four types of wetlands all decreased with the increasing soil depth, and S. anglica wetland ranked the first in the contents of soil DOC, followed by the barren wetland, S. glauca wetland and P. australis wetland. Five fluorescence peaks including B, T, A, D and C were found in the three-dimensional fluorescence spectrum (3DEEMs), indicating tyrosine-like, tryptophan-like, phenol-like, soluble microbial byproduct-like and humic acid-like- substances, respectively. Fluorescence integration (FRI) was applied in the qualitative analysis of five components. The results showed that tryptophan-like, phenol-like and tyrosine-like substances ranked in top three in content, followed by soluble microbial byproduct-like and humic acid-like substances which were not significantly different. Pearson correlation analysis demonstrated that a positive correlation existed between any two of the five components of DOM, and they were all positively related to DOC content. In addition, there existed different correlations between the five components of DOM and total phosphorus (TP), available phosphorus (AP) and total nitrogen (TN). The soil DOM in the four types of wetlands was mainly produced by biotic interactions, and the degree of humification was relatively low.

NeuronRead, an open source semi-automated tool for morphometric analysis of phase contrast and fluorescence neuronal images.

PubMed

Dias, Roberto A; Gonçalves, Bruno P; da Rocha, Joana F; da Cruz E Silva, Odete A B; da Silva, Augusto M F; Vieira, Sandra I

2017-12-01

Neurons are specialized cells of the Central Nervous System whose function is intricately related to the neuritic network they develop to transmit information. Morphological evaluation of this network and other neuronal structures is required to establish relationships between neuronal morphology and function, and may allow monitoring physiological and pathophysiologic alterations. Fluorescence-based microphotographs are the most widely used in cellular bioimaging, but phase contrast (PhC) microphotographs are easier to obtain, more affordable, and do not require invasive, complicated and disruptive techniques. Despite the various freeware tools available for fluorescence-based images analysis, few exist that can tackle the more elusive and harder-to-analyze PhC images. To surpass this, an interactive semi-automated image processing workflow was developed to easily extract relevant information (e.g. total neuritic length, average cell body area) from both PhC and fluorescence neuronal images. This workflow, named 'NeuronRead', was developed in the form of an ImageJ macro. Its robustness and adaptability were tested and validated on rat cortical primary neurons under control and differentiation inhibitory conditions. Validation included a comparison to manual determinations and to a golden standard freeware tool for fluorescence image analysis. NeuronRead was subsequently applied to PhC images of neurons at distinct differentiation days and exposed or not to DAPT, a pharmacological inhibitor of the γ-secretase enzyme, which cleaves the well-known Alzheimer's amyloid precursor protein (APP) and the Notch receptor. Data obtained confirms a neuritogenic regulatory role for γ-secretase products and validates NeuronRead as a time- and cost-effective useful monitoring tool. Copyright © 2017. Published by Elsevier Inc.

Spectral and Temporal Laser Fluorescence Analysis Such as for Natural Aquatic Environments

NASA Technical Reports Server (NTRS)

Chekalyuk, Alexander (Inventor)

2015-01-01

An Advanced Laser Fluorometer (ALF) can combine spectrally and temporally resolved measurements of laser-stimulated emission (LSE) for characterization of dissolved and particulate matter, including fluorescence constituents, in liquids. Spectral deconvolution (SDC) analysis of LSE spectral measurements can accurately retrieve information about individual fluorescent bands, such as can be attributed to chlorophyll-a (Chl-a), phycobiliprotein (PBP) pigments, or chromophoric dissolved organic matter (CDOM), among others. Improved physiological assessments of photosynthesizing organisms can use SDC analysis and temporal LSE measurements to assess variable fluorescence corrected for SDC-retrieved background fluorescence. Fluorescence assessments of Chl-a concentration based on LSE spectral measurements can be improved using photo-physiological information from temporal measurements. Quantitative assessments of PBP pigments, CDOM, and other fluorescent constituents, as well as basic structural characterizations of photosynthesizing populations, can be performed using SDC analysis of LSE spectral measurements.

Determination of Tinopal CBS-X in rice papers and rice noodles using HPLC with fluorescence detection and LC-MS/MS.

PubMed

Ko, Kyung Yuk; Lee, Chae A; Choi, Jae Chon; Kim, Meehye

2014-01-01

To date there have been no reports of methods to determine Tinopal CBS-X. We developed a rapid and simple method to determine the Tinopal CBS-X content in rice noodles and rice papers using HPLC equipped with fluorescence detection. Heating the rice noodles and rice papers to 80°C after adding 75% methanol solution induced the release of Tinopal CBS-X from processed rice products. Tinopal CBS-X was separated using an isocratic mobile phase comprising 50% acetonitrile/water containing 0.4% tetrabutyl ammonium hydrogen sulphate at pH 8.0. The samples suspected to be positive by HPLC analysis were then confirmed by LC-MS/MS analysis. This study also investigated the Tinopal CBS-X content of three rice noodle products and two rice papers. The limits of quantification for rice papers and rice noodles were 1.58 and 1.51 µg kg(-1), respectively, and their correlation curves showed good linearity with r(2) ≥ 0.9997 and ≥ 0.9998, respectively. Moreover, rice papers had recoveries of 70.3-83.3% with precision ranging from 5.0% to 7.9%, whereas rice noodles had slightly lower recoveries of 63.4-78.7% and precisions of 8.5-11.5%. Only one rice noodle product contained Tinopal CBS-X, at around 2.1 mg kg(-1), whereas it was not detected in four other samples. Consequently, Tinopal CBS-X from rice noodles and rice papers can be successfully detected using the developed pre-treatment and ion-pairing HPLC system coupled with fluorescence detection.

75 FR 22031 - Energy Conservation Program for Consumer Products and Certain Commercial and Industrial Equipment...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-27

..., including induction and fluorescent. Non-HID technologies are achieving market penetration in lighting... the electromagnetic (magnetic) type. The industry-accepted life of magnetic ballasts is 50,000 hours... analysis for today's proposed determination, DOE only considered magnetic ballasts because they are the...

Dissolved organic matter fluorescence at wavelength 275/342 nm as a key indicator for detection of point-source contamination in a large Chinese drinking water lake.

PubMed

Zhou, Yongqiang; Jeppesen, Erik; Zhang, Yunlin; Shi, Kun; Liu, Xiaohan; Zhu, Guangwei

2016-02-01

Surface drinking water sources have been threatened globally and there have been few attempts to detect point-source contamination in these waters using chromophoric dissolved organic matter (CDOM) fluorescence. To determine the optimal wavelength derived from CDOM fluorescence as an indicator of point-source contamination in drinking waters, a combination of field campaigns in Lake Qiandao and a laboratory wastewater addition experiment was used. Parallel factor (PARAFAC) analysis identified six components, including three humic-like, two tryptophan-like, and one tyrosine-like component. All metrics showed strong correlation with wastewater addition (r(2) > 0.90, p < 0.0001). Both the field campaigns and the laboratory contamination experiment revealed that CDOM fluorescence at 275/342 nm was the most responsive wavelength to the point-source contamination in the lake. Our results suggest that pollutants in Lake Qiandao had the highest concentrations in the river mouths of upstream inflow tributaries and the single wavelength at 275/342 nm may be adapted for online or in situ fluorescence measurements as an early warning of contamination events. This study demonstrates the potential utility of CDOM fluorescence to monitor water quality in surface drinking water sources. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modeling of transdermal fluorescence measurements from first-in-human clinical trials for renal function determination using fluorescent tracer agent MB-102

NASA Astrophysics Data System (ADS)

Shultz, Kimberly M.; Debreczeny, Martin P.; Dorshow, Richard B.; Keating, Jennifer E.; Bechtel, Kate L.

2017-02-01

The fluorescent tracer agent 3,6-diamino-2,5-bisN-[(1R)-1-carboxy-2-hydroxyethyl]carbamoylpyrazine, designated MB-102, is cleared from the body solely by the kidneys. A prototype noninvasive fluorescence detection device has been developed for monitoring transdermal fluorescence after bolus intravenous injection of MB-102 in order to measure kidney function. A mathematical model of the detected fluorescence signal was created for evaluation of observed variations in agent kinetics across body locations and for analysis of candidate instrument geometries. The model comprises pharmacokinetics of agent distribution within body compartments, local diffusion of the agent within the skin, Monte Carlo photon transport through tissue, and ray tracing of the instrument optics. Data from eight human subjects with normal renal function and a range of skin colors shows good agreement with simulated data. Body site dependence of equilibration kinetics was explored using the model to find the local vasculature-to-interstitial diffusion time constant, blood volume fraction, and interstitial volume fraction. Finally, candidate instrument geometries were evaluated using the model. While an increase in source-detector separation was found to increase sensitivity to tissue optical properties, it reduced the relative intensity of the background signal with minimal effect on the measured equilibration kinetics.

Multivariate methods on the excitation emission matrix fluorescence spectroscopic data of diesel-kerosene mixtures: a comparative study.

PubMed

Divya, O; Mishra, Ashok K

2007-05-29

Quantitative determination of kerosene fraction present in diesel has been carried out based on excitation emission matrix fluorescence (EEMF) along with parallel factor analysis (PARAFAC) and N-way partial least squares regression (N-PLS). EEMF is a simple, sensitive and nondestructive method suitable for the analysis of multifluorophoric mixtures. Calibration models consisting of varying compositions of diesel and kerosene were constructed and their validation was carried out using leave-one-out cross validation method. The accuracy of the model was evaluated through the root mean square error of prediction (RMSEP) for the PARAFAC, N-PLS and unfold PLS methods. N-PLS was found to be a better method compared to PARAFAC and unfold PLS method because of its low RMSEP values.

Fluorescence lifetime imaging of endogenous molecules in live mouse cancer models (Conference Presentation)

NASA Astrophysics Data System (ADS)

Svindrych, Zdenek; Wang, Tianxiong; Hu, Song; Periasamy, Ammasi

2017-02-01

NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

Ozonation effects on emerging micropollutants and effluent organic matter in wastewater: characterization using changes of three-dimensional HP-SEC and EEM fluorescence data.

PubMed

Liu, Chen; Li, Penghui; Tang, Xiangyu; Korshin, Gregory V

2016-10-01

The degradation of effluent organic matter (EfOM) in a municipal wastewater treated by ozonation was characterized using the methods of high-performance size-exclusion chromatography (HP-SEC) and excitation/emission matrix (EEM) fluorescence combined with parallel factor analysis (PARAFAC). The removal of 40 diverse trace-level contaminants of emerging concern (CEC) present in the wastewater was determined as well. Ozonation caused a rapid decrease of the absorbance and fluorescence of the wastewater, which was associated primarily with the oxidation of high and low apparent molecular weight (AMW) EfOM fractions. PARAFAC analysis also showed that components C1 and C2 decreased prominently in these conditions. The EfOM fraction of intermediate molecular weight ascribable to a terrestrial humic-like component (C3) tended to be less reactive toward ozone. Relative changes of EEM fluorescence were quantified using F max values of PARAFAC-identified components (∆F/F 0 max ). Unambiguous relationships between ∆F/F 0 max values and the extent of the degradation of the examined CECs (∆C/C 0 ) were established. This allowed correlating main parameters of the ∆C/C 0 vs. ∆F/F 0 max relationships with the rates of oxidation of these CECs. The results demonstrate the potential of online measurements of EEM fluorescence for quantitating effects of ozonation on EfOM and micropollutants in wastewater effluents.

Water Quality Monitoring Using Tryptophan-like Fluorescence.

NASA Astrophysics Data System (ADS)

Hudson, N.; Urquhart, G.; Baker, A.; Ward, D.; Reynolds, D.; Carliell-Marquet, C.

2006-12-01

The Biochemical Oxygen Demand (BOD) test is recognised as being credible with over 90 years of application in water analysis. However it is easily affected by environmental constraints and requires the presence of a viable biological community. The BOD test takes 5-21 days and is laboratory based and so is unsuitable for rapid responses to potential pollution incidents. Analysis of fluorescence excitation emission matrices (EEM) of natural waters gives a rapid determination of the proportions of labile and refractory organic matter present. It facilitates a greater understanding of the oxygen depleting potential of organic matter in unfiltered samples in a shorter timescale than would be the case using BOD, the conventional water quality assessment method. The research presented assesses the relationship between 5-day BOD (BOD5) and the fluorescent amino acid tryptophan-like peak for a range of waters. The research is undertaken with a view to using fluorescence spectroscopy as an alternative to the BOD5 test for on-site monitoring or lab based, rapid indication of organic pollution in natural waters. A significant relationship is observed between the analytical parameters in line with the findings of previous research in which waste waters including synthetic sewage, and polluted surface waters were studied. This research demonstrates that for a large, variable data set tryptophan-like fluorescence is a strong indicator of BOD5 and may be used as a water quality monitoring tool particularly for high BOD5 samples.

Investigation of injection dose and camera integration time on quantifying pharmacokinetics of a Cy5.5-GX1 probe with dynamic fluorescence imaging in vivo

NASA Astrophysics Data System (ADS)

Dai, Yunpeng; Chen, Xueli; Yin, Jipeng; Kang, Xiaoyu; Wang, Guodong; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

2016-08-01

The aim of this article is to investigate the influence of a tracer injection dose (ID) and camera integration time (IT) on quantifying pharmacokinetics of Cy5.5-GX1 in gastric cancer BGC-823 cell xenografted mice. Based on three factors, including whether or not to inject free GX1, the ID of Cy5.5-GX1, and the camera IT, 32 mice were randomly divided into eight groups and received 60-min dynamic fluorescence imaging. Gurfinkel exponential model (GEXPM) and Lammertsma simplified reference tissue model (SRTM) combined with a singular value decomposition analysis were used to quantitatively analyze the acquired dynamic fluorescent images. The binding potential (Bp) and the sum of the pharmacokinetic rate constants (SKRC) of Cy5.5-GX1 were determined by the SRTM and EXPM, respectively. In the tumor region, the SKRC value exhibited an obvious trend with change in the tracer ID, but the Bp value was not sensitive to it. Both the Bp and SKRC values were independent of the camera IT. In addition, the ratio of the tumor-to-muscle region was correlated with the camera IT but was independent of the tracer ID. Dynamic fluorescence imaging in conjunction with a kinetic analysis may provide more quantitative information than static fluorescence imaging, especially for a priori information on the optimal ID of targeted probes for individual therapy.

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

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

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Determination of rare-earth elements in Luna 16 regolith sample by chemical spectral method

NASA Technical Reports Server (NTRS)

Stroganova, N. S.; Ryabukhin, V. A.; Laktinova, N. V.; Ageyeva, L. V.; Galkina, I. P.; Gatinskaya, N. G.; Yermakov, A. N.; Karyakin, A. V.

1974-01-01

An analysis was made of regolith from layer A of the Luna 16 sample for rare earth elements, by a chemical spectral method. Chemical and ion exchange concentrations were used to determine the content of 12 elements and Y at the level 0.001 to 0.0001 percent with 10 to 15 percent reproducibility of the emission determination. Results within the limits of reproducibility agree with data obtained by mass spectra, activation, and X-ray fluorescent methods.

Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

PubMed

Barlow, Andrew L; Macleod, Alasdair; Noppen, Samuel; Sanderson, Jeremy; Guérin, Christopher J

2010-12-01

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

Some Aspects of the Wavelength Dispersive X-Ray Determination of Fluorine Content in Various Matrices

NASA Astrophysics Data System (ADS)

Boča, M.; Gurišová, V.; Šimko, F.

2017-05-01

X-ray fluorescent signals of F Kα, Na Kα, Cl Kα, K Kα, and Ta Lα were measured by WD-XRF for various fluorine-containing systems: K2TaF7, Na3AlF6, K2ZrF6, NaF, and LiF (with NaCl and wax as additional additives). The data were recorded for 41 samples (in the form of pellets prepared in the laboratory) by more than 200 scans. The analysis of the measured fluorine X-ray fluorescence intensities demonstrated that the balance between absorption and enhancement effects depends strongly on the presence and concentration of other elements in the system. The experimental intensities of X-ray fluorescent radiation of fluorine for different systems with comparable fluorine content could differ by as much as 500%.

Investigating the intersystem crossing rate and triplet quantum yield of Protoporphyrin IX by means of pulse train fluorescence technique

NASA Astrophysics Data System (ADS)

Gotardo, Fernando; Cocca, Leandro H. Z.; Acunha, Thiago V.; Longoni, Ana; Toldo, Josene; Gonçalves, Paulo F. B.; Iglesias, Bernardo A.; De Boni, Leonardo

2017-04-01

Photophysical investigations of PPIX were described in order to determine the triplet conversion efficiency. Time resolved fluorescence and pulse train fluorescence were employed to characterize the main mechanism responsible for deactivation of the first singlet excited state (excited singlet and triplet states). Single pulse and Z-Scan analysis were employed to measure the singlet excited state absorption cross-sections. Theoretical calculations were performed in order to get some properties of PPIX in ground state, first singlet and triplet excited state. A TD-DFT result shows a great possibility of ISC associated to out-of-plane distortions in porphyrinic ring. Furthermore, the B and Q bands in the calculated spectrum are assigned to the four frontier molecular orbitals as proposed by Gouterman for free-based porphyrins.

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains☆

PubMed Central

Teague, Heather; Ross, Ron; Harris, Mitchel; Mitchell, Drake C.; Shaikh, Saame Raza

2012-01-01

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo. PMID:22841541

Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis▿

PubMed Central

Duffy, Ellen B.; Barquera, Blanca

2006-01-01

The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na+-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed. PMID:17041063

Determination of the carmine content based on spectrum fluorescence spectral and PSO-SVM

NASA Astrophysics Data System (ADS)

Wang, Shu-tao; Peng, Tao; Cheng, Qi; Wang, Gui-chuan; Kong, De-ming; Wang, Yu-tian

2018-03-01

Carmine is a widely used food pigment in various food and beverage additives. Excessive consumption of synthetic pigment shall do harm to body seriously. The food is generally associated with a variety of colors. Under the simulation context of various food pigments' coexistence, we adopted the technology of fluorescence spectroscopy, together with the PSO-SVM algorithm, so that to establish a method for the determination of carmine content in mixed solution. After analyzing the prediction results of PSO-SVM, we collected a bunch of data: the carmine average recovery rate was 100.84%, the root mean square error of prediction (RMSEP) for 1.03e-04, 0.999 for the correlation coefficient between the model output and the real value of the forecast. Compared with the prediction results of reverse transmission, the correlation coefficient of PSO-SVM was 2.7% higher, the average recovery rate for 0.6%, and the root mean square error was nearly one order of magnitude lower. According to the analysis results, it can effectively avoid the interference caused by pigment with the combination of the fluorescence spectrum technique and PSO-SVM, accurately determining the content of carmine in mixed solution with an effect better than that of BP.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

PubMed

Gallo, Alessandra; Boni, Raffaele; Tosti, Elisabetta

2018-01-01

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

A method for trace element determination of single Daphnia specimens using total reflection X-ray fluorescence spectrometry

NASA Astrophysics Data System (ADS)

Mages, Margarete; Woelfl, Stefan; v. Tümpling jun, Wolf

2001-11-01

Two new preparation techniques for total-reflection X-ray fluorescence (TXRF) element determination of single freshwater crustacean specimens (dry weight: 3-40 μg ind -1) have been developed and tested using Daphnia pulex from a deep, oligotrophic freshwater lake located in southern Chile. Dry method: Specimens were washed with 0.2 μm filtered lake water and frozen in liquid nitrogen. The freeze-dried Daphnia specimens were weighed using an ultra-fine microbalance and placed on quartz glass carriers for TXRF analysis. Wet method: Specimens were washed with 0.2 μm filtered lake water and placed on quartz glass carriers for TXRF analysis and dried in air. The dry weight was determined using the previously established body length-dry weight relationship. Method validation for both the dry and the wet preparation method in combination with TXRF spectrometry for the element determination in small single freshwater crustaceans showed that both methods can be used for routine investigations. There were no significant differences between the dry and the wet methods concerning the elements Ca, K, Fe, Zn, Br, P, Cu, but the determination of Mn, S and Sr revealed significant differences between the two methods. It seems that the dry method yields more precise results, but the wet method is easier to handle in the field when samples cannot be fixed with liquid nitrogen.

Rapid and sensitive detection of ketamine in blood using novel fluorescence genosensor.

PubMed

Ding, Yanjun; Li, Xingmei; Guo, Yadong; Yan, Jie; Ling, Jiang; Li, Weichen; Lan, Lingmei; Chang, Yunfeng; Cai, Jifeng; Zha, Lagabaiyla

2017-12-01

In recent years, drug abuse has been considered as a most challenging social problem that aroused public attention. Ketamine has increased in unregulated use as a 'recreational drug' in teenagers. However, there is no suitable and maneuverable detection method for ketamine in situ at the moment. Fluorescence sensor technique, with predominant recognition and simple operation, is a good potential application in drug detection. Here, we first reported a highly sensitive and selective fluorescence genosensor for rapid detection of ketamine based on DNA-templated silver nanoclusters (DNA-AgNCs) probes, in which the DNA sequence could specially recognize ketamine with high affinity. Parameters affecting detection efficiency were investigated and optimized. Under optimum conditions, the as-prepared genosensor can allow for the determination of ketamine in the concentration range of 0.0001-20 μg/mL with two linear equations: one is y = 2.84x-7.139 (R 2 = 0.987) for 0.0001-0.1 μg/mL, and the other is y = 1.87x-0.091 (R 2 = 0.962) for 0.1-20 μg/mL, and the estimated detection limit of ketamine is 0.06 ng/mL. Moreover, the feasibility of this proposed method was also demonstrated by analyzing forensic blood samples. Compared with official gas chromatography/mass spectrometry (GC/MS), this fluorescence genosensor is simple, rapid, and accurate for quantitative determination of ketamine in blood for pharmaceutical and forensic analysis. Overall, it is the first report on a fluorescence genosensor for detecting ketamine directly in blood. This research may provide a new insight for the analyst to band fluorescence genosensor technology together with drug monitoring in the battle against drug abuse and forensic examination. Graphical abstract High selectively detection of ketamine using a novel fluorescence genosensor based on DNA-AgNCs probe.

Diversity and Evolution of Coral Fluorescent Proteins

PubMed Central

Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Beltran-Ramirez, Victor; Miller, David J.; Wiedenmann, Jörg; Salih, Anya; Matz, Mikhail V.

2008-01-01

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors. PMID:18648549

ANAlyte: A modular image analysis tool for ANA testing with indirect immunofluorescence.

PubMed

Di Cataldo, Santa; Tonti, Simone; Bottino, Andrea; Ficarra, Elisa

2016-05-01

The automated analysis of indirect immunofluorescence images for Anti-Nuclear Autoantibody (ANA) testing is a fairly recent field that is receiving ever-growing interest from the research community. ANA testing leverages on the categorization of intensity level and fluorescent pattern of IIF images of HEp-2 cells to perform a differential diagnosis of important autoimmune diseases. Nevertheless, it suffers from tremendous lack of repeatability due to subjectivity in the visual interpretation of the images. The automatization of the analysis is seen as the only valid solution to this problem. Several works in literature address individual steps of the work-flow, nonetheless integrating such steps and assessing their effectiveness as a whole is still an open challenge. We present a modular tool, ANAlyte, able to characterize a IIF image in terms of fluorescent intensity level and fluorescent pattern without any user-interactions. For this purpose, ANAlyte integrates the following: (i) Intensity Classifier module, that categorizes the intensity level of the input slide based on multi-scale contrast assessment; (ii) Cell Segmenter module, that splits the input slide into individual HEp-2 cells; (iii) Pattern Classifier module, that determines the fluorescent pattern of the slide based on the pattern of the individual cells. To demonstrate the accuracy and robustness of our tool, we experimentally validated ANAlyte on two different public benchmarks of IIF HEp-2 images with rigorous leave-one-out cross-validation strategy. We obtained overall accuracy of fluorescent intensity and pattern classification respectively around 85% and above 90%. We assessed all results by comparisons with some of the most representative state of the art works. Unlike most of the other works in the recent literature, ANAlyte aims at the automatization of all the major steps of ANA image analysis. Results on public benchmarks demonstrate that the tool can characterize HEp-2 slides in terms of intensity and fluorescent pattern with accuracy better or comparable with the state of the art techniques, even when such techniques are run on manually segmented cells. Hence, ANAlyte can be proposed as a valid solution to the problem of ANA testing automatization. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Use of fluorescence spectroscopy to control ozone dosage in recirculating aquaculture systems.

PubMed

Spiliotopoulou, Aikaterini; Martin, Richard; Pedersen, Lars-Flemming; Andersen, Henrik R

2017-03-15

The aim of this study was to investigate the potential of fluorescence spectroscopy to be used as an ozone dosage determination tool in recirculating aquaculture systems (RASs), by studying the relationship between fluorescence intensities and dissolved organic matter (DOM) degradation by ozone, in order to optimise ozonation treatment. Water samples from six different Danish facilities (two rearing units from a commercial trout RAS, a commercial eel RAS, a pilot RAS and two marine water aquariums) were treated with different O 3 dosages (1.0-20.0 mg/L ozone) in bench-scale experiments, following which fluorescence intensity degradation was eventually determined. Ozonation kinetic experiments showed that RAS water contains fluorescent organic matter, which is easily oxidised upon ozonation in relatively low concentrations (0-5 mg O 3 /L). Fluorescence spectroscopy has a high level of sensitivity and selectivity in relation to associated fluorophores, and it is able to determine accurately the ozone demand of each system. The findings can potentially be used to design offline or online sensors based on the reduction by ozone of natural fluorescent-dissolved organic matter in RAS. The suggested indirect determination of ozone delivered into water can potentially contribute to a safer and more adequate ozone-based treatment to improve water quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dual x-ray fluorescence spectrometer and method for fluid analysis

DOEpatents

Wilson, Bary W.; Shepard, Chester L.

2005-02-22

Disclosed are an X-ray fluorescence (SRF) spectrometer and method for on-site and in-line determination of contaminant elements in lubricating oils and in fuel oils on board a marine vessel. An XRF source block 13 contains two radionuclide sources 16, 17 (e.g. Cd 109 and Fe 55), each oriented 180 degrees from the other to excite separate targets. The Cd 109 source 16 excites sample lube oil flowing through a low molecular weight sample line 18. The Fe 55 source 17 excites fuel oil manually presented to the source beam inside a low molecular weight vial 26 or other container. Two separate detectors A and B are arranged to detect the fluorescent x-rays from the targets, photons from the analyte atoms in the lube oil for example, and sulfur identifying x-rays from bunker fuel oil for example. The system allows both automated in-line and manual on-site analysis using one set of signal processing and multi-channel analyzer electronics 34, 37 as well as one computer 39 and user interface 43.

PubMed

Miranda, Geraldo Elias; Melani, Rodolfo Francisco Haltenhoff; Francisquini, Luiz; Daruge, Eduardo

2017-01-01

The aim of this study was to identify the combination of wavelength and filter that best detects tooth and bone, and to determine which biological materials (enamel, dental root or bone) have highest fluorescence intensity when exposed to an alternate light source (ALS). Tooth and bone samples were lighted with ALS and photographed. Adobe Photoshop™ and ImageJ™ softwares were used for image analysis. Data obtained by measuring the photograph pixels were subjected to analysis of variance. The mean values of significant effects were compared by the Tukey test. In all tests, the significance level was set at p≤0.05 and the values calculated by the SAS system. The results showed that the best combination for detecting tooth and bone is an illumination wavelength of 455 nm with an orange filter. The fluorescence of dental root is greater than that of enamel, which in turn is greater than that of bone. The biological material had markedly higher fluorescence than the inert material. This knowledge can help the forensic expert to screen and detect biological materials, for example in situations where there are fragmented teeth and small bones, both at the scene and in the laboratory.

[Rapid determination of major and trace elements in the salt lake clay minerals by X-ray fluorescence spectrometry].

PubMed

Wang, Xiao-Huan; Meng, Qing-Fen; Dong, Ya-Ping; Chen, Mei-Da; Li, Wu

2010-03-01

A rapid multi-element analysis method for clay mineral samples was described. This method utilized a polarized wave-length dispersive X-ray fluorescence spectrometer--Axios PW4400, which had a maximum tube power of 4 000 watts. The method was developed for the determination of As, Mn, Co, Cu, Cr, Dy, Ga, Mo, P, Pb, Rb, S, Sr, Ni, ,Cs, Ta, Th, Ti, U, V, Y, Zn, Zr, MgO, K2O, Na2O, CaO, Fe2O3, Al2O3, SiO2 and so on. Thirty elements in clay mineral species were measured by X-ray fluorescence spectrometry with pressed powder pellets. Spectral interferences, in particular the indirect interferences of each element, were studied. A method to distinguish the interference between each other periodic elements in element periodic table was put forward. The measuring conditions and existence were mainly investigated, and the selected background position as well as corrected spectral overlap for the trace elements were also discussed. It was found that the indirect spectral overlap line was the same important as direct spectral overlap line. Due to inducing the effect of indirect spectral overlap, some elements jlike Bi, Sn, W which do not need analysis were also added to the elements channel. The relative standard deviation (RSD) was in the range of 0.01% to 5.45% except three elements Mo, Cs and Ta. The detection limits, precisions and accuracies for most elements using this method can meet the requirements of sample analysis in clay mineral species.

Controlling photophysics of styrylnaphthalimides through TICT, fluorescence and E,Z-photoisomerization interplay.

PubMed

Panchenko, Pavel A; Arkhipova, Antonina N; Fedorova, Olga A; Fedorov, Yuri V; Zakharko, Marina A; Arkhipov, Dmitry E; Jonusauskas, Gediminas

2017-01-04

The photophysical properties of naphthalimide dyes NI1-3 with electron releasing 4-methoxy- (NI1), 3,4-dimethoxystyryl- (NI2) and dimethylaminostyryl (NI3) groups are examined in a variety of protic and aprotic solvents. All compounds demonstrate positive solvatochromism in the steady-state absorption and fluorescence spectra. The analysis of the dependence of the Stokes shift on the polarity of the solvent using the Lippert-Mataga equation allowed us to determine the change in the dipole moment upon excitation. The obtained data correspond to the formation of highly polar charge transfer states. Based on the transient absorption spectra and time-resolved fluorescence measurements, the presence of two different emissive states was definitely proved. The primarily formed planar Local Excited (LE) state dominates in non-polar solvents like cyclohexane and toluene where it relaxes mostly through fluorescence and E,Z-isomerisation pathways. In polar solvents, an alternative relaxation channel emerges that consists of twisting around single bond between styryl and naphthalimide fragments, which leads to the formation of a Twisted Intramolecular Charge Transfer (TICT) state. The factors affecting the fluorescence of TICT states are discussed. The observed spectral effects are rationalized using quantum-chemical calculations, X-ray data and NMR spectroscopy.

Fifteen years of PARAFAC application to organic matter fluorescence - progress, problems and possibilities

NASA Astrophysics Data System (ADS)

Murphy, K.; Stedmon, C. A.; Wunsch, U.

2017-12-01

The study of dissolved organic matter in aquatic milieu frequently involves measuring and interpreting fluorescence excitation emission matrices (EEMs) as a proxy for studying the total organic matter pool. Parallel Factor Analysis (PARAFAC) is used widely to identify and track independent organic matter fractions. This approach assumes that each EEM reflects the combined fluorescence signal from a limited number of unique, non-interacting chemical components, which are determined via a fitting algorithm. During the past fifteen years, considerable progress in understanding dissolved organic matter fluorescence has been achieved with the aid of PARAFAC; however, very few identical or ubiquitous fluorescence spectra have been independently identified. We studied the influence of wavelength selection on PARAFAC models and found this factor to have a decisive impact on PARAFAC spectra despite receiving little attention in most studies. Because large, chemically-diverse datasets may be too complex to analyse with PARAFAC, we are exploring novel methods for increasing variability in small datasets in order to reduce biases and increase interpretability. Our results suggest that spectral variability in PARAFAC models between studies are in many cases due to artefacts that could be minimised by careful experimental and modelling approaches.

Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study

NASA Astrophysics Data System (ADS)

Singh, T. Sanjoy; Mitra, Sivaprasad

2011-03-01

Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern-Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant ( κq), Stern-Volmer constant ( KSV) and also the ligand-protein association constant ( Ka). The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 10 4 dm 3 mol -1. In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure.

Fluorescence quenching as an indirect detection method for nitrated explosives.

PubMed

Goodpaster, J V; McGuffin, V L

2001-05-01

A novel approach based on fluorescence quenching is presented for the analysis of nitrated explosives. Seventeen common explosives and their degradation products are shown to be potent quenchers of pyrene, having Stern-Volmer constants that generally increase with the degree of nitration. Aromatic explosives such as 2,4,6-trinitrotoluene (2,4,6-TNT) are more effective quenchers than aliphatic or nitramine explosives. In addition, nitroaromatic explosives are found to have unique interactions with pyrene that lead to a wavelength dependence of their Stern-Volmer constants. This phenomenon allows for their differentiation from other nitrated explosives. The fluorescence quenching method is then applied to the determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine(HMX), 2,4,6-TNT, nitromethane, and ammonium nitrate in various commercial explosive samples. The samples are separated by capillary liquid chromatography with post-column addition of the pyrene solution and detection by laser-induced fluorescence. The indirect fluorescence quenching method shows increased sensitivity and selectivity over traditional UV-visible absorbance as well as the ability to detect a wider range of organic and inorganic nitrated compounds.

Spectral properties of the association nanoparticle system of ciprofloxacin-phloxine and its application to fluorescence analysis.

PubMed

Zou, Jieming; Jiang, Zhiliang; Wang, Lisheng; Li, Tingsheng; Liu, Qinye

2004-06-01

There is a fluorescence peak at 570 nm, and a maximum absorption peak at 560 nm for phloxine (PHLO) in a pH 7 water solution. Under these conditions, the ciprofloxacin cation (CPFX+) and PHLO- combine into hydrophobic CPFX-PHLO association molecule by means of static gravitation. There are stronger van der Waals forces and hydrophobic forces among the CPFX-PHLO molecules. Thus, they aggregate automatically to the (CPFX-PHLO)n association nanoparticle in red-violet color. That was characterized by scan electron microscopy (SEM), hyperfiltration and dialysis tests. In 0.04 M HCl, the red-violet nanoparticles exhibited a Rayleigh scattering peak at 470 nm, a resonance scattering peak at 580 nm, a maximum absorption wavelength at 565 nm, and a fluorescence peak at 450 nm. The fluorescence analytical conditions of CPFX have been considered. The CPFX concentration in the range of 1.0 x 10(-6)-4.0 x 10(-5) M is linear to the fluorescence intensity, F450nm. The detection limit was achieved at 4.0 x 10(-7) M CPFX. The CPFX in real samples was determined with satisfactory results.

Solid surface fluorescence immunosensor for ultrasensitive detection of hepatitis B virus surface antigen using PAMAM/CdTe@CdS QDs nanoclusters.

PubMed

Babamiri, Bahareh; Hallaj, Rahman; Salimi, Abdollah

2018-06-20

In the present study, we constructed an ultrasensitive solid surface fluorescence-immunosensor based on highly luminescent CdTe@CdS-PAMAM structures as nanoprobe for determination of HBsAg by monitoring fluorescence intensity. This strategy was achieved by using PAMAM as a signal amplifier; the PAMAM dendrimer with the many functional amine groups can amplify the fluorescence signal of QDs by covalent attachment of CdTe@CdS on PAMAM and hence, improve the sensitivity of the proposed method significantly. A sandwich type immunosensor was formed after the addition of HBsAg and the PAMAM-QD-Ab 2 , respectively. Under optimal conditions, the designed immunosensor demonstrates a good analytical performance for the HBsAg detection in an excellent linear range from 5 fg ml -1 to 0.15 ng ml -1 with the detection limit (LOD) of 0.6 fg ml -1 at a S/N ratio of 3. In addition, the analysis of human serum samples shows that the fluorescent immunoassay has the great potential for early diagnosis of hepatitis B and can be used for the detection of other tumor markers in clinical applications.

[Fluorescence spectra and quantum yield of TiO2 nanocrystals synthesized by alcohothermal method].

PubMed

Song, Cui-Hong; Li, Yan-Ting; Li, Jing; Wei, Yong-Ju; Hu, Yu-Zhu; Wei, Yu

2008-01-01

Fluorescence spectra and fluorescence quantum yield of TiO2 nanocrystals were studied. Using tetra n-butyl titanate as a starting material, a facile alcohothermal technique was used to synthesize TiO2 nanocrystals. As can be seen from the transmittance electron microscopy (TEM) image, TiO2 nanocrystals with a relatively uniform particle size distribution of < 10 nm are present in the transparent sol. The transparent sol presents a strong stable fluorescence emission with a maximum at 450 nm, which is greatly dependent on the size quantization effects, defect energy level and the surface state of TiO2 nanocrystals. The quantum yield (gamma) of TiO2 was determined by the relative comparison procedure, using freshly prepared analytical purity quinine sulfate in 0.05 mol x L(-1) H2SO4 as a relative quantum yield standard. The emission quantum yield of TiO2 nanocrystals prepared in alcoholic media was calculated to be about 0.20 at wavelengths ranging from 330 to 370 nm, which was much higher than the values reported in previous works. So, it is supposed that nano-TiO2 will be applied as a potential quantum dots fluorescence probe in biological analysis.

Determination of lysergic acid diethylamide (LSD) in mouse blood by capillary electrophoresis/ fluorescence spectroscopy with sweeping techniques in micellar electrokinetic chromatography.

PubMed

Fang, Ching; Liu, Ju-Tsung; Chou, Shiu-Huey; Lin, Cheng-Huang

2003-03-01

The separation and on-line concentration of lysergic acid diethylamide (LSD) in mouse blood was achieved by means of capillary electrophoresis/fluorescence spectroscopy using sodium dodecyl sulfate (SDS) as the surfactant. Techniques involving on-line sample concentration, including sweeping micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were applied; the optimum on-line concentration and separation conditions were determined. In the analysis of an actual sample, LSD was found in a blood sample from a test mouse (0.1 mg LSD fed to a 20 g mouse; approximately 1/10 to the value of LD(50)). As a result, 120 and 30 ng/mL of LSD was detected at 20 and 60 min, respectively, after ingestion of the doses.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Hyeonggon; Attota, Ravikiran, E-mail: ravikiran.attota@nist.gov; Tondare, Vipin

We present a method that uses conventional optical microscopes to determine the number of nanoparticles in a cluster, which is typically not possible using traditional image-based optical methods due to the diffraction limit. The method, called through-focus scanning optical microscopy (TSOM), uses a series of optical images taken at varying focus levels to achieve this. The optical images cannot directly resolve the individual nanoparticles, but contain information related to the number of particles. The TSOM method makes use of this information to determine the number of nanoparticles in a cluster. Initial good agreement between the simulations and the measurements ismore » also presented. The TSOM method can be applied to fluorescent and non-fluorescent as well as metallic and non-metallic nano-scale materials, including soft materials, making it attractive for tag-less, high-speed, optical analysis of nanoparticles down to 45 nm diameter.« less

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

PubMed

Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter

2018-01-01

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Rainwater analysis by synchrotron radiation-total reflection X-ray fluorescence

NASA Astrophysics Data System (ADS)

López, María L.; Ceppi, Sergio A.; Asar, María L.; Bürgesser, Rodrigo E.; Ávila, Eldo E.

2015-11-01

Total reflection X-ray fluorescence analysis excited with synchrotron radiation was used to quantify the elemental concentration of rainwater in Córdoba, Argentina. Standard solutions with gallium as internal standard were prepared for the calibration curves. Rainwater samples of 5 μl were added to an acrylic reflector, allowed to dry, and analyzed for 200 s measuring time. The elemental concentrations of As, Ca, Co, Cr, Cu, Fe, K, Mn, Ni, Pb, S, Sr, V, and Zn were determined. The electrical conductivity, pH, and elemental concentrations were compared to data previously reported for the soluble fraction of rainwater at different sites. A factor analysis was performed in order to determine the sources that contributed to the elemental concentration in rainwater. Anthropogenic sources were identified as traffic pollution, vehicular emissions, and metallurgical factories. The quality of rainwater was analyzed by comparing the concentrations of all the elements in rainwater samples with the WHO guideline values for drinking water. The results show the need to control the atmospheric emissions in order to preserve the quality of rainwater. SR-TXRF analysis of chemical composition of rainwater in Córdoba represents the very first contribution in the region to the knowledge of the concentration of trace metals in the soluble fraction of rainwater. These data are scarce, especially in the Southern Hemisphere.

Determination of U, Th and K in bricks by gamma-ray spectrometry, X-ray fluorescence analysis and neutron activation analysis

NASA Astrophysics Data System (ADS)

Bártová, H.; Kučera, J.; Musílek, L.; Trojek, T.; Gregorová, E.

2017-11-01

Knowledge of the content of natural radionuclides in bricks can be important in some cases in dosimetry and application of ionizing radiation. Dosimetry of naturally occurring radionuclides in matter (NORM) in general is one of them, the other one, related to radiation protection, is radon exposure evaluation, and finally, it is needed for the thermoluminescence (TL) dating method. The internal dose rate inside bricks is caused mostly by contributions of the natural radionuclides 238U, 232Th, radionuclides of their decay chains, and 40K. The decay chain of 235U is usually much less important. The concentrations of 238U, 232Th and 40K were measured by various methods, namely by gamma-ray spectrometry, X-ray fluorescence analysis (XRF), and neutron activation analysis (NAA) which was used as a reference method. These methods were compared from the point of view of accuracy, limit of detection (LOD), amount of sample needed and sample handling, time demands, and instrument availability.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

PubMed

Sorio, Daniela; De Palo, Elio Franco; Bertaso, Anna; Bortolotti, Federica; Tagliaro, Franco

2017-02-01

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

Comparison of visual microscopic and computer-automated fluorescence detection of rabies virus neutralizing antibodies.

PubMed

Péharpré, D; Cliquet, F; Sagné, E; Renders, C; Costy, F; Aubert, M

1999-07-01

The rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVNT) are both diagnostic tests for determining levels of rabies neutralizing antibodies. An automated method for determining fluorescence has been implemented to reduce the work time required for fluorescent visual microscopic observations. The automated method offers several advantages over conventional visual observation, such as the ability to rapidly test many samples. The antibody titers obtained with automated techniques were similar to those obtained with both the RFFIT (n = 165, r = 0.93, P < 0.001) and the FAVNT (n = 52, r = 0.99, P < 0.001).

Development of dual-emission ratiometric probe-based on fluorescent silica nanoparticle and CdTe quantum dots for determination of glucose in beverages and human body fluids.

PubMed

Zhai, Hong; Feng, Ting; Dong, Lingyu; Wang, Liyun; Wang, Xiangfeng; Liu, Hailing; Liu, Yuan; Chen, Luan; Xie, MengXia

2016-08-01

A novel dual emission ratiometric fluorescence probe for determination of glucose has been developed. The reference dye fluorescence isothiocyanate (FITC) has been encapsulated in the silica nanoparticles and then the red emission CdTe QDs were grafted on the surface of the silica particles to obtain the fluorescence probe. With glucose and dopamine as substrates, the glucose level was proportional to the fluorescence ratio change of above probe caused by dopamine oxidation, which was produced via bienzyme catalysis (glucose oxidase and horseradish peroxidase). The established approach was sensitive and selective, and has been applied to determine the glucose in beverage, urine and serum samples. The average recoveries of the glucose at various spiking levels ranged from 95.5% to 108.9% with relative standard deviations from 1.5% to 4.3%. The results provided a clue to develop sensors for rapid determination of the target analytes from complex matrices. Copyright © 2016 Elsevier Ltd. All rights reserved.

Error minimization algorithm for comparative quantitative PCR analysis: Q-Anal.

PubMed

OConnor, William; Runquist, Elizabeth A

2008-07-01

Current methods for comparative quantitative polymerase chain reaction (qPCR) analysis, the threshold and extrapolation methods, either make assumptions about PCR efficiency that require an arbitrary threshold selection process or extrapolate to estimate relative levels of messenger RNA (mRNA) transcripts. Here we describe an algorithm, Q-Anal, that blends elements from current methods to by-pass assumptions regarding PCR efficiency and improve the threshold selection process to minimize error in comparative qPCR analysis. This algorithm uses iterative linear regression to identify the exponential phase for both target and reference amplicons and then selects, by minimizing linear regression error, a fluorescence threshold where efficiencies for both amplicons have been defined. From this defined fluorescence threshold, cycle time (Ct) and the error for both amplicons are calculated and used to determine the expression ratio. Ratios in complementary DNA (cDNA) dilution assays from qPCR data were analyzed by the Q-Anal method and compared with the threshold method and an extrapolation method. Dilution ratios determined by the Q-Anal and threshold methods were 86 to 118% of the expected cDNA ratios, but relative errors for the Q-Anal method were 4 to 10% in comparison with 4 to 34% for the threshold method. In contrast, ratios determined by an extrapolation method were 32 to 242% of the expected cDNA ratios, with relative errors of 67 to 193%. Q-Anal will be a valuable and quick method for minimizing error in comparative qPCR analysis.

Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest.

PubMed

Libregts, S F W M; Arkesteijn, G J A; Németh, A; Nolte-'t Hoen, E N M; Wauben, M H M

2018-05-20

Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation. © 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ziqiang

1999-12-10

Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based onmore » monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10 -8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.« less

Study of marbles from Middle Atlas (Morocco): elemental, mineralogical and structural analysis

NASA Astrophysics Data System (ADS)

Khrissi, S.; Bejjit, L.; Haddad, M.; Falguères, C.; Ait Lyazidi, S.; El Amraoui, M.

2018-05-01

A series of marbles sampled from the region of Middle Atlas (Morocco), are characterized by different complementary spectroscopic techniques. X-Ray fluorescence is used to determine elemental composition of rock while X-Ray diffraction and the Raman spectroscopy are used to determine major crystalline phases (calcite and dolomite) and minor ones (quartz).The samples display typical EPR spectra of Mn2+ in calcite and reveal the presence of Fe3+ ions.

Time-resolved confocal fluorescence microscopy: novel technical features and applications for FLIM, FRET and FCS using a sophisticated data acquisition concept in TCSPC

NASA Astrophysics Data System (ADS)

Koberling, Felix; Krämer, Benedikt; Kapusta, Peter; Patting, Matthias; Wahl, Michael; Erdmann, Rainer

2007-05-01

In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full experimental information. This format allows us not only to easily study dependencies between various fluorescence parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example, cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing performance can be significantly increased by using new SPAD detectors which enable, in conjunction with new TCSPC electronics, an overall IRF width of less than 120 ps maintaining single molecule sensitivity.

Photolysis of Indole-Containing Mycotoxins to Fluorescent Products

USDA-ARS?s Scientific Manuscript database

Photochemical reaction of the non-fluorescent mycotoxin cyclopiazonic acid (CPA) to fluorescent products was recently reported. Because CPA contains an indole moiety, believed to contribute to the fluorescence, it was of interest to determine whether the effect might be more generally applicable to ...

The determination of nanogram amounts of Chromium in urine by x-ray fluorescence spectroscopy

USGS Publications Warehouse

Beyermann, K.; Rose, H.J.; Christian, R.P.

1969-01-01

Nanogram amounts of chromium can be extracted as oxinate into chloform. By treatment of the chloroform layer 3 M hydrochloric acid, oxinates of other elements and excess of reagent are removed, leaving a chloroform solution of the chromium chelate only. This solution is concentrated and transferred to the top of a small brass rod acting as sample holder. The intensity of the X-ray fluorescence of the Cr K?? line is measured with curved crystal optics. Chromium amounts greater than 5 ng can be detected. The application of the procedure to the analysis of the chromium content of urine is demonstrated. ?? 1969.

DETERMINATION OF ALIPHATIC AMINES IN WATER USING DERIVATIZATION WITH FLUORESCEIN ISOTHIOCYANATE AND CAPILLARY ELECTROPHORESIS/LASER-INDUCED FLUORESCENCE DETECTION.

EPA Science Inventory

Detection-oriented derivatization of aliphatic amines and amine functional groups in coumpounds of environmental interest was studied using fluorescein isothiocyanate (FITC) with separation/determination by capillary electrophoresis/laser-induced fluorescence. Determinative level...

Dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells.

PubMed

Sakai, Tatsuya; Ohuchi, Masanobu; Imai, Masaki; Mizuno, Takafumi; Kawasaki, Kazunori; Kuroda, Kazumichi; Yamashina, Shohei

2006-02-01

Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.

Extraction and determination of biogenic amines in fermented sausages and other meat products using reversed-phase-HPLC.

PubMed

Straub, B; Schollenberger, M; Kicherer, M; Luckas, B; Hammes, W P

1993-09-01

A convenient method is described for the analysis of biogenic amines (BA) by means of reversed-phase-HPLC. The method is characterized by multi-channel UV detection (diodearray), subsequent post-column derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid, and fluorescence detection. For the analysis of meat products and especially fermented sausages an optimized perchloric acid extraction process was introduced to determine putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. BA recoveries from meat ranged between 96 and 113% with a detection limit for amines of 0.5 mg/kg.

Quantitative method of determining beryllium or a compound thereof in a sample

DOEpatents

McCleskey, T. Mark; Ehler, Deborah S.; John, Kevin D.; Burrell, Anthony K.; Collis, Gavin E.; Minogue, Edel M.; Warner, Benjamin P.

2006-10-31

A method of determining beryllium or a beryllium compound thereof in a sample, includes providing a sample suspected of comprising beryllium or a compound thereof, extracting beryllium or a compound thereof from the sample by dissolving in a solution, adding a fluorescent indicator to the solution to thereby bind any beryllium or a compound thereof to the fluorescent indicator, and determining the presence or amount of any beryllium or a compound thereof in the sample by measuring fluorescence.

Quantitative method of determining beryllium or a compound thereof in a sample

DOEpatents

McCleskey, T. Mark; Ehler, Deborah S.; John, Kevin D.; Burrell, Anthony K.; Collis, Gavin E.; Minogue, Edel M.; Warner, Benjamin P.

2010-08-24

A method of determining beryllium or a beryllium compound thereof in a sample, includes providing a sample suspected of comprising beryllium or a compound thereof, extracting beryllium or a compound thereof from the sample by dissolving in a solution, adding a fluorescent indicator to the solution to thereby bind any beryllium or a compound thereof to the fluorescent indicator, and determining the presence or amount of any beryllium or a compound thereof in the sample by measuring fluorescence.

Determining the size of nanoparticles in the example of magnetic iron oxide core-shell systems

NASA Astrophysics Data System (ADS)

Jarzębski, Maciej; Kościński, Mikołaj; Białopiotrowicz, Tomasz

2017-08-01

The size of nanoparticles is one of the most important factors for their possible applications. Various techniques for the nanoparticle size characterization are available. In this paper selected techniques will be considered base on the prepared core-shell magnetite nanoparticles. Magnetite is one of the most investigated and developed magnetic material. It shows interesting magnetic properties which can be used for biomedical applications, such as drug delivery, hypothermia and also as a contrast agent. To reduce the toxic effects of Fe3O4, magnetic core was covered by dextran and gelatin. Moreover, the shell was doped by fluorescent dye for confocal microscopy investigation. The main investigation focused on the methods for particles size determination of modified magnetite nanoparticles prepared with different techniques. The size distribution were obtained by nanoparticle tracking analysis, dynamic light scattering and transmission electron microscopy. Furthermore, fluorescent correlation spectroscopy (FCS) and confocal microscopy were used to compare the results for particle size determination of core-shell systems.

Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Liu, Xin; Li, Dian-Fan; Wang, Yun; Lu, Ying-Tang

2004-12-17

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ-ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20 mM borate buffer (pH 9.35) containing 40 mM sodium dodecyl sulfate and 10 mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 microM with a correlation of 0.9967. The concentration detection limit for ACC was 10 nM (signal-to-noise = 3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crawford, Andrew M.; Kurecka, Patrick; Yim, Tsz Kwan

An X-ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X-ray beam. X-ray fluorescence was then used to determine the mass of metal in each cell. By making single-cell measurements, the population heterogeneity for metals in the µ M to m M concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approachmore » has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ~4 cells min –1. These data show evidence for surprisingly broad metal distributions. Lastly, details of the device design, data analysis and opportunities for further sensitivity improvement are described.« less

De facto molecular weight distributions of glucans by size-exclusion chromatography combined with mass/molar-detection of fluorescence labeled terminal hemiacetals.

PubMed

Praznik, Werner; Huber, Anton

2005-09-25

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).

INVESTIGATION OF CE/LIF AS A TOOL IN THE CHARACTERIZATION OF SEWAGE EFFLUENT FOR FLUORESCENT ACIDICS: DETERMINATION OF SALICYLIC ACID

EPA Science Inventory

The investigation of emerging contaminant issues is a proactive effort in environmental analysis. As a part of this effort, sewage effluent is of current analytical interest because of the presence of pharmaceuticals and their metabolites and personal care products The en...

IDENTIFICATION OF SEX CHROMOSOME MOLECULAR MARKERS USING RAPDS AND FLUORESCENT IN SITU HYBRIDIZATION IN RAINBOW TROUT

EPA Science Inventory

The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphi...

Determination of arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid in cereals by hydride generation atomic fluorescence spectrometry

NASA Astrophysics Data System (ADS)

Matos Reyes, M. N.; Cervera, M. L.; Campos, R. C.; de la Guardia, M.

2007-09-01

A fast, sensitive and simple non-chromatographic analytical method was developed for the speciation analysis of toxic arsenic species in cereal samples, namely rice and wheat semolina. An ultrasound-assisted extraction of the toxic arsenic species was performed with 1 mol L - 1 H 3PO 4 and 0.1% (m/v) Triton XT-114. After extraction, As(III), As(V), dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) concentrations were determined by hydride generation atomic fluorescence spectrometry using a series of proportional equations corresponding to four different experimental reduction conditions. The detection limits of the method were 1.3, 0.9, 1.5 and 0.6 ng g - 1 for As(III), As(V), DMA and MMA, respectively, expressed in terms of sample dry weight. Recoveries were always greater than 90%, and no species interconversion occurred. The speciation analysis of a rice flour reference material certified for total arsenic led to coherent results, which were also in agreement with other speciation studies made on the same certified reference material.

New approaches to the analysis of complex samples using fluorescence lifetime techniques and organized media

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hertz, P.R.

Fluorescence spectroscopy is a highly sensitive and selective tool for the analysis of complex systems. In order to investigate the efficacy of several steady state and dynamic techniques for the analysis of complex systems, this work focuses on two types of complex, multicomponent samples: petrolatums and coal liquids. It is shown in these studies dynamic, fluorescence lifetime-based measurements provide enhanced discrimination between complex petrolatum samples. Additionally, improved quantitative analysis of multicomponent systems is demonstrated via incorporation of organized media in coal liquid samples. This research provides the first systematic studies of (1) multifrequency phase-resolved fluorescence spectroscopy for dynamic fluorescence spectralmore » fingerprinting of complex samples, and (2) the incorporation of bile salt micellar media to improve accuracy and sensitivity for characterization of complex systems. In the petroleum studies, phase-resolved fluorescence spectroscopy is used to combine spectral and lifetime information through the measurement of phase-resolved fluorescence intensity. The intensity is collected as a function of excitation and emission wavelengths, angular modulation frequency, and detector phase angle. This multidimensional information enhances the ability to distinguish between complex samples with similar spectral characteristics. Examination of the eigenvalues and eigenvectors from factor analysis of phase-resolved and steady state excitation-emission matrices, using chemometric methods of data analysis, confirms that phase-resolved fluorescence techniques offer improved discrimination between complex samples as compared with conventional steady state methods.« less

Laser-induced fluorescence measurement of the oil film thickness in an internal combustion engine

NASA Astrophysics Data System (ADS)

Ostroski, Greg M.; Ghandhi, Jaal B.

1997-11-01

The use of a fluorescent dopant molecule to enhance the natural fluorescence of motor oils, and allow quantitative determination of temperature and film thickens in internal combustion engines has been investigated. Measurement of the fluorescence as a function of temperature were made with neat Mobil 1, and solutions of the dopant BTBP in mineral oil and Mobil 1. The fluorescence yield of neat Mobil 1 was found to vary by 30 percent over the temperature range explored, but the spectral characteristics, as measured with bandpass filters, were unaffected by temperature. The BTBP fluorescence was found to increase significantly with temperature, and it was found the narrower regions in the spectrum increased proportionally more than the fluorescence collected over the entire spectrum, allowing a determination of temperature to be made which can then be used to correct for the change in fluorescence yield. Solutions in Mobil 1 showed a smaller increase than that observed in mineral oil.

Determination of ethambutol by a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wu, Wen-Ying; Yang, Ji-Yuan; Du, Li-Ming; Wu, Hao; Li, Chang-Feng

2011-08-01

The competitive reaction between ethambutol and two fluorescent probes (i.e., berberine and palmatine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry. The CB[7] reacts with these probes to form stable complexes, and the fluorescence intensity of the complexes is greatly enhanced. In addition, the excitation and emission wavelengths of their complexes moved to wavelengths of 343 nm and 495 nm, respectively. However, the addition of ethambutol dramatically quenches the fluorescence intensity of the two complexes. Accordingly, a couple of new fluorescence quenching methods for the determination of ethambutol were established. The methods can be applied for quantifying ethambutol. A linear relationship between the fluorescence quenching values (Δ F) and ethambutol concentration exists in the range of 5.0-1000.0 ng mL -1, with a correlation coefficient ( r) of 0.9997. The detection limit is 1.7 ng mL -1. The fluorescent probe of berberine has higher sensitivity than palmatine. This paper also discusses the mechanism of fluorescence indicator probes.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

PubMed

Xian, Jian-An; Guo, Hui; Li, Bin; Miao, Yu-Tao; Ye, Jian-Min; Zhang, Sheng-Peng; Pan, Xun-Bin; Ye, Chao-Xia; Wang, An-Li; Hao, Xuan-Ming

2013-12-01

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 μM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe

NASA Astrophysics Data System (ADS)

Mathejczyk, Julia Eva; Pauli, Jutta; Dullin, Christian; Resch-Genger, Ute; Alves, Frauke; Napp, Joanna

2012-07-01

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Fluorescence lifetime imaging of calcium flux in neurons in response to pulsed infrared light

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Sedelnikova, Anna; Tolstykh, Gleb P.; Ibey, Bennett L.; Beier, Hope T.

2017-02-01

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye

PubMed Central

Klemm, Matthias; Schweitzer, Dietrich; Peters, Sven; Sauer, Lydia; Hammer, Martin; Haueisen, Jens

2015-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX’s applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation. PMID:26192624

Method and apparatus for simultaneously measuring temperature and pressure

DOEpatents

Hirschfeld, Tomas B.; Haugen, Gilbert R.

1988-01-01

Method and apparatus are provided for simultaneously measuring temperature and pressure in a class of crystalline materials having anisotropic thermal coefficients and having a coefficient of linear compression along the crystalline c-axis substantially the same as those perpendicular thereto. Temperature is determined by monitoring the fluorescence half life of a probe of such crystalline material, e.g., ruby. Pressure is determined by monitoring at least one other fluorescent property of the probe that depends on pressure and/or temperature, e.g., absolute fluorescent intensity or frequency shifts of fluorescent emission lines.

Lead foil in dental X-ray film: Backscattering rejection or image intensifier?

NASA Astrophysics Data System (ADS)

Hönnicke, M. G.; Delben, G. J.; Godoi, W. C.; Swinka-Filho, V.

2014-11-01

Dental X-ray films are still largely used due to sterilization issues, simplicity and, mainly, economic reasons. These films almost always are double coated (double emulsion) and have a lead foil in contact with the film for X-ray backscattering rejection. Herein we explore the use of the lead foil as an image intensifier. In these studies, spatial resolution was investigated when images were acquired on the dental X-ray films with and without the lead foil. Also, the lead foil was subjected to atomic analysis (fluorescent measurements) and structure analysis (X-ray diffraction). We determined that the use of the lead foil reduces the exposure time, however, does not affect the spatial resolution on the acquired images. This suggests that the fluorescent radiation spread is smaller than the grain sizes of the dental X-ray films.

Development and validation of a reversed-phase fluorescence HPLC method for determination of bucillamine in human plasma using pre-column derivatization with monobromobimane.

PubMed

Lee, Kang Choon; Chun, Young Goo; Kim, Insoo; Shin, Beom Soo; Park, Eun-Seok; Yoo, Sun Dong; Youn, Yu Seok

2009-07-15

A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50 ng/mL to 10 microg/mL) in human plasma (r(2)=0.9998). The lower limit of quantitation (LLOQ) was 50 ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300 mg) of bucillamine to 20 healthy Korean volunteers.

A quantum dot-based immunoassay for screening of tylosin and tilmicosin in edible animal tissues.

PubMed

Le, Tao; Zhu, Liqian; Yang, Xian

2015-01-01

A rapid, indirect competitive fluorescence-linked immunosorbent assay (ic-FLISA) based on quantum dots (QDs) as the fluorescent marker was developed for the detection of tylosin and tilmicosin in edible animal tissues. The end point fluorescent detection system was carried out using QDs conjugated with goat anti-mouse secondary antibody. The limits of detection (LODs) for the determination of tylosin and tilmicosin were 0.02 and 0.04 μg kg(-1), respectively. This detection method was used to analyse spiked samples and the recoveries ranged from 83.5% to 98.7% for tylosin and from 81.8% to 98.2% for tilmicosin. In real porcine tissue sample analysis, the results of ic-FLISA were similar to those obtained from an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to an HPLC method indicating its potential for tylosin and tilmicosin screening in edible animal tissues.

Supramolecular interactions of nonsteroidal anti-inflammatory drug in nanochannels of molecular containers: a spectroscopic, thermogravimetric and microscopic investigation.

PubMed

Maity, Banibrata; Chatterjee, Aninda; Ahmed, Sayeed Ashique; Seth, Debabrata

2014-11-10

Supramolecular host-guest complexation between the nonsteroidal anti-inflammatory drug indomethacin (IMC) and molecular containers were investigated. The weakly fluorescent drug molecule becomes highly fluorescent on complexation with different molecular containers, and time-resolved fluorescence emission spectroscopy reveals that the lifetime components of IMC significantly increase in the presence of molecular containers, compared with the lifetimes in neat water. The respective solid host-guest complexes were synthesised and characterised by Fourier transform infrared and (1) H nuclear magnetic resonance spectroscopic analysis. Microscopy techniques were used to analyse modifications of the surface morphology, owing to the formation of supramolecular complexes. The effect of the molecular container on the optical properties of IMC has also been investigated to determine the effect of nanochannels of different size and structure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of microgravity to improve the efficiency and power output of Nd-doped laser glasses

NASA Technical Reports Server (NTRS)

Ray, Chandra S.

1992-01-01

The objectives of this research are to: (1) obtain further evidence and understand the science for the reported improvement in chemical homogeneity in glasses prepared in microgravity; and (2) study the feasibility of improving the optical and fluorescence properties, particularly, the limit for Nd(+3) concentration quenching and threshold energy for laser action for laser glasses prepared in microgravity. Attention was directed to ground based investigation whose primary purpose was to determine the suitability and conditions for processing these laser glasses in space. This report describes that the scientific and technical information required for planning flight experiments for these glasses have been obtained, and the preparation for handling and analyzing post flight samples have also been taken. Instruments required for measuring the fluorescence properties of interest have been constructed. The optical and fluorescence properties for the glasses have been measured and made available for comparative property analysis.

Arsenic species analysis in porewaters and sediments using hydride generation atomic fluorescence spectrometry.

PubMed

Liao, Meng-xia; Deng, Tian-long

2006-01-01

It was observed that the atomic fluorescence emission due to As(V) could has a 10% to 40% of fluorescence emission signal during the determination of As(III) in the mixture of As(III) and As(V). Besides, interferes from heavy metals such as Pb(lIl), Cu(ll) can cause severe increase of the signals as compared to the insignificant effects caused by Cd(II), Zn(ll), Mn(II) and Fe(Ill). On the basis of further studies, the masking agent of 8-hydroxyquinoline was used as an efficient agent to eliminate interference of As(V) emission and the heavy metal of Cu2+ and Pb2+ in the measurements of arsenic species. After a series standard additions and CRM researches, a sensitive and interference-free analytical procedure was developed for the speciation of arsenic in samples of porewaters and sediments in Poyang Lake, China.

Seasonal Variation of Eutrophication in Some Lakes of Danube Delta Biosphere Reserve.

PubMed

Török, Liliana; Török, Zsolt; Carstea, Elfrida M; Savastru, Dan

2017-01-01

  To understand the trophic state of lakes, this study aims to determine the dynamics of phytoplankton assemblages and the main factors that influence their seasonal variation. Sampling campaigns were carried out in three lakes from the Danube Delta Biosphere Reserve. Spectral analysis of specific phytoplankton pigments was applied as a diagnostic marker to establish the distribution and composition of phytoplankton taxonomic groups. Fluorescence spectroscopy was used to quantify changes in dissolved organic matter (DOM). The relative contribution of the main phytoplankton groups to the total phytoplankton biomass and the trend of development during succession of the seasons showed that cyanobacteria could raise potential ecological or human health problems. Moreover, fluorescence spectroscopy revealed that Cryptophyta and cyanobacteria were the main contributors to the protein-like components of DOM. It was concluded that fluorescence could be used to provide a qualitative evaluation of the eutrophication degree in Danube Delta lakes.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

PubMed

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Apparatus and method for determining the optical power passing through an optical fiber

DOEpatents

Toeppen, John S.

1995-01-01

An apparatus and method for determining the optical power transmitted through an optical fiber. The invention is based on measuring the intensity of the fluorescence produced by a doped segment of an optical fiber. The dopant is selected so that it emits light at a different wavelength than that responsible for producing the fluorescence. The doped segment is of sufficient length and dopant concentration to provide a detectable signal, but short enough to prevent the doped segment from serving as a gain medium, resulting in amplified spontaneous emission and excess fluorescence traveling along the optical fiber. The dopant material is excited by the optical signal carried by the fiber, causing a fluorescence. In the preferred embodiment the intensity of the fluorescence is proportional to the intensity of the propagating light. The signal power is then determined from the intensity of the fluorescence. The intensity of the fluorescent signal is measured by a photodetector placed so as to detect the light emitted through the side of the doped segment. The detector may wrap around the circumference of the fiber, or be placed to one side and used in conjunction with a reflector placed on the opposing side of the fiber. Filters may be used to shield the detector from other light sources and assist with accurately determining the optical power of the signal propagating within the fiber.

Apparatus and method for determining the optical power passing through an optical fiber

DOEpatents

Toeppen, John S.

1995-04-04

An apparatus and method for determining the optical power transmitted through an optical fiber. The invention is based on measuring the intensity of the fluorescence produced by a doped segment of an optical fiber. The dopant is selected so that it emits light at a different wavelength than that responsible for producing the fluorescence. The doped segment is of sufficient length and dopant concentration to provide a detectable signal, but short enough to prevent the doped segment from serving as a gain medium, resulting in amplified spontaneous emission and excess fluorescence traveling along the optical fiber. The dopant material is excited by the optical signal carried by the fiber, causing a fluorescence. In the preferred embodiment the intensity of the fluorescence is proportional to the intensity of the propagating light. The signal power is then determined from the intensity of the fluorescence. The intensity of the fluorescent signal is measured by a photodetector placed so as to detect the light emitted through the side of the doped segment. The detector may wrap around the circumference of the fiber, or be placed to one side and used in conjunction with a reflector placed on the opposing side of the fiber. Filters may be used to shield the detector from other light sources and assist with accurately determining the optical power of the signal propagating within the fiber.

Real-time analysis of endogenous protoporphyrin IX fluorescence from δ-aminolevulinic acid and its derivatives reveals distinct time- and dose-dependent characteristics in vitro

NASA Astrophysics Data System (ADS)

Kiesslich, Tobias; Helander, Linda; Illig, Romana; Oberdanner, Christian; Wagner, Andrej; Lettner, Herbert; Jakab, Martin; Plaetzer, Kristjan

2014-08-01

Photodynamic therapy (PDT) and photodiagnosis based on the intracellular production of the photosensitizer protoporphyrin IX (PPIX) by administration of its metabolic precursor δ-aminolevulinic acid (ALA) achieved their breakthrough upon the clinical approval of MAL (ALA methyl ester) and HAL (ALA hexyl ester). For newly developed ALA derivatives or application in new tumor types, in vitro determination of PPIX formation involves multiparametric experiments covering variable pro-drug concentrations, medium composition, time points of analysis, and cell type(s). This study uses a fluorescence microplate reader with a built-in temperature and atmosphere control to investigate the high-resolution long-term kinetics (72 h) of cellular PPIX fueled by administration of either ALA, MAL, or HAL for each 10 different concentrations. For simultaneous proliferation correction, A431 cells were stably transfected with green fluorescent protein. The results indicate that the peak PPIX level is a function of both, incubation concentration and period: maximal PPIX is generated with 1 to 2-mM ALA/MAL or 0.125-mM HAL; also, the PPIX peak shifts to longer incubation periods with increasing pro-drug concentrations. The results underline the need for detailed temporal analysis of PPIX formation to optimize ALA (derivative)-based PDT or photodiagnosis and highlight the value of environment-controlled microplate readers for automated in vitro analysis.

Determination of amantadine and rimantadine using a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wang, Guang-Quan; Qin, Yan-Fang; Du, Li-Ming; Li, Jun-Fei; Jing, Xu; Chang, Yin-Xia; Wu, Hao

2012-12-01

Amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) are non-fluorescent in aqueous solutions. This property makes their determination through direct fluorescent method difficult. The competing reactions and the supramolecular interaction mechanisms between the two drugs and coptisine (COP) as they fight for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AMA or RIM in their pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.0040 to 1.0 μg mL-1 with a detection limit ranging from 0.0012 to 0.0013 μg mL-1. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red

NASA Astrophysics Data System (ADS)

Shao-Qin, Lin; Xuan, Lin; Shi-Rong, Hu; Li-Qing, Zeng; Yan, Wang; Li, Chen; Jia-Ming, Liu; Long-Di, Li

2005-11-01

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 °C for 30 min and Al 3+ can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ng l -1 with a detection limit of 5.3 pg l -1. The regression equation can be expressed as Δ If = 8.731 + 21.73 c (ng l -1), with the correlation coefficient r = 0.9992 ( n = 6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red.

PubMed

Shao-Qin, Lin; Xuan, Lin; Shi-Rong, Hu; Li-Qing, Zeng; Yan, Wang; Li, Chen; Jia-Ming, Liu; Long-Di, Li

2005-11-01

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 degrees C for 30 min and Al(3+) can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ngl(-1) with a detection limit of 5.3 pgl(-1). The regression equation can be expressed as DeltaI(f)=8.731+21.73c(Al(3+)) (ngl(-1)), with the correlation coefficient r=0.9992 (n=6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.

Increased fluorescence intensity in CaTiO3:Pr3+ phosphor due to NH3 treatment and Nb Co-doping

NASA Astrophysics Data System (ADS)

Holliday, K. S.; Kohlgruber, T. A.; Tran, I. C.; Åberg, D.; Seeley, Z. M.; Bagge-Hansen, M.; Srivastava, A. M.; Cherepy, N. J.; Payne, S. A.

2016-10-01

Development of next generation red phosphors for commercial lighting requires understanding of how increased luminescence is achieved by various treatment strategies. In this work, we compare co-doping with Nb to NH3 treatment of CaTiO3:Pr phosphors to reveal a general mechanism responsible for the increased luminescence. The phosphors were synthesized using standard solid-state synthesis techniques and the fluorescence was characterized for potential use in fluorescent lighting, with 254 nm excitation. The lifetime of the fluorescence was determined and used to identify a change in a trap state by the co-doping of Nb5+ in the phosphor. The oxidation state of the Pr was probed by NEXAFS and revealed that both Nb5+ co-doping and NH3 treatment reduced the number of non-fluorescing Pr4+ centers. Calculations were performed to determine the energetically favorable defects. Vacuum annealing was also used to further probe the nature of the trap state. It was determined that NH3 treatments reduce the number of Pr4+ non-fluorescing centers, while Nb5+ co-doping additionally reduces the number of excess oxygen trap states that quench the fluorescence.

Determination of copper (II) in foodstuffs based on its quenching effect on the fluorescence of N,N'-bis(pyridoxal phosphate)-o-phenylenediamine.

PubMed

Xu, Canhui; Liao, Lifu; He, Yunfei; Wu, Rurong; Li, Shijun; Yang, Yanyan

2015-01-01

A Schiff base-type fluorescence probe was prepared for the detection of copper (II) in foodstuffs. The probe is N,N'-bis(pyridoxal phosphate)-o-phenylenediamine (BPPP). It was synthesized by utilizing the Schiff base condensation reaction of pyridoxal 5-phosphate with 1,2-phenylenediamine. BPPP has the properties of high fluorescence stability, good water solubility and low toxicity. Its maximum excitation wavelength and maximum fluorescence emission wavelength are at 389 and 448 nm, respectively. When BPPP coexists with copper (II), its fluorescence is dramatically quenched. Under a certain condition, the fluorescence intensity decreased proportionally to the concentration of copper (II) by the quenching effect. Based on this fact, we established a fluorescence quenching method for the determination of copper (II). Under optimal conditions a linear range was found to be 0.5-50 ng/mL with a detection limit of 0.2 ng/mL. The method has been applied to determine copper (II) in foodstuff samples and the analytical results show good agreement with that obtained from atomic absorption spectrometry method. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative and qualitative 5-aminolevulinic acid–induced protoporphyrin IX fluorescence in skull base meningiomas

PubMed Central

Bekelis, Kimon; Valdés, Pablo A.; Erkmen, Kadir; Leblond, Frederic; Kim, Anthony; Wilson, Brian C.; Harris, Brent T.; Paulsen, Keith D.; Roberts, David W.

2011-01-01

Object Complete resection of skull base meningiomas provides patients with the best chance for a cure; however, surgery is frequently difficult given the proximity of lesions to vital structures, such as cranial nerves, major vessels, and venous sinuses. Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative assessment of protoporphyrin IX (PpIX) fluorescence following the exogenous administration of 5-aminolevulinic acid (ALA) has demonstrated utility in malignant glioma resection but limited use in meningiomas. Here the authors demonstrate the use of ALA-induced PpIX fluorescence guidance in resecting a skull base meningioma and elaborate on the advantages and disadvantages provided by both quantitative and qualitative fluorescence methodologies in skull base meningioma resection. Methods A 52-year-old patient with a sphenoid wing WHO Grade I meningioma underwent tumor resection as part of an institutional review board–approved prospective study of fluorescence-guided resection. A surgical microscope modified for fluorescence imaging was used for the qualitative assessment of visible fluorescence, and an intraoperative probe for in situ fluorescence detection was utilized for quantitative measurements of PpIX. The authors assessed the detection capabilities of both the qualitative and quantitative fluorescence approaches. Results The patient harboring a sphenoid wing meningioma with intraorbital extension underwent radical resection of the tumor with both visibly and nonvisibly fluorescent regions. The patient underwent a complete resection without any complications. Some areas of the tumor demonstrated visible fluorescence. The quantitative probe detected neoplastic tissue better than the qualitative modified surgical microscope. The intraoperative probe was particularly useful in areas that did not reveal visible fluorescence, and tissue from these areas was confirmed as tumor following histopathological analysis. Conclusions Fluorescence-guided resection may be a useful adjunct in the resection of skull base meningiomas. The use of a quantitative intraoperative probe to detect PpIX concentration allows more accurate determination of neoplastic tissue in meningiomas than visible fluorescence and is readily applicable in areas, such as the skull base, where complete resection is critical but difficult because of the vital structures surrounding the pathology. PMID:21529179

Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

PubMed Central

Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

2015-01-01

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

An image-processing method to detect sub-optical features based on understanding noise in intensity measurements.

PubMed

Bhatia, Tripta

2018-07-01

Accurate quantitative analysis of image data requires that we distinguish between fluorescence intensity (true signal) and the noise inherent to its measurements to the extent possible. We image multilamellar membrane tubes and beads that grow from defects in the fluid lamellar phase of the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine dissolved in water and water-glycerol mixtures by using fluorescence confocal polarizing microscope. We quantify image noise and determine the noise statistics. Understanding the nature of image noise also helps in optimizing image processing to detect sub-optical features, which would otherwise remain hidden. We use an image-processing technique "optimum smoothening" to improve the signal-to-noise ratio of features of interest without smearing their structural details. A high SNR renders desired positional accuracy with which it is possible to resolve features of interest with width below optical resolution. Using optimum smoothening, the smallest and the largest core diameter detected is of width [Formula: see text] and [Formula: see text] nm, respectively, discussed in this paper. The image-processing and analysis techniques and the noise modeling discussed in this paper can be used for detailed morphological analysis of features down to sub-optical length scales that are obtained by any kind of fluorescence intensity imaging in the raster mode.

Analysis of low levels of rare earths by radiochemical neutron activation analysis

USGS Publications Warehouse

Wandless, G.A.; Morgan, J.W.

1985-01-01

A procedure for the radiochemical neutron-activation analysis for the rare earth elements (REE) involves the separation of the REE as a group by rapid ion-exchange methods and determination of yields by reactivation or by energy dispersive X-ray fluorescence (EDXRF) spectrometry. The U. S. Geological Survey (USGS) standard rocks, BCR-1 and AGV-1, were analyzed to determine the precision and accuracy of the method. We found that the precision was ??5-10% on the basis of replicate analysis and that, in general the accuracy was within ??5% of accepted values for most REE. Data for USGS standard rocks BIR-1 (Icelandic basalt) and DNC-1 (North Carolina diabase) are also presented. ?? 1985 Akade??miai Kiado??.

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, M.S.

1998-08-18

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device. 27 figs.

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.; Wang, Chunwei; Jevons, Luis C.; Bernhart, Derek H.; Lipshutz, Robert J.

2004-05-11

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

1998-08-18

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

Computer-aided visualization and analysis system for sequence evaluation

DOEpatents

Chee, Mark S.

2003-08-19

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

On-line capillary electrophoresis/laser-induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump-based nanospray ion source.

PubMed

Khan, Shaheer; Liu, Jenkuei; Szabo, Zoltan; Kunnummal, Baburaj; Han, Xiaorui; Ouyang, Yilan; Linhardt, Robert J; Xia, Qiangwei

2018-06-15

N-linked glycan analysis of recombinant therapeutic proteins, such as monoclonal antibodies, Fc-fusion proteins, and antibody-drug conjugates, provides valuable information regarding protein therapeutics glycosylation profile. Both qualitative identification and quantitative analysis of N-linked glycans on recombinant therapeutic proteins are critical analytical tasks in the biopharma industry during the development of a biotherapeutic. Currently, such analyses are mainly carried out using capillary electrophoresis/laser-induced fluorescence (CE/LIF), liquid chromatography/fluorescence (LC/FLR), and liquid chromatography/fluorescence/mass spectrometry (LC/FLR/MS) technologies. N-linked glycans are first released from glycoproteins by enzymatic digestion, then labeled with fluorescence dyes for subsequent CE or LC separation, and LIF or MS detection. Here we present an on-line CE/LIF/MS N-glycan analysis workflow that incorporates the fluorescent Teal™ dye and an electrokinetic pump-based nanospray sheath liquid capillary electrophoresis/mass spectrometry (CE/MS) ion source. Electrophoresis running buffer systems using ammonium acetate and ammonium hydroxide were developed for the negative ion mode CE/MS analysis of fluorescence-labeled N-linked glycans. Results show that on-line CE/LIF/MS analysis can be readily achieved using this versatile CE/MS ion source on common CE/MS instrument platforms. This on-line CE/LIF/MS method using Teal™ fluorescent dye and electrokinetic pump-based nanospray sheath liquid CE/MS coupling technology holds promise for on-line quantitation and identification of N-linked glycans on recombinant therapeutic proteins. Copyright © 2018 John Wiley & Sons, Ltd.

Effects of laser irradiation on immature olfactory neuroepithelial explants from the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mester, A.F.; Snow, J.B. Jr.

1988-07-01

The photobiological effect of low-output laser irradiation on the maturation and regeneration of immature olfactory bipolar receptor cells of the rat was studied. The maturation and regeneration of the receptor cells of rat fetuses were quantified in neuroepithelial explants with morphometric analysis. The number of explants with outgrowth and the number and length of neuritic outgrowths were determined on a regular basis for 12 days. Explants in the experimental group were irradiated with a helium-neon laser using different incident energy densities (IED). Explants in the fluorescent light control group were exposed to fluorescent light for the same periods of timemore » as those in the experimental group were exposed to laser irradiation. Explants in another control group were not exposed to laser or fluorescent light irradiation. The IED of 0.5 J/cm2 laser irradiation has been found to increase significantly the number of explants with outgrowth and the number and length of the outgrowths. Other laser IEDs or fluorescent light irradiation did not influence maturation or regeneration.« less

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

PubMed

Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

2016-12-15

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors.

PubMed

Zhang, Qin; Tang, Xiuwen; Hou, Fenghua; Yang, Jianping; Xie, Zhiyong; Cheng, Zhiyi

2013-10-01

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC50 value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery. Copyright © 2013 Elsevier Inc. All rights reserved.

Dyes assay for measuring physicochemical parameters.

PubMed

Moczko, Ewa; Meglinski, Igor V; Bessant, Conrad; Piletsky, Sergey A

2009-03-15

A combination of selective fluorescent dyes has been developed for simultaneous quantitative measurements of several physicochemical parameters. The operating principle of the assay is similar to electronic nose and tongue systems, which combine nonspecific or semispecific elements for the determination of diverse analytes and chemometric techniques for multivariate data analysis. The analytical capability of the proposed mixture is engendered by changes in fluorescence signal in response to changes in environment such as pH, temperature, ionic strength, and presence of oxygen. The signal is detected by a three-dimensional spectrofluorimeter, and the acquired data are processed using an artificial neural network (ANN) for multivariate calibration. The fluorescence spectrum of a solution of selected dyes allows discreet reading of emission maxima of all dyes composing the mixture. The variations in peaks intensities caused by environmental changes provide distinctive fluorescence patterns which can be handled in the same way as the signals collected from nose/tongue electrochemical or piezoelectric devices. This optical system opens possibilities for rapid, inexpensive, real-time detection of a multitude of physicochemical parameters and analytes of complex samples.

Electron Shuttling by Dissolved Humic Substances: Using Fluorescence Spectroscopy to Move Beyond the Laboratory to Natural Lakes, Streams and Groundwaters

NASA Astrophysics Data System (ADS)

McKnight, D. M.

2017-12-01

Humic substances are an important class of reactive chemical species in natural waters, and one important role is their capacity to as an electron acceptor and/or electron shuttle to ferric iron present as solid phase ferric oxides. Several lines of evidence point to quinone-like moieties being the main redox active moieties that can be used by microbes in respiration. Concomitantly, the humic fraction of dissolved organic mater (DOM) contains the dominant fluorophores in many natural waters. Examination of excitation emission matrices (EEMs) across redox gradients in diverse aquatic systems show that the EEMs are generally red-shifted under reducing conditions, such as anoxic bottom waters in lakes and hypoxic waters in riparian wetlands. Furthermore, there is striking similarity between the humic fluorophores that are resolved by statistical analysis and the fluorescence spectra of model quinone compounds, with the more reduced species having red-shifted fluorescence spectra. This apparent red-shift can be quantified based on the distribution of apparently "quinone-like", "semi-quinone-like" and "hydroquinone-like" fluorophores determined by the PARAFAC statistical analysis. Because fluorescence spectroscopy can be applied at ambient DOM concentrations for samples that have been maintained in an anoxic condition, fluorescence spectroscopy can provide insight into the role of humic electron shuttling in natural systems. Examples are presented demosntrating the changing EEMs in anoxic bottomwaters in a lake in the McMurdo Dry Valleys following a major flood event and the role of organic material in the mobilization of arsenic in shallow groundwater in South East Asia.

Development, validation, and application of a method for selected avermectin determination in rural waters using high performance liquid chromatography and fluorescence detection.

PubMed

Lemos, Maria Augusta Travassos; Matos, Camila Alves; de Resende, Michele Fabri; Prado, Rachel Bardy; Donagemma, Raquel Andrade; Netto, Annibal Duarte Pereira

2016-11-01

Avermectins (AVM) are macrocyclic lactones used in livestock and agriculture. A quantitative method of high performance liquid chromatography with fluorescence detection for the determination of eprinomectin, abamectin, doramectin and ivermectin in rural water samples was developed and validated. The method was employed to study samples collected in the Pito Aceso River microbasin, located in the Bom Jardim municipality, Rio de Janeiro State, Brazil. Samples were extracted by solid phase extraction using a polymeric stationary phase, the eluted fraction was re-concentrated under a gentle N2 flow and derivatized to allow AVM determination using liquid chromatography with fluorescence detection. The excitation and emission wavelengths of the derivatives were 365 and 470nm, respectively, and a total chromatographic run of 12min was achieved. Very low limits of quantification (22-58ngL(-1)) were found after re-concentration using N2. Recovery values varied from 85.7% to 119.2% with standard deviations between 1.2% and 10.2%. The validated method was applied in the determination of AVM in 15 water samples collected in the Pito Aceso River microbasin, but most of them were free of AVM or showed only trace levels of these compounds, except for a sample that contained doramectin (9.11µgL(-1)). The method is suitable for routine analysis with satisfactory recovery, sensitivity, and selectivity. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluorescent Approaches to High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

2006-01-01

We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells.

PubMed

Berg, R J; de Bueger, S C; Guikers, K; van Weelden, H; van Vloten, W A; van der Leun, J C; de Gruijl, F R

1995-12-01

We have earlier reported on determining UV-induced DNA damage in murine epidermal cell suspensions by flow cytometric analysis of the fluorescence from a fluorescein isothiocyanate-labeled antibody (H3) directed against thymine dimers (T < > T). Here we present an optimization of the technique for analysis of epidermal cell suspensions from 4 mm biopsies from human skin. Cells with different DNA contents can easily be distinguished in flow cytometry by the intensity of DNA-specific 7-amino-actinomycin D fluorescence. Genuine G2-M-phase cells can further be distinguished from cell doublets by pulse-shape discrimination. Thus, T < > T levels in individual cells with different DNA contents (i.e. G0-G1, S or G2-M phases) can be determined after in vivo exposure of human skin to environmentally relevant UVB (280-315 nm) doses. The method was applied to measure the decrease of T < > T in nonreplicating cells (G0-G1 phase) and replicating cells (S phase or G2-M phase) from seven volunteers exposed to twice their minimal erythema dose. The reduction in the average T < > T-specific fluorescence at 24 h after exposure was 46% (ranging between 16% and 66%) for the G0-G1 cells and 70% (ranging between 37% and 100%) for the S + G2-M cells. The difference was statistically highly significant. Determination of individual DNA repair capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to identify individuals with low repair proficiencies and increased risk of developing skin cancers.

[Simultaneous determination of multiple elements in airborne particulate samples by X-ray fluorescence spectrometry].

PubMed

Takada, T; Hitosugi, M; Kadowaki, T; Kudo, M

1983-07-01

An energy dispersive X-ray fluorescence spectrometer (EDX) has been applied to determine multielements in the workplace air. The standards for X-ray fluorescence analysis were prepared by the chelate precipitation method on polyvinyl chloride (PVC) membrane filter. And, the specimens were prepared to deposit various metal compounds of different chemical forms by the suspension method on PVC membrane filter, and they were determined with EDX and atomic absorption spectrometer (AAS). The results obtained were as follows. Though there is a difference by each element, an amount less than 3 microgram/cm2 per unit area makes it possible to undergo multielement analysis, that is, is has no influence on fine particle effect (particle size; under 5 microns). Then, effects of the X-ray intensity by different chemical forms are negligible. At the presence the neighboring element and other elements this technique showed greater precision by carrying out on corrective treatment, etc. The coefficient of variation of this technique was in the range of 2.5-6.5% at DDTC-Cu of 0.5-5.0 micrograms/cm2, with the limit of detection for As : 0.002 microgram/cm2, Zn : 0.003 microgram/cm2, Pb : 0.003 microgram/cm2, Cu : 0.004 microgram/cm2, Ni : 0.003 microgram/cm2, Fe : 0.005 microgram/cm2, Mn : 0.008 microgram/cm2, Cr : 0.013 microgram/cm2, respectively. Aerosols collected at the workplace were analyzed with EDX and AAS, and the obtained results showed good agreement with such regression line as y = 1.04 chi + 0.04, the coefficient of correlation being r = 0.995. From these results, this technique was found to be a very excellent method for monitoring of multielements in the workplace air.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Preparation and characterization of polymer layer systems for validation of 3D Micro X-ray fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Schaumann, Ina; Malzer, Wolfgang; Mantouvalou, Ioanna; Lühl, Lars; Kanngießer, Birgit; Dargel, Rainer; Giese, Ulrich; Vogt, Carla

2009-04-01

For the validation of the quantification of the newly-developed method of 3D Micro X-ray fluorescence spectroscopy (3D Micro-XRF) samples with a low average Z matrix and minor high Z elements are best suited. In a light matrix the interferences by matrix effects are minimized so that organic polymers are appropriate as basis for analytes which are more easily detected by X-ray fluorescence spectroscopy. Polymer layer systems were assembled from single layers of ethylene-propylene-diene rubber (EPDM) filled with changing concentrations of silica and zinc oxide as inorganic additives. Layer thicknesses were in the range of 30-150 μm. Before the analysis with 3D Micro-XRF all layers have been characterized by scanning micro-XRF with regard to filler dispersion, by infrared microscopy and light microscopy in order to determine the layer thicknesses and by ICP-OES to verify the concentration of the X-ray sensitive elements in the layers. With the results obtained for stacked polymer systems the validity of the analytical quantification model for the determination of stratified materials by 3D Micro-XRF could be demonstrated.

An efficient spectrofluorimetric method adopts doxazosin, terazosin and alfuzosin coupling with orthophthalaldehyde: Application in human plasma

NASA Astrophysics Data System (ADS)

Omar, Mahmoud A.; Mohamed, Abdel-Maaboud I.; Derayea, Sayed M.; Hammad, Mohamed A.; Mohamed, Abobakr A.

2018-04-01

A new, selective and sensitive spectrofluorimetric method was designed for the quantitation of doxazosin (DOX), terazosin (TER) and alfuzosin (ALF) in their dosage forms and human plasma. The method adopts efficient derivatization of the studied drugs with ortho-phthalaldehyde (OPA), in the presence of 2-mercaptoethanol in borate buffer (pH 9.7) to generate a highly fluorescent isoindole derivatives, which can strongly enhance the fluorescence intensities of the studied drugs, allowing their sensitive determination at 430 nm after excitation at 337 nm. The fluorescence-concentration plots were rectilinear over the ranges (10.0-400.0) ng/mL. Detection and quantification limits were found to be (0.52-3.88) and (1.59-11.76) ng/mL, respectively. The proposed method was validated according to ICH guidelines, and successfully applied for the determination of pharmaceutical preparations of the studied drugs. Moreover, the high sensitivity of the proposed method permits its successful application to the analysis of the studied drugs in spiked human plasma with % recovery (96.12 ± 1.34-100.66 ± 0.57, n = 3). A proposal for the reaction mechanism was presented.

Green analytical determination of emerging pollutants in environmental waters using excitation-emission photoinduced fluorescence data and multivariate calibration.

PubMed

Hurtado-Sánchez, María Del Carmen; Lozano, Valeria A; Rodríguez-Cáceres, María Isabel; Durán-Merás, Isabel; Escandar, Graciela M

2015-03-01

An eco-friendly strategy for the simultaneous quantification of three emerging pharmaceutical contaminants is presented. The proposed analytical method, which involves photochemically induced fluorescence matrix data combined with second-order chemometric analysis, was used for the determination of carbamazepine, ofloxacin and piroxicam in water samples of different complexity without the need of chromatographic separation. Excitation-emission photoinduced fluorescence matrices were obtained after UV irradiation, and processed with second-order algorithms. Only one of the tested algorithms was able to overcome the strong spectral overlapping among the studied pollutants and allowed their successful quantitation in very interferent media. The method sensitivity in superficial and underground water samples was enhanced by a simple solid-phase extraction with C18 membranes, which was successful for the extraction/preconcentration of the pollutants at trace levels. Detection limits in preconcentrated (1:125) real water samples ranged from 0.04 to 0.3 ng mL(-1). Relative prediction errors around 10% were achieved. The proposed strategy is significantly simpler and greener than liquid chromatography-mass spectrometry methods, without compromising the analytical quality of the results. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of aristolochic acids by high-performance liquid chromatography with fluorescence detection.

PubMed

Wang, Yinan; Chan, Wan

2014-06-25

Nephrotoxic and carcinogenic aristolochic acids (AAs) are naturally occurring nitrophenanthrene carboxylic acids in the herbal genus Aristolochia. The misuse of AA-containing herbs in preparing slimming drugs has caused hundred of cases of kidney disease in Belgium women in a slimming regime in the early 1990s. Accumulating evidence also suggested that prolong dietary intake of AA-contaminated food is one of the major causes to the Balkan endemic nephropathy that was first observed in the late 1950s. Therefore, analytical methods of high sensitivity are extremely important for safeguarding human exposure to AA-containing herbal medicines, herbal remedies, and food composites. In this paper, we describe the development of a new high-performance liquid chromatography coupled fluorescence detector (HPLC-FLD) method for the sensitive determination of AAs. The method makes use of a novel cysteine-induced denitration reaction that "turns on" the fluorescence of AAs for fluorometric detections. Our results showed that the combination of cysteine-induced denitration and HPLC-FLD analysis allows for sensitive quantification of AA-I and AA-II at detection limits of 27.1 and 25.4 ng/g, respectively. The method was validated and has been successfully applied in quantifying AAs in Chinese herbal medicines.

Determination of copper nanoparticle size distributions with total reflection X-ray fluorescence spectroscopy

DOE PAGES

Singh, Andy; Luening, Katharina; Brennan, Sean; ...

2017-01-01

Total reflection X-ray fluorescence (TXRF) analysis is extensively used by the semiconductor industry for measuring trace metal contamination on silicon surfaces. In addition to determining the quantity of impurities on a surface, TXRF can reveal information about the vertical distribution of contaminants by measuring the fluorescence signal as a function of the angle of incidence. In this study, two samples were intentionally contaminated with copper in non-deoxygenated and deoxygenated ultrapure water (UPW) resulting in impurity profiles that were either atomically dispersed in a thin film or particle-like, respectively. The concentration profile of the samples immersed into deoxygenated UPW was calculatedmore » using a theoretical concentration profile representative of particles, yielding a mean particle height of 16.1 nm. However, the resulting theoretical profile suggested that a distribution of particle heights exists on the surface. The fit of the angular distribution data was further refined by minimizing the residual error of a least-squares fit employing a model with a Gaussian distribution of particle heights about the mean height. The presence of a height distribution was also confirmed with atomic force microscopy measurements.« less

Determination of copper nanoparticle size distributions with total reflection X-ray fluorescence spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Andy; Luening, Katharina; Brennan, Sean

Total reflection X-ray fluorescence (TXRF) analysis is extensively used by the semiconductor industry for measuring trace metal contamination on silicon surfaces. In addition to determining the quantity of impurities on a surface, TXRF can reveal information about the vertical distribution of contaminants by measuring the fluorescence signal as a function of the angle of incidence. In this study, two samples were intentionally contaminated with copper in non-deoxygenated and deoxygenated ultrapure water (UPW) resulting in impurity profiles that were either atomically dispersed in a thin film or particle-like, respectively. The concentration profile of the samples immersed into deoxygenated UPW was calculatedmore » using a theoretical concentration profile representative of particles, yielding a mean particle height of 16.1 nm. However, the resulting theoretical profile suggested that a distribution of particle heights exists on the surface. The fit of the angular distribution data was further refined by minimizing the residual error of a least-squares fit employing a model with a Gaussian distribution of particle heights about the mean height. The presence of a height distribution was also confirmed with atomic force microscopy measurements.« less

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

A high resolution Passive Flux Meter approach based on colorimetric responses

NASA Astrophysics Data System (ADS)

Chardi, K.; Dombrowski, K.; Cho, J.; Hatfield, K.; Newman, M.; Annable, M. D.

2016-12-01

Subsurface water and contaminant mass flux measurements are critical in determining risk, optimizing remediation strategies, and monitoring contaminant attenuation. The standard Passive Flux Meter, hereafter knows as a (PFM), is a well-developed device used for determining and monitoring rates of groundwater and contaminant mass flux in screened wells. The current PFM is a permeable device that contains granular activated carbon impregnated with alcohol tracers which is deployed in a flow field for a designated period of time. Once extracted, sampling requires laboratory analysis to quantify Darcy flux, which can be time consuming and have significant cost. To expedite test results, a modified PFM based on the image analysis of colorimetric responses, herein referred to as a colorimetric Passive Flux Meter (cPFM), was developed. Various dyes and sorbents were selected and evaluated to determine colorimetric response to water flow. Rhodamine, fluorescent yellow, fluorescent orange, and turmeric were the dye candidates while 100% wool and a 35% wool blend with 65% rayon were the sorbent candidates selected for use in the cPFM. Ultraviolet light image analysis was used to calculate average color intensity using ImageJ, a Java-based image processing program. These results were then used to quantify Darcy flux. Error ranges evaluated for Darcy flux using the cPFM are comparable to those with the standard, activated carbon based, PFM. The cPFM has the potential to accomplish the goal of obtaining high resolution Darcy flux data while eliminating high costs and analysis time. Implications of groundwater characteristics, such as PH and contaminant concentrations, on image analysis are to be tested through laboratory analysis followed by field testing of the cPFM.

The Rate Constant for Fluorescence Quenching

ERIC Educational Resources Information Center

Legenza, Michael W.; Marzzacco, Charles J.

1977-01-01

Describes an experiment that utilizes fluorescence intensity measurements from a Spectronic 20 to determine the rate constant for the fluorescence quenching of various aromatic hydrocarbons by carbon tetrachloride in an ethanol solvent. (MLH)

Correcting for surface topography in X-ray fluorescence imaging

PubMed Central

Geil, E. C.; Thorne, R. E.

2014-01-01

Samples with non-planar surfaces present challenges for X-ray fluorescence imaging analysis. Here, approximations are derived to describe the modulation of fluorescence signals by surface angles and topography, and suggestions are made for reducing this effect. A correction procedure is developed that is effective for trace element analysis of samples having a uniform matrix, and requires only a fluorescence map from a single detector. This procedure is applied to fluorescence maps from an incised gypsum tablet. PMID:25343805

Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2 in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique.

PubMed

Gui, Z; Liu, H Q; Wang, Y; Yuan, Q H; Xin, N; Zhang, X; Li, X L; Pi, Y S; Gao, J M

2014-12-04

In this study, 2 approaches were adopted to obtain good single-strand conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP conditions for a 331-bp fragment in the coding region of polygalacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized, and the results showed that the best SSCP gel pattern could be obtained when the loading mixture was made by mixing 1 μL PCR products, 0.2 to 0.8 μL unlabeled primers (50 μM) and 4 to 16 μL loading buffer. Furthermore, the use of the fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2 complementary single DNA strands, thus making the determination of alleles and idiotypes a relatively easy task. In addition, the results of sequencing prove that the determination of alleles and idiotypes were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed great genetic variation, and the addition of primers to the PCR products in combination with the fluorescent labeling of primers could significantly improve the sensitivity and resolution of SSCP analysis. This technique could be used for genetic diversity detection and marker-assisted breeding of useful genes in autopolyploid species such as alfalfa.

Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging

PubMed Central

Crane, Jonathan M.; Verkman, Alan S.

2009-01-01

Summary We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length `long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the `short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23. PMID:19240114

Fluorescent quantification of terazosin hydrochloride content in human plasma and tablets using second-order calibration based on both parallel factor analysis and alternating penalty trilinear decomposition.

PubMed

Zou, Hong-Yan; Wu, Hai-Long; OuYang, Li-Qun; Zhang, Yan; Nie, Jin-Fang; Fu, Hai-Yan; Yu, Ru-Qin

2009-09-14

Two second-order calibration methods based on the parallel factor analysis (PARAFAC) and the alternating penalty trilinear decomposition (APTLD) method, have been utilized for the direct determination of terazosin hydrochloride (THD) in human plasma samples, coupled with the excitation-emission matrix fluorescence spectroscopy. Meanwhile, the two algorithms combing with the standard addition procedures have been applied for the determination of terazosin hydrochloride in tablets and the results were validated by the high-performance liquid chromatography with fluorescence detection. These second-order calibrations all adequately exploited the second-order advantages. For human plasma samples, the average recoveries by the PARAFAC and APTLD algorithms with the factor number of 2 (N=2) were 100.4+/-2.7% and 99.2+/-2.4%, respectively. The accuracy of two algorithms was also evaluated through elliptical joint confidence region (EJCR) tests and t-test. It was found that both algorithms could give accurate results, and only the performance of APTLD was slightly better than that of PARAFAC. Figures of merit, such as sensitivity (SEN), selectivity (SEL) and limit of detection (LOD) were also calculated to compare the performances of the two strategies. For tablets, the average concentrations of THD in tablet were 63.5 and 63.2 ng mL(-1) by using the PARAFAC and APTLD algorithms, respectively. The accuracy was evaluated by t-test and both algorithms could give accurate results, too.

Determination of ochratoxin A in wine by means of immunoaffinity and aminopropyl solid-phase column cleanup and fluorometric detection.

PubMed

Longobardi, Francesco; Iacovelli, Vito; Catucci, Lucia; Panzarini, Giuseppe; Pascale, Michelangelo; Visconti, Angelo; Agostiano, Angela

2013-02-27

A new analytical method for the determination of ochratoxin A (OTA) in red wine has been developed by using a double-extract cleanup and a fluorometric measurement after spectral deconvolution. Wine samples were diluted with a solution containing 1% polyethylene glycol and 5% sodium hydrogencarbonate, filtered, and purified by immunoaffinity and aminopropyl solid-phase column. OTA contents in the purified extract were determined by a spectrofluorometer (excitation wavelength, 330 nm; emission wavelength, 470 nm) after deconvolution of fluorescence spectra. Average recoveries from wine samples spiked with OTA at levels ranging from 0.5 to 3.0 ng/mL were 94.5-105.4% with relative standard deviations (RSD) of <15% (n = 4). The limit of detection (LOD) was 0.2 ng/mL, and the total time of analysis was 30 min. The developed method was tested on 18 red wine samples (naturally contaminated and spiked with OTA at levels ranging from 0.4 to 3.0 ng/mL) and compared with AOAC Official Method 2001.01, based on immunoaffinity column cleanup and HPLC with fluorescence detector. A good correlation (r(2) = 0.9765) was observed between OTA levels obtained with the two methods, highlighting the reliability of the proposed method, the main advantage of which is the simple OTA determination by a benchtop fluorometer with evident reductions of cost and time of analysis.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Detection of bacterial infection of agave plants by laser-induced fluorescence

NASA Astrophysics Data System (ADS)

Cervantes-Martinez, Jesus; Flores-Hernandez, Ricardo; Rodriguez-Garay, Benjamin; Santacruz-Ruvalcaba, Fernando

2002-05-01

Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

Detection of bacterial infection of agave plants by laser-induced fluorescence.

PubMed

Cervantes-Martínez, Jesús; Flores-Hernández, Ricardo; Rodríguez-Garay, Benjamin; Santacruz-Ruvalcaba, Fernando

2002-05-01

Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

PubMed

Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

2016-03-01

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

[Resolving characteristic of CDOM by excitation-emission matrix spectroscopy combined with parallel factor analysis in the seawater of outer Yangtze Estuary in Autumn in 2010].

PubMed

Yan, Li-Hong; Chen, Xue-Jun; Su, Rong-Guo; Han, Xiu-Rong; Zhang, Chuan-Song; Shi, Xiao-Yong

2013-01-01

The distribution and estuarine behavior of fluorescent components of chromophoric dissolved organic matter in the seawater of outer Yangtze Estuary were determined by fluorescence excitation emission matrix spectra combined with parallel factor analysis. Six individual fluorescent components were identified by PARAFAC models, including three terrestrial humic-like components C1 [330 nm/390(430) nm], C2 (390 nm/480 nm), C3 (360 nm/440 nm), marine biological production component C5 (300 nm/400 nm) and protein-like components C4 (290 nm/350 nm) and C6 (275 nm/300 nm). The results indicated that C1, C2, and C3 showed a conservative mixing behavior in the whole estuarine region, especially in high-salinity region. And the fluorescence intensity proportion of C1 and C3 decreased with increase of salinity and fluorescence intensity proportion of C2 kept constant with increase of salinity in the whole estuarine region. While C4 showed conservative mixing behavior in low-salinity region and non-conservative mixing behavior in high-salinity region, and fluorescence intensity proportion of C4 increased with increase of salinity. However, C5 and C6 showed a non-conservative mixing behavior and fluorescence intensity proportion increased with increase of salinity in high-salinity region. Significantly spatial difference was recorded for CDOM absorption coefficient in the coastal region and in the open water areas with the highest value in coastal region and the lowest value in the open water areas. The scope of absorption coefficient and absorption slope was higher in coastal region than that in the open water areas. Significantly positive correlations were found between CDOM absorption coefficient and the fluorescence intensities of C1, C2, C3, and C4, but no significant correlation was found between C5 and C6, suggesting that the river inputs contributed to the coastal areas, while CDOM in the open water areas was affected by terrestrial inputs and phytoplankton degradation.