Position-sensitive scanning fluorescence correlation spectroscopy.

PubMed

Skinner, Joseph P; Chen, Yan; Müller, Joachim D

2005-08-01

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Non-Uniformly Sampled MR Correlated Spectroscopic Imaging in Breast Cancer and Nonlinear Reconstruction

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-16-1-0524 TITLE: Non-Uniformly Sampled MR Correlated Spectroscopic Imaging in Breast Cancer and Nonlinear Reconstruction...author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other...COVERED 30 Sep 2016 - 29 Sep 2017 5a. CONTRACT NUMBER 4. TITLE AND SUBTITLE Non-Uniformly Sampled MR Correlated Spectroscopic Imaging in Breast

Viscosity-dependent diffusion of fluorescent particles using fluorescence correlation spectroscopy.

PubMed

Jung, Chanbae; Lee, Jaeran; Kang, Manil; Kim, Sok Won

2014-11-01

Fluorescent particles show the variety characteristics by the interaction with other particles and solvent. In order to investigate the relationship between the dynamic properties of fluorescent particles and solvent viscosity, particle diffusion in various solvents was evaluated using a fluorescence correlation spectroscopy. Upon analyzing the correlation functions of AF-647, Q-dot, and beads with different viscosity values, the diffusion time of all particles was observed to increase with increasing solvent viscosity, and the ratio of diffusion time to solvent viscosity, τ D /η, showed a linear dependence on particle size. The particle diffusion coefficients calculated from the diffusion time decreased with increasing solvent viscosity. Further, the hydrodynamic radii of AF-647, Q-dot, and beads were 0.98 ± 0.1 nm, 64.8 ± 3.23 nm, and 89.8 ± 4.91 nm, respectively, revealing a linear dependence on τ D /η, which suggests that the hydrodynamic radius of a particle strongly depends on both the physical size of the particle and solvent viscosity.

Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

2015-07-01

Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Steady-state fluorescence and phosphorescence spectroscopic studies of bacterial luciferase tryptophan mutants.

PubMed

Li, Z; Meighen, E A

1994-09-01

Bacterial luciferase, which catalyzes the bioluminescence reaction in luminous bacteria, consists of two nonidentical polypeptides, α and β. Eight mutants of luciferase with each of the tryptophans replaced by tyrosine were generated by site-directed mutagenesis and purified to homogeneity. The steady-state tryptophan fluorescence and low-temperature phosphorescence spectroscopic properties of these mutants were characterized. In some instances, mutation of only a single tryptophan residue resulted in large spectral changes. The tryptophan residues conserved in both the α and the β subunits exhibited distinct fluorescence emission properties, suggesting that these tryptophans have different local enviroments. The low-temperature phosphorescence data suggest that the tryptophans conserved in bot the α and the β subunits are not located at the subunit interface and/or involved in subunit interactions. The differences in the spectral properties of the mutants have provided useful information on the local environment of the individual tryptophan residues as well as on the quaternary structure of the protein.

Fluorescent-Spectroscopic Research of in Vivo Tissues Pathological Conditions

NASA Astrophysics Data System (ADS)

Giraev, K. M.; Ashurbekov, N. A.; Medzhidov, R. T.

The steady-state spectra of autofluorescence and the reflection coefficient on the excitation wavelength of some stomach tissues in vivo with various pathological conditions (surface gastritis, displasia, cancer) are measured under excitation by the nitrogen laser irradiation (λex=337.1 nm). The contour expansion of obtained fluorescence spectra into contributions of components is conducted by the Gaussian-Lorentzian curves method. It is shown that at least 7 groups of fluorophores forming a total luminescence spectrum can be distinguished during the development of displasia and tumor processes. The correlation of intensities of flavins and NAD(P)·H fluorescence is determined and the degree of respiratory activity of cells for the functional condition considered is estimated. The evaluations of the fluorescence quantum yield of the tissue's researched are given.

Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye

NASA Astrophysics Data System (ADS)

Patra, Digambara; Barakat, Christelle

2011-09-01

Curcumin, the main yellow bioactive component of turmeric, has recently acquired attention by chemists due its wide range of potential biological applications as an antioxidant, an anti-inflammatory, and an anti-carcinogenic agent. This molecule fluoresces weakly and poorly soluble in water. In this detailed study of curcumin in thirteen different solvents, both the absorption and fluorescence spectra of curcumin was found to be broad, however, a narrower and simple synchronous fluorescence spectrum of curcumin was obtained at Δ λ = 10-20 nm. Lippert-Mataga plot of curcumin in different solvents illustrated two sets of linearity which is consistent with the plot of Stokes' shift vs. the ET30. When Stokes's shift in wavenumber scale was replaced by synchronous fluorescence maximum in nanometer scale, the solvent polarity dependency measured by λSFSmax vs. Lippert-Mataga plot or ET30 values offered similar trends as measured via Stokes' shift for protic and aprotic solvents for curcumin. Better linear correlation of λSFSmax vs. π* scale of solvent polarity was found compared to λabsmax or λemmax or Stokes' shift measurements. In Stokes' shift measurement both absorption/excitation as well as emission (fluorescence) spectra are required to compute the Stokes' shift in wavenumber scale, but measurement could be done in a very fast and simple way by taking a single scan of SFS avoiding calculation and obtain information about polarity of the solvent. Curcumin decay properties in all the solvents could be fitted well to a double-exponential decay function.

Synchronous fluorescence spectroscopic study of solvatochromic curcumin dye.

PubMed

Patra, Digambara; Barakat, Christelle

2011-09-01

Curcumin, the main yellow bioactive component of turmeric, has recently acquired attention by chemists due its wide range of potential biological applications as an antioxidant, an anti-inflammatory, and an anti-carcinogenic agent. This molecule fluoresces weakly and poorly soluble in water. In this detailed study of curcumin in thirteen different solvents, both the absorption and fluorescence spectra of curcumin was found to be broad, however, a narrower and simple synchronous fluorescence spectrum of curcumin was obtained at Δλ=10-20 nm. Lippert-Mataga plot of curcumin in different solvents illustrated two sets of linearity which is consistent with the plot of Stokes' shift vs. the ET30. When Stokes's shift in wavenumber scale was replaced by synchronous fluorescence maximum in nanometer scale, the solvent polarity dependency measured by λSFSmax vs. Lippert-Mataga plot or ET30 values offered similar trends as measured via Stokes' shift for protic and aprotic solvents for curcumin. Better linear correlation of λSFSmax vs. π* scale of solvent polarity was found compared to λabsmax or λemmax or Stokes' shift measurements. In Stokes' shift measurement both absorption/excitation as well as emission (fluorescence) spectra are required to compute the Stokes' shift in wavenumber scale, but measurement could be done in a very fast and simple way by taking a single scan of SFS avoiding calculation and obtain information about polarity of the solvent. Curcumin decay properties in all the solvents could be fitted well to a double-exponential decay function. Copyright © 2011 Elsevier B.V. All rights reserved.

New fluorescence spectroscopic method for the simultaneous determination of alkaloids in aqueous extract of green coffee beans.

PubMed

Yisak, Hagos; Redi-Abshiro, Mesfin; Chandravanshi, Bhagwan Singh

2018-05-11

There is no fluorescence spectroscopic method for the determination of trigonelline and theobromine in green coffee beans. Therefore, the objective of this study was to develop a new fluorescence spectroscopic method to determine the alkaloids simultaneously in the aqueous extract of green coffee beans. The calibration curves were linear in the range 2-6, 1-6, 1-5 mg/L for caffeine, theobromine and trigonelline, respectively, with R 2  ≥ 0.9987. The limit of detection and limit of quantification were 2, 6 and 7 µg/L and 40, 20 and 20 µg/L for caffeine, theobromine and trigonelline, respectively. Caffeine and trigonelline exhibited well separated fluorescence excitation spectra and therefore the two alkaloids were selectively quantified in the aqueous extract of green coffee. While theobromine showed overlapping fluorescence excitation spectra with caffeine and hence theobromine could not be determined in the aqueous extract of green coffee beans. The amount of caffeine and trigonelline in the three samples of green coffee beans were found to be 0.95-1.10 and 1.00-1.10% (w/w), respectively. The relative standard deviations (RSD ≤ 4%) of the method for the three compounds of interest were of very good. The accuracy of the developed analytical method was evaluated by spiking standard caffeine and trigonelline to green coffee beans and the average recoveries were 99 ± 2% for both the alkaloids. A fast, sensitive and reliable fluorescence method for the simultaneous determination of caffeine and trigonelline in the aqueous extract of green coffee beans was developed and validated. The developed method reflected an effective performance to the direct determination of the two alkaloids in the aqueous extract of green coffee beans.

Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing

NASA Astrophysics Data System (ADS)

Saha, Dipika; Negi, Devendra P. S.

2018-01-01

The purpose of the present work was to develop a method for the sensing of thioacetamide by using spectroscopic techniques. Thioacetamide is a carcinogen and it is important to detect its presence in food-stuffs. Semiconductor quantum dots are frequently employed as sensing probes since their absorption and fluorescence properties are highly sensitive to the interaction with substrates present in the solution. In the present work, the interaction between thioacetamide and ZnO quantum dots has been investigated by using UV-visible, fluorescence and infrared spectroscopy. Besides, dynamic light scattering (DLS) has also been utilized for the interaction studies. UV-visible absorption studies indicated the bonding of the lone pair of sulphur atom of thioacetamide with the surface of the semiconductor. The fluorescence band of the ZnO quantum dots was found to be quenched in the presence of micromolar concentrations of thioacetamide. The quenching was found to follow the Stern-Volmer relationship. The Stern-Volmer constant was evaluated to be 1.20 × 105 M- 1. Infrared spectroscopic measurements indicated the participation of the sbnd NH2 group and the sulphur atom of thioacetamide in bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the surface charge of the semiconductor was shielded by the thioacetamide molecules.

Fluorescence correlation spectroscopy: the case of subdiffusion.

PubMed

Lubelski, Ariel; Klafter, Joseph

2009-03-18

The theory of fluorescence correlation spectroscopy is revisited here for the case of subdiffusing molecules. Subdiffusion is assumed to stem from a continuous-time random walk process with a fat-tailed distribution of waiting times and can therefore be formulated in terms of a fractional diffusion equation (FDE). The FDE plays the central role in developing the fluorescence correlation spectroscopy expressions, analogous to the role played by the simple diffusion equation for regular systems. Due to the nonstationary nature of the continuous-time random walk/FDE, some interesting properties emerge that are amenable to experimental verification and may help in discriminating among subdiffusion mechanisms. In particular, the current approach predicts 1), a strong dependence of correlation functions on the initial time (aging); 2), sensitivity of correlation functions to the averaging procedure, ensemble versus time averaging (ergodicity breaking); and 3), that the basic mean-squared displacement observable depends on how the mean is taken.

Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing.

PubMed

Saha, Dipika; Negi, Devendra P S

2018-01-15

The purpose of the present work was to develop a method for the sensing of thioacetamide by using spectroscopic techniques. Thioacetamide is a carcinogen and it is important to detect its presence in food-stuffs. Semiconductor quantum dots are frequently employed as sensing probes since their absorption and fluorescence properties are highly sensitive to the interaction with substrates present in the solution. In the present work, the interaction between thioacetamide and ZnO quantum dots has been investigated by using UV-visible, fluorescence and infrared spectroscopy. Besides, dynamic light scattering (DLS) has also been utilized for the interaction studies. UV-visible absorption studies indicated the bonding of the lone pair of sulphur atom of thioacetamide with the surface of the semiconductor. The fluorescence band of the ZnO quantum dots was found to be quenched in the presence of micromolar concentrations of thioacetamide. The quenching was found to follow the Stern-Volmer relationship. The Stern-Volmer constant was evaluated to be 1.20×10 5 M -1 . Infrared spectroscopic measurements indicated the participation of the NH 2 group and the sulphur atom of thioacetamide in bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the surface charge of the semiconductor was shielded by the thioacetamide molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Chiral alkylated-aniline as a noninvasive fluorescence sensor: Spectroscopic and molecular modeling studies

NASA Astrophysics Data System (ADS)

Sengupta, Bidisha; Mukherjee, Chirantan Sen; Chakraborty, Sandipan; Muhammad, Maria Jones; Gladney, William; Armstrong, George

2017-12-01

Aniline, heterocyclic aromatic amines, and arylamines are known carcinogens. Recently aniline mustard has come into prominence as a novel anticancer agent. In this project, microwave irradiation has been used to synthesize an optically active alkylated aniline namely 2,6-dimethyl-4-(1-(p-tolyl)ethyl)aniline (abbreviated DMPA). The presence of quartet and doublet peaks in NMR and a single chromatogram in HPLC verified that the final product DMPA, prepared from the synthesis reactions, had no major impurities. By using a Lux chiral column in HPLC, two peaks have been detected in the chromatogram, which correspond to two enantiomers of the chiral aniline derivative. Fluorescence spectroscopic measurements on DMPA indicated conspicuous dependence of its emission behavior on the polarity (in terms of the empirical polarity parameter ET(30)) of the homogeneous solvents used, a property important for an optical sensor. The nature of the emission profiles, along with the relevant parameter namely wavelength at emission maximum (λemmax) is used to infer the distribution, binding and microenvironment of the DMPA molecules in human serum albumin protein (HSA). DMPA is weakly fluorescent in aqueous buffer medium, with a dramatic enhancement in the fluorescence emission in the presence of HSA. Molecular modeling studies have been carried out on the two enantiomers (R and S) of DMPA with HSA. The implications of these findings are examined in relation to the potentialities of DMPA as a novel fluorescence sensor for biological systems.

Fluorescence correlation spectroscopy: principles and applications.

PubMed

Bacia, Kirsten; Haustein, Elke; Schwille, Petra

2014-07-01

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed. © 2014 Cold Spring Harbor Laboratory Press.

Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

PubMed

Ishii, Kunihiko; Tahara, Tahei

2013-10-03

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Fluorescence correlation spectroscopy: novel variations of an established technique.

PubMed

Haustein, Elke; Schwille, Petra

2007-01-01

Fluorescence correlation spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine.

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

PubMed Central

Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela, Lorena; Orte, Angel

2012-01-01

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. PMID:22949804

Chiral alkylated-aniline as a noninvasive fluorescence sensor: Spectroscopic and molecular modeling studies.

PubMed

Sengupta, Bidisha; Mukherjee, Chirantan Sen; Chakraborty, Sandipan; Muhammad, Maria Jones; Gladney, William; Armstrong, George

2017-12-05

Aniline, heterocyclic aromatic amines, and arylamines are known carcinogens. Recently aniline mustard has come into prominence as a novel anticancer agent. In this project, microwave irradiation has been used to synthesize an optically active alkylated aniline namely 2,6-dimethyl-4-(1-(p-tolyl)ethyl)aniline (abbreviated DMPA). The presence of quartet and doublet peaks in NMR and a single chromatogram in HPLC verified that the final product DMPA, prepared from the synthesis reactions, had no major impurities. By using a Lux chiral column in HPLC, two peaks have been detected in the chromatogram, which correspond to two enantiomers of the chiral aniline derivative. Fluorescence spectroscopic measurements on DMPA indicated conspicuous dependence of its emission behavior on the polarity (in terms of the empirical polarity parameter E T (30)) of the homogeneous solvents used, a property important for an optical sensor. The nature of the emission profiles, along with the relevant parameter namely wavelength at emission maximum (λ em max ) is used to infer the distribution, binding and microenvironment of the DMPA molecules in human serum albumin protein (HSA). DMPA is weakly fluorescent in aqueous buffer medium, with a dramatic enhancement in the fluorescence emission in the presence of HSA. Molecular modeling studies have been carried out on the two enantiomers (R and S) of DMPA with HSA. The implications of these findings are examined in relation to the potentialities of DMPA as a novel fluorescence sensor for biological systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Spatial fluorescence cross-correlation spectroscopy between core and ring pinholes

NASA Astrophysics Data System (ADS)

Blancquaert, Yoann; Delon, Antoine; Derouard, Jacques; Jaffiol, Rodolphe

2006-04-01

Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.

Multifunction Imaging and Spectroscopic Instrument

NASA Technical Reports Server (NTRS)

Mouroulis, Pantazis

2004-01-01

A proposed optoelectronic instrument would perform several different spectroscopic and imaging functions that, heretofore, have been performed by separate instruments. The functions would be reflectance, fluorescence, and Raman spectroscopies; variable-color confocal imaging at two different resolutions; and wide-field color imaging. The instrument was conceived for use in examination of minerals on remote planets. It could also be used on Earth to characterize material specimens. The conceptual design of the instrument emphasizes compactness and economy, to be achieved largely through sharing of components among subsystems that perform different imaging and spectrometric functions. The input optics for the various functions would be mounted in a single optical head. With the exception of a targeting lens, the input optics would all be aimed at the same spot on a specimen, thereby both (1) eliminating the need to reposition the specimen to perform different imaging and/or spectroscopic observations and (2) ensuring that data from such observations can be correlated with respect to known positions on the specimen. The figure schematically depicts the principal components and subsystems of the instrument. The targeting lens would collect light into a multimode optical fiber, which would guide the light through a fiber-selection switch to a reflection/ fluorescence spectrometer. The switch would have four positions, enabling selection of spectrometer input from the targeting lens, from either of one or two multimode optical fibers coming from a reflectance/fluorescence- microspectrometer optical head, or from a dark calibration position (no fiber). The switch would be the only moving part within the instrument.

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

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.

Binding-Induced Fluorescence of Serotonin Transporter Ligands: A Spectroscopic and Structural Study of 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) and APP+ Analogues

PubMed Central

2014-01-01

The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP+) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP+) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP+), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

Fluorescence-correlation spectroscopy study of molecular transport within reversed-phase chromatographic particles compared to planar model surfaces.

PubMed

Cooper, Justin; Harris, Joel M

2014-12-02

Reversed-phase liquid chromatography (RPLC) is a widely used technique for molecular separations. Stationary-phase materials for RPLC generally consist of porous silica-gel particles functionalized with n-alkane ligands. Understanding motions of molecules within the interior of these particles is important for developing efficient chromatographic materials and separations. To characterize these dynamics, time-resolved spectroscopic methods (photobleach recovery, fluorescence correlation, single-molecule imaging) have been adapted to measure molecular diffusion rates, typically at n-alkane-modified planar silica surfaces, which serve as models of chromatographic interfaces. A question arising from these studies is how dynamics of molecules on a planar surface relate to motions of molecules within the interior of a porous chromatographic particle. In this paper, imaging-fluorescence-correlation spectroscopy is used to measure diffusion rates of a fluorescent probe molecule 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) within authentic RPLC porous silica particles and compared with its diffusion at a planar C18-modified surface. The results show that surface diffusion on the planar C18 substrate is much faster than the diffusion rate of the probe molecule through a chromatographic particle. Surface diffusion within porous particles, however, is governed by molecular trajectories along the tortuous contours of the interior surface of the particles. By accounting for the greater surface area that a molecule must explore to diffuse macroscopic distances through the particle, the molecular-scale diffusion rates on the two surfaces can be compared, and they are virtually identical. These results provide support for the relevance of surface-diffusion measurements made on planar model surfaces to the dynamic behavior of molecules on the internal surfaces of porous chromatographic particles.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy

PubMed Central

Ridgeway, William K; Millar, David P; Williamson, James R

2013-01-01

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. PMID:23525193

Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs

PubMed Central

Bujalowski, Wlodzimierz; Jezewska, Maria J.

2011-01-01

Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand–macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein–nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein–nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein–nucleic acid interactions, it can generally be applied to any ligand–macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. PMID:21195223

Spectroscopic and photochemical properties of open-chain carotenoids.

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

Frank, H. A.; Josue, J. S.; Bautista, J. A.

2002-02-28

The spectroscopic properties of open-chain, all-trans-C{sub 30} carotenoids having seven, eight and nine {pi}-electron conjugated carbon-carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation and time-resolved absorption spectroscopy. These diapocarotenes were purified by high performance liquid chromatography (HPLC) prior to the spectroscopic experiments. The fluorescence data show a systematic crossover from dominant S{sub 1} {yields} S{sub 0} (2{sup 1}Ag{yields} 1{sup 1}Ag) emission to dominant S{sub 2} {yields} S{sub 0} (1{sup 1}Bu {yields} 1{sup 1}Ag) with increasing extent of conjugation. The low temperatures facilitated the determination of the spectral origins of the S{sub 1} {yields} S{sub 0} (2{sup 1}Agmore » {yields} 1{sup 1}Ag) emissions, which were assigned by Gaussian deconvolution of the experimental line shapes. The lifetimes of the S{sub 1} states of the molecules were measured by transient absorption spectroscopy and were found to decrease as the conjugated chain length increases. The energy gap law for radiationless transitions is used to correlate the S{sub 1} energies with the dynamics. These molecules provide a systematic series for understanding the structural features that control the photochemical properties of open-chain, diapocarotenoids. The implications of these results on the roles of carotenoids in photosynthetic organisms are discussed.« less

Fluorescence correlation spectroscopy of diffusion probed with a Gaussian Lorentzian spatial distribution

NASA Astrophysics Data System (ADS)

Marrocco, Michele

2007-11-01

Fluorescence correlation spectroscopy is fundamental in many physical, chemical and biological studies of molecular diffusion. However, the concept of fluorescence correlation is founded on the assumption that the analytical description of the correlation decay of diffusion can be achieved if the spatial profile of the detected volume obeys a three-dimensional Gaussian distribution. In the present Letter, the analytical result is instead proven for the fundamental Gaussian-Lorentzian profile.

Two-Photon Fluorescence Correlation Spectroscopy

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory A.; Fischer, David G.

2002-01-01

We will describe a two-photon microscope currently under development at the NASA Glenn Research Center. It is composed of a Coherent Mira 900 tunable, pulsed Titanium:Sapphire laser system, an Olympus Fluoview 300 confocal scanning head, and a Leica DM IRE inverted microscope. It will be used in conjunction with a technique known as fluorescence correlation spectroscopy (FCS) to study intracellular protein dynamics. We will briefly explain the advantages of the two-photon system over a conventional confocal microscope, and provide some preliminary experimental results.

Correlation Between Bulk Material Defects and Spectroscopic Response in Cadmium Zinc Telluride Detectors

NASA Technical Reports Server (NTRS)

Parker, Bradford H.; Stahle, C. M.; Barthelmy, S. D.; Parsons, A. M.; Tueller, J.; VanSant, J. T.; Munoz, B. F.; Snodgrass, S. J.; Mullinix, R. E.

1999-01-01

One of the critical challenges for large area cadmium zinc telluride (CdZnTe) detector arrays is obtaining material capable of uniform imaging and spectroscopic response. Two complementary nondestructive techniques for characterizing bulk CdZnTe have been developed to identify material with a uniform response. The first technique, infrared transmission imaging, allows for rapid visualization of bulk defects. The second technique, x-ray spectral mapping, provides a map of the material spectroscopic response when it is configured as a planar detector. The two techniques have been used to develop a correlation between bulk defect type and detector performance. The correlation allows for the use of infrared imaging to rapidly develop wafer mining maps. The mining of material free of detrimental defects has the potential to dramatically increase the yield and quality of large area CdZnTe detector arrays.

Whole-brain spectroscopic MRI biomarkers identify infiltrating margins in glioblastoma patients

PubMed Central

Cordova, James S.; Shu, Hui-Kuo G.; Liang, Zhongxing; Gurbani, Saumya S.; Cooper, Lee A. D.; Holder, Chad A.; Olson, Jeffrey J.; Kairdolf, Brad; Schreibmann, Eduard; Neill, Stewart G.; Hadjipanayis, Constantinos G.; Shim, Hyunsuk

2016-01-01

Background The standard of care for glioblastoma (GBM) is maximal safe resection followed by radiation therapy with chemotherapy. Currently, contrast-enhanced MRI is used to define primary treatment volumes for surgery and radiation therapy. However, enhancement does not identify the tumor entirely, resulting in limited local control. Proton spectroscopic MRI (sMRI), a method reporting endogenous metabolism, may better define the tumor margin. Here, we develop a whole-brain sMRI pipeline and validate sMRI metrics with quantitative measures of tumor infiltration. Methods Whole-brain sMRI metabolite maps were coregistered with surgical planning MRI and imported into a neuronavigation system to guide tissue sampling in GBM patients receiving 5-aminolevulinic acid fluorescence-guided surgery. Samples were collected from regions with metabolic abnormalities in a biopsy-like fashion before bulk resection. Tissue fluorescence was measured ex vivo using a hand-held spectrometer. Tissue samples were immunostained for Sox2 and analyzed to quantify the density of staining cells using a novel digital pathology image analysis tool. Correlations among sMRI markers, Sox2 density, and ex vivo fluorescence were evaluated. Results Spectroscopic MRI biomarkers exhibit significant correlations with Sox2-positive cell density and ex vivo fluorescence. The choline to N-acetylaspartate ratio showed significant associations with each quantitative marker (Pearson's ρ = 0.82, P < .001 and ρ = 0.36, P < .0001, respectively). Clinically, sMRI metabolic abnormalities predated contrast enhancement at sites of tumor recurrence and exhibited an inverse relationship with progression-free survival. Conclusions As it identifies tumor infiltration and regions at high risk for recurrence, sMRI could complement conventional MRI to improve local control in GBM patients. PMID:26984746

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Nakashima, K.; Xing, Shaoyong; Gong, Yongkuan; Miyajima, Toru

2008-07-01

We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda's rule.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Ridgeway, William K.; Millar, David P.; Williamson, James R.

2013-04-01

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantify reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. Catalogue identifier: AEOP_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOP_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 50189 No. of bytes in distributed program, including test data, etc.: 6135283 Distribution format: tar.gz Programming language: C/Assembly. Computer: Any with GCC and

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation

NASA Astrophysics Data System (ADS)

Weger, Lukas; Hoffmann-Jacobsen, Kerstin

2017-09-01

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction

NASA Astrophysics Data System (ADS)

Gärtner, Maria; Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

2009-07-01

In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

CFHTLenS and RCSLenS: testing photometric redshift distributions using angular cross-correlations with spectroscopic galaxy surveys

NASA Astrophysics Data System (ADS)

Choi, A.; Heymans, C.; Blake, C.; Hildebrandt, H.; Duncan, C. A. J.; Erben, T.; Nakajima, R.; Van Waerbeke, L.; Viola, M.

2016-12-01

We determine the accuracy of galaxy redshift distributions as estimated from photometric redshift probability distributions p(z). Our method utilizes measurements of the angular cross-correlation between photometric galaxies and an overlapping sample of galaxies with spectroscopic redshifts. We describe the redshift leakage from a galaxy photometric redshift bin j into a spectroscopic redshift bin I using the sum of the p(z) for the galaxies residing in bin j. We can then predict the angular cross-correlation between photometric and spectroscopic galaxies due to intrinsic galaxy clustering when I ≠ j as a function of the measured angular cross-correlation when I = j. We also account for enhanced clustering arising from lensing magnification using a halo model. The comparison of this prediction with the measured signal provides a consistency check on the validity of using the summed p(z) to determine galaxy redshift distributions in cosmological analyses, as advocated by the Canada-France-Hawaii Telescope Lensing Survey (CFHTLenS). We present an analysis of the photometric redshifts measured by CFHTLenS, which overlaps the Baryon Oscillation Spectroscopic Survey (BOSS). We also analyse the Red-sequence Cluster Lensing Survey, which overlaps both BOSS and the WiggleZ Dark Energy Survey. We find that the summed p(z) from both surveys are generally biased with respect to the true underlying distributions. If unaccounted for, this bias would lead to errors in cosmological parameter estimation from CFHTLenS by less than ˜4 per cent. For photometric redshift bins which spatially overlap in 3D with our spectroscopic sample, we determine redshift bias corrections which can be used in future cosmological analyses that rely on accurate galaxy redshift distributions.

Sentinel lymph nodes and lymphatic vessels: noninvasive dual-modality in vivo mapping by using indocyanine green in rats--volumetric spectroscopic photoacoustic imaging and planar fluorescence imaging.

PubMed

Kim, Chulhong; Song, Kwang Hyun; Gao, Feng; Wang, Lihong V

2010-05-01

To noninvasively map sentinel lymph nodes (SLNs) and lymphatic vessels in rats in vivo by using dual-modality nonionizing imaging-volumetric spectroscopic photoacoustic imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission-of indocyanine green (ICG). Institutional animal care and use committee approval was obtained. Healthy Sprague-Dawley rats weighing 250-420 g (age range, 60-120 days) were imaged by using volumetric photoacoustic imaging (n = 5) and planar fluorescence imaging (n = 3) before and after injection of 1 mmol/L ICG. Student paired t tests based on a logarithmic scale were performed to evaluate the change in photoacoustic signal enhancement of SLNs and lymphatic vessels before and after ICG injection. The spatial resolutions of both imaging systems were compared at various imaging depths (2-8 mm) by layering additional biologic tissues on top of the rats in vivo. Spectroscopic photoacoustic imaging was applied to identify ICG-dyed SLNs. In all five rats examined with photoacoustic imaging, SLNs were clearly visible, with a mean signal enhancement of 5.9 arbitrary units (AU) + or - 1.8 (standard error of the mean) (P < .002) at 0.2 hour after injection, while lymphatic vessels were seen in four of the five rats, with a signal enhancement of 4.3 AU + or - 0.6 (P = .001). In all three rats examined with fluorescence imaging, SLNs and lymphatic vessels were seen. The average full width at half maximum (FWHM) of the SLNs in the photoacoustic images at three imaging depths (2, 6, and 8 mm) was 2.0 mm + or - 0.2 (standard deviation), comparable to the size of a dissected lymph node as measured with a caliper. However, the FWHM of the SLNs in fluorescence images widened from 8 to 22 mm as the imaging depth increased, owing to strong light scattering. SLNs were identified spectroscopically in photoacoustic images. These two modalities, when used together with ICG, have the potential to help map SLNs in

LaPO4:Eu fluorescent nanorods, synthesis, characterization and spectroscopic studies on interaction with human serum albumin

NASA Astrophysics Data System (ADS)

Guo, Xingjia; Yao, Jie; Liu, Xuehui; Wang, Hongyan; Zhang, Lizhi; Xu, Liping; Hao, Aijun

2018-06-01

Eu3+ doped LaPO4 fluorescent nanorods (LaPO4:Eu) was successfully fabricated by a hydrothermal process. The obtained LaPO4:Eu nanorods under the optimal conditions were characterized by means of transmission electron microscopy (TEM), X-ray diffraction (XRD) technique, Fourier transform infrared (FTIR), UV-vis absorption and fluorescence spectroscopy. The nanorods with a length of 50-100 nm and a diameter of about 10 nm, can emit strong red fluorescence upon excitation at 241 nm. The FTIR result confirmed that there are lots of phosphate groups on the surfaces of nanorods. In order to better understand the physiological behavior of nanorods in human body, multiple spectroscopic methods were used to study the interaction between the LaPO4:Eu nanorods and human serum albumin (HSA) in the simulated physiological conditions. The results indicated that the nanorods can effectively quench the intrinsic fluorescence of HSA through a dynamic quenching mode with the association constants of the order of 103 L mol-1. The values of the thermodynamic parameters suggested that the binding of the nanorods to HSA was a spontaneous process and van der Waals forces and hydrogen bonds played a predominant role. The displacement experiments verified that the binding site of nanorods on HSA was mainly located in the hydrophobic pocket of subdomain IIA (site I) of HSA. The binding distance between nanorods and HSA was calculated to be 4.2 nm according to the theory of Förster non-radiation energy transfer. The analysis of synchronous fluorescence, three-dimensional fluorescence (3D) and circular dichroism (CD) spectra indicated that there the addition of LaPO4:Eu nanorods did not caused significant alterations in conformation of HSA secondary structure and the polarity around the amino acid residues.

LaPO4:Eu fluorescent nanorods, synthesis, characterization and spectroscopic studies on interaction with human serum albumin.

PubMed

Guo, Xingjia; Yao, Jie; Liu, Xuehui; Wang, Hongyan; Zhang, Lizhi; Xu, Liping; Hao, Aijun

2018-06-05

Eu 3+ doped LaPO 4 fluorescent nanorods (LaPO 4 :Eu) was successfully fabricated by a hydrothermal process. The obtained LaPO 4 :Eu nanorods under the optimal conditions were characterized by means of transmission electron microscopy (TEM), X-ray diffraction (XRD) technique, Fourier transform infrared (FTIR), UV-vis absorption and fluorescence spectroscopy. The nanorods with a length of 50-100nm and a diameter of about 10nm, can emit strong red fluorescence upon excitation at 241nm. The FTIR result confirmed that there are lots of phosphate groups on the surfaces of nanorods. In order to better understand the physiological behavior of nanorods in human body, multiple spectroscopic methods were used to study the interaction between the LaPO 4 :Eu nanorods and human serum albumin (HSA) in the simulated physiological conditions. The results indicated that the nanorods can effectively quench the intrinsic fluorescence of HSA through a dynamic quenching mode with the association constants of the order of 10 3 Lmol -1 . The values of the thermodynamic parameters suggested that the binding of the nanorods to HSA was a spontaneous process and van der Waals forces and hydrogen bonds played a predominant role. The displacement experiments verified that the binding site of nanorods on HSA was mainly located in the hydrophobic pocket of subdomain IIA (site I) of HSA. The binding distance between nanorods and HSA was calculated to be 4.2nm according to the theory of Förster non-radiation energy transfer. The analysis of synchronous fluorescence, three-dimensional fluorescence (3D) and circular dichroism (CD) spectra indicated that there the addition of LaPO 4 :Eu nanorods did not caused significant alterations in conformation of HSA secondary structure and the polarity around the amino acid residues. Copyright © 2018 Elsevier B.V. All rights reserved.

Transient Fluorescence Spectroscopy and laser induced fluorescence lifetimes of terbium doped dipicolinic acid

NASA Astrophysics Data System (ADS)

Makoui, Anali

We have investigated the use of deep UV laser induced fluorescence for the sensitive detection and spectroscopic lifetime studies of terbium doped dipicolinic acid (DPA-Tb) and used this to study the optical characteristics of DPA which is a chemical surrounding most bacterial spores. Background absorption spectra, fluorescence spectra, and Excitation Emission Matrix (EEM) spectra were made of the DPA-Tb complex, using both fixed 266 nm wavelength and tunable (220 nm--280 nm) UV laser excitations. Of importance, the fluorescence lifetimes of the four main fluorescence peaks (488 nm, 543 nm, 581 nm, and 618 nm) of the DPA-Tb complex have been measured for the first time to our knowledge. The lifetimes of all the fluorescing lines have been measured as a function of DPA-Tb concentration, solvent pH, and solvent composition, including that for the weakest fluorescing line of DPA-Tb at 618 nm. In addition, a new spectroscopic lifetime measurement technique, which we call "Transient Fluorescence Spectroscopy", was developed. In this technique, a weak, quasi-CW, amplitude modulated UV laser (8.5 kHz) was used to measure the lifetimes of the fluorescence lines, and yields insight into energy transfer and excitation lifetimes within the system. This technique is especially useful when a high power laser is not either available or not suitable. In the latter case, this would be when a high power pulsed deep-UV laser could produce bleaching or destruction of the biological specimen. In addition, this technique simulated the excitation and fluorescence emission of the DPA-Tb using a 4-level energy model, and solved the dynamic transient rate equations to predict the temporal behavior of the DPA-Tb emitted fluorescence. Excellent agreement between the experiments and the simulation were found. This technique has the potential to provide a more accurate value for the fluorescence lifetime values. In addition, with the use of asymmetric excitation waveforms, the dynamic

A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

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

Gong, S.; Labanca, I.; Rech, I.

2014-10-15

Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments.more » However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.« less

Computer Modeling of the Structure and Spectra of Fluorescent Proteins

PubMed Central

Grigorenko, B.L.; Savitsky, A.P.

2009-01-01

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers. PMID:22649601

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

[Vermicomposting of different organic materials and three-dimensional excitation emission matrix fluorescence spectroscopic characterization of their dissolved organic matter].

PubMed

Yang, Wei; Wang, Dong-sheng; Liu, Man-qiang; Hu, Feng; Li, Hui-xin; Huang, Zhong-yang; Chang, Yi-jun; Jiao, Jia-guo

2015-10-01

In this experiment, different proportions of the cattle manure, tea-leaf, herb and mushroom residues, were used as food for earthworm (Eisenia fetida) to study the growth of the earth-worm. Then the characteristics and transformation of nutrient content and three-dimensional excitation emission matrix fluorescence (3DEEM) of dissolved organic matter (DOM) during vermistabilization were investigated by means of chemical and spectroscopic methods. The result showed that the mixture of different ratios of cattle manure with herb residue, and cattle manure with tea-leaf were conducive to the growth of earthworm, while the materials compounded with mushroom residue inhibited the growth of earthworm. With the increasing time of verimcomposting, the pH in vermicompost tended to be circumneutral and weakly acidic, and there were increases in electrical conductivity, and the contents of total nitrogen, total phosphorus, available nitrogen, and available phosphorus, while the total potassium and available potassium increased first and then decreased, and the organic matter content decreased. 3DEEM and fluorescence regional integration results indicated that, the fluorescence of protein-like fluorescence peaks declined significantly, while the intensity of humic-like fluorescence peak increased significantly in DOM. Vermicomposting process might change the compositions of DOM with elevated concentrations of humic acid and fulvic acid in the organics. In all, this study suggested the suitability of 3DEEM for monitoring the organics transformation and assessing the maturity in the vermicomposting.

Spectroscopic identification of individual fluorophores using photoluminescence excitation spectra.

PubMed

Czerski, J; Colomb, W; Cannataro, F; Sarkar, S K

2018-01-25

The identity of a fluorophore can be ambiguous if other fluorophores or nonspecific fluorescent impurities have overlapping emission spectra. The presence of overlapping spectra makes it difficult to differentiate fluorescent species using discrete detection channels and unmixing of spectra. The unique absorption and emission signatures of fluorophores provide an opportunity for spectroscopic identification. However, absorption spectroscopy may be affected by scattering, whereas fluorescence emission spectroscopy suffers from signal loss by gratings or other dispersive optics. Photoluminescence excitation spectra, where excitation is varied and emission is detected at a fixed wavelength, allows hyperspectral imaging with a single emission filter for high signal-to-background ratio without any moving optics on the emission side. We report a high throughput method for measuring the photoluminescence excitation spectra of individual fluorophores using a tunable supercontinuum laser and prism-type total internal reflection fluorescence microscope. We used the system to measure and sort the photoluminescence excitation spectra of individual Alexa dyes, fluorescent nanodiamonds (FNDs), and fluorescent polystyrene beads. We used a Gaussian mixture model with maximum likelihood estimation to objectively separate the spectra. Finally, we spectroscopically identified different species of fluorescent nanodiamonds with overlapping spectra and characterized the heterogeneity of fluorescent nanodiamonds of varying size. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Spectroscopic characterization of dissolved organic matter in coking wastewater during bio-treatment: full-scale plant study.

PubMed

Xu, Ronghua; Ou, Huase; Yu, Xubiao; He, Runsheng; Lin, Chong; Wei, Chaohai

2015-01-01

This paper taking a full-scale coking wastewater (CWW) treatment plant as a case study aimed to characterize removal behaviors of dissolved organic matter (DOM) by UV spectra and fluorescence excitation-emission matrix-parallel factor analysis (PARAFAC), and investigate the correlations between spectroscopic indices and water quality parameters. Efficient removal rates of chemical oxygen demand (COD), dissolved organic carbon (DOC) and total nitrogen (TN) after the bio-treatment were 91.3%, 87.3% and 69.1%, respectively. UV270 was proven to be a stable UV absorption peak of CWW that could reflect the mixture of phenols, heterocyclics, polynuclear aromatic hydrocarbons and their derivatives. Molecular weight and aromaticity were increased, and also the content of polar functional groups was greatly reduced after bio-treatment. Three fluorescent components were identified by PARAFAC: C1 (tyrosine-like), C2 (tryptophan-like) and C3 (humic-like). The removal rate of protein-like was higher than that of humic-like and C1 was identified as biodegradable substance. Correlation analysis showed UV270 had an excellent correlation with COD (r=0.921, n=60, P<0.01) and DOC (r=0.959, n=60, P<0.01) and significant correlation (r=0.875, n=60, P<0.01) was also found between C2 and TN. Therefore, spectroscopic characterization could provide novel insights into removal behaviors of DOM and potential to monitor water quality real-time during CWW bio-treatment.

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Spectroscopic study of honey from Apis mellifera from different regions in Mexico

NASA Astrophysics Data System (ADS)

Frausto-Reyes, C.; Casillas-Peñuelas, R.; Quintanar-Stephano, JL; Macías-López, E.; Bujdud-Pérez, JM; Medina-Ramírez, I.

2017-05-01

The objective of this study was to analyze by Raman and UV-Vis-NIR Spectroscopic techniques, Mexican honey from Apis Mellífera, using representative samples with different botanic origins (unifloral and multifloral) and diverse climates. Using Raman spectroscopy together with principal components analysis, the results obtained represent the possibility to use them for determination of floral origin of honey, independently of the region of sampling. For this, the effect of heat up the honey was analyzed in relation that it was possible to greatly reduce the fluorescence background in Raman spectra, which allowed the visualization of fructose and glucose peaks. Using UV-Vis-NIR, spectroscopy, a characteristic spectrum profile of transmittance was obtained for each honey type. In addition, to have an objective characterization of color, a CIE Yxy and CIE L*a*b* colorimetric register was realized for each honey type. Applying the principal component analysis and their correlation with chromaticity coordinates allowed classifying the honey samples in one plot as: cutoff wavelength, maximum transmittance, tones and lightness. The results show that it is possible to obtain a spectroscopic record of honeys with specific characteristics by reducing the effects of fluorescence.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy.

PubMed

Haustein, Elke; Schwille, Petra

2003-02-01

Fluorescence correlation spectroscopy (FCS) extracts information about molecular dynamics from the tiny fluctuations that can be observed in the emission of small ensembles of fluorescent molecules in thermodynamic equilibrium. Employing a confocal setup in conjunction with highly dilute samples, the average number of fluorescent particles simultaneously within the measurement volume (approximately 1 fl) is minimized. Among the multitude of chemical and physical parameters accessible by FCS are local concentrations, mobility coefficients, rate constants for association and dissociation processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resolution of less than 0.5 microm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local molecular dynamics provides access to environmental parameters such as local oxygen concentrations, pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quantitative assessment of interactions and dynamics of small molecular quantities in biologically relevant systems.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

Fluorescence detection of esophageal neoplasia

NASA Astrophysics Data System (ADS)

Borisova, E.; Vladimirov, B.; Avramov, L.

2008-06-01

White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

On the Development, Characterization, and Use of Protein Fluorescence and Infrared Spectroscopic Probes

NASA Astrophysics Data System (ADS)

Hilaire, Mary Rose

Proteins possess unique physical and chemical properties that allow them to carry out a wide variety of biological activities and functions. While it is generally understood that a protein's function is dictated by its structure and dynamics, arriving at a molecule-level understanding of the underlying structure-dynamics-function relationship still poses a challenging task in many cases. This is due, at least in part, to the fact that we lack the ability to take snapshots along the reaction coordinate of proteins with sufficient temporal and structural resolution. Therefore, to improve one's ability to acquire site-specific structural and/or environmental information of proteins via either infrared (IR) or fluorescence spectroscopy, the main focus of this thesis is to develop and characterize amino acid-based spectroscopic probes as well as to use such probes to study important biological questions. Specifically, we show that (1) p-cyanophenylalanine and selenomethionine constitute an efficient fluorophore-quencher pair, useful for characterizing protein conformational changes that occur on a short distance; (2) 4-cyanotryptophan is a novel blue fluorescent amino acid, applicable for biological imaging due to its unique photophysical properties; (3) the dielectric constant inside the hydrophobic interior of staphylococcal nuclease is about 10-15, significantly larger than previously assumed; and (4) a single mutation in a short segment of the protein transthyretin (i.e., 110-115) induces formation of amyloid fibrils consisting of both beta- and alpha-sheets, where the latter is a proposed structure in proteins, but has never been observed previously.

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.

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

Synthesis and spectroscopic properties of some new difluoroboron bis-β-diketonate derivatives.

PubMed

Pi, Yan; Wang, Dun-Jia; Liu, Hua; Hu, Yan-Jun; Wei, Xian-Hong; Zheng, Jing

2014-10-15

Six new bis-β-diketones (RCOCH2CO-C7H7N-COCH2COR) were synthesized from 3,5-diacetyl-2,6-dimethylpyridine via Claisen condensation with the corresponding esters, and then reacted with boron trifluoride etherate to afford difluoroboron bis-β-diketonate derivatives. Their spectroscopic properties were investigated by UV-vis, FTIR, (1)H NMR and fluorescence spectroscopic techniques. It was found that these boron complexes exhibited violet or blue fluorescence emission at 422-445nm and possessed high extinction coefficients. The results indicate that the extending π-conjugation can increase the fluorescence intensity and quantum yield for these boron complexes. Especially, the compound 2b displayed the stronger fluorescence intensity and the highest fluorescence quantum yield (Φu=0.94) in these boron compounds. However, compounds 2c and 2d had the lower fluorescence intensity and quantum yield as a result of the heavy atom effect of the chlorine atom in the molecules. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis and spectroscopic properties of some new difluoroboron bis-β-diketonate derivatives

NASA Astrophysics Data System (ADS)

Pi, Yan; Wang, Dun-Jia; Liu, Hua; Hu, Yan-Jun; Wei, Xian-Hong; Zheng, Jing

2014-10-01

Six new bis-β-diketones (RCOCH2CO-C7H7N-COCH2COR) were synthesized from 3,5-diacetyl-2,6-dimethylpyridine via Claisen condensation with the corresponding esters, and then reacted with boron trifluoride etherate to afford difluoroboron bis-β-diketonate derivatives. Their spectroscopic properties were investigated by UV-vis, FTIR, 1H NMR and fluorescence spectroscopic techniques. It was found that these boron complexes exhibited violet or blue fluorescence emission at 422-445 nm and possessed high extinction coefficients. The results indicate that the extending π-conjugation can increase the fluorescence intensity and quantum yield for these boron complexes. Especially, the compound 2b displayed the stronger fluorescence intensity and the highest fluorescence quantum yield (Φu = 0.94) in these boron compounds. However, compounds 2c and 2d had the lower fluorescence intensity and quantum yield as a result of the heavy atom effect of the chlorine atom in the molecules.

Antiphase dual-color correlation in a reactant-product pair imparts ultrasensitivity in reaction-linked double-photoswitching fluorescence imaging.

PubMed

Wan, Wei; Zhu, Ming-Qiang; Tian, Zhiyuan; Li, Alexander D Q

2015-04-08

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude.

Tracking quasi-stationary flow of weak fluorescent signals by adaptive multi-frame correlation.

PubMed

Ji, L; Danuser, G

2005-12-01

We have developed a novel cross-correlation technique to probe quasi-stationary flow of fluorescent signals in live cells at a spatial resolution that is close to single particle tracking. By correlating image blocks between pairs of consecutive frames and integrating their correlation scores over multiple frame pairs, uncertainty in identifying a globally significant maximum in the correlation score function has been greatly reduced as compared with conventional correlation-based tracking using the signal of only two consecutive frames. This approach proves robust and very effective in analysing images with a weak, noise-perturbed signal contrast where texture characteristics cannot be matched between only a pair of frames. It can also be applied to images that lack prominent features that could be utilized for particle tracking or feature-based template matching. Furthermore, owing to the integration of correlation scores over multiple frames, the method can handle signals with substantial frame-to-frame intensity variation where conventional correlation-based tracking fails. We tested the performance of the method by tracking polymer flow in actin and microtubule cytoskeleton structures labelled at various fluorophore densities providing imagery with a broad range of signal modulation and noise. In applications to fluorescent speckle microscopy (FSM), where the fluorophore density is sufficiently low to reveal patterns of discrete fluorescent marks referred to as speckles, we combined the multi-frame correlation approach proposed above with particle tracking. This hybrid approach allowed us to follow single speckles robustly in areas of high speckle density and fast flow, where previously published FSM analysis methods were unsuccessful. Thus, we can now probe cytoskeleton polymer dynamics in living cells at an entirely new level of complexity and with unprecedented detail.

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

PubMed

Pandžić, Elvis; Abu-Arish, Asmahan; Whan, Renee M; Hanrahan, John W; Wiseman, Paul W

2018-02-16

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Fluorescence Lifetime Imaging Microscopy Using Near-Infrared Contrast Agents

PubMed Central

Nothdurft, Ralph; Sarder, Pinaki; Bloch, Sharon; Culver, Joseph; Achilefu, Samuel

2013-01-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labeled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes’ relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. PMID:22788550

Cross-correlation redshift calibration without spectroscopic calibration samples in DES Science Verification Data

NASA Astrophysics Data System (ADS)

Davis, C.; Rozo, E.; Roodman, A.; Alarcon, A.; Cawthon, R.; Gatti, M.; Lin, H.; Miquel, R.; Rykoff, E. S.; Troxel, M. A.; Vielzeuf, P.; Abbott, T. M. C.; Abdalla, F. B.; Allam, S.; Annis, J.; Bechtol, K.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Burke, D. L.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Crocce, M.; Cunha, C. E.; D'Andrea, C. B.; da Costa, L. N.; Desai, S.; Diehl, H. T.; Doel, P.; Drlica-Wagner, A.; Fausti Neto, A.; Flaugher, B.; Fosalba, P.; Frieman, J.; García-Bellido, J.; Gaztanaga, E.; Gerdes, D. W.; Giannantonio, T.; Gruen, D.; Gruendl, R. A.; Gutierrez, G.; Honscheid, K.; Jain, B.; James, D. J.; Jeltema, T.; Krause, E.; Kuehn, K.; Kuhlmann, S.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Lima, M.; March, M.; Marshall, J. L.; Martini, P.; Melchior, P.; Ogando, R. L. C.; Plazas, A. A.; Romer, A. K.; Sanchez, E.; Scarpine, V.; Schindler, R.; Schubnell, M.; Sevilla-Noarbe, I.; Smith, M.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Thomas, D.; Vikram, V.; Walker, A. R.; Wechsler, R. H.

2018-06-01

Galaxy cross-correlations with high-fidelity redshift samples hold the potential to precisely calibrate systematic photometric redshift uncertainties arising from the unavailability of complete and representative training and validation samples of galaxies. However, application of this technique in the Dark Energy Survey (DES) is hampered by the relatively low number density, small area, and modest redshift overlap between photometric and spectroscopic samples. We propose instead using photometric catalogues with reliable photometric redshifts for photo-z calibration via cross-correlations. We verify the viability of our proposal using redMaPPer clusters from the Sloan Digital Sky Survey (SDSS) to successfully recover the redshift distribution of SDSS spectroscopic galaxies. We demonstrate how to combine photo-z with cross-correlation data to calibrate photometric redshift biases while marginalizing over possible clustering bias evolution in either the calibration or unknown photometric samples. We apply our method to DES Science Verification (DES SV) data in order to constrain the photometric redshift distribution of a galaxy sample selected for weak lensing studies, constraining the mean of the tomographic redshift distributions to a statistical uncertainty of Δz ˜ ±0.01. We forecast that our proposal can, in principle, control photometric redshift uncertainties in DES weak lensing experiments at a level near the intrinsic statistical noise of the experiment over the range of redshifts where redMaPPer clusters are available. Our results provide strong motivation to launch a programme to fully characterize the systematic errors from bias evolution and photo-z shapes in our calibration procedure.

Cross-correlation redshift calibration without spectroscopic calibration samples in DES Science Verification Data

DOE PAGES

Davis, C.; Rozo, E.; Roodman, A.; ...

2018-03-26

Galaxy cross-correlations with high-fidelity redshift samples hold the potential to precisely calibrate systematic photometric redshift uncertainties arising from the unavailability of complete and representative training and validation samples of galaxies. However, application of this technique in the Dark Energy Survey (DES) is hampered by the relatively low number density, small area, and modest redshift overlap between photometric and spectroscopic samples. We propose instead using photometric catalogs with reliable photometric redshifts for photo-z calibration via cross-correlations. We verify the viability of our proposal using redMaPPer clusters from the Sloan Digital Sky Survey (SDSS) to successfully recover the redshift distribution of SDSS spectroscopic galaxies. We demonstrate how to combine photo-z with cross-correlation data to calibrate photometric redshift biases while marginalizing over possible clustering bias evolution in either the calibration or unknown photometric samples. We apply our method to DES Science Verification (DES SV) data in order to constrain the photometric redshift distribution of a galaxy sample selected for weak lensing studies, constraining the mean of the tomographic redshift distributions to a statistical uncertainty ofmore » $$\\Delta z \\sim \\pm 0.01$$. We forecast that our proposal can in principle control photometric redshift uncertainties in DES weak lensing experiments at a level near the intrinsic statistical noise of the experiment over the range of redshifts where redMaPPer clusters are available. Here, our results provide strong motivation to launch a program to fully characterize the systematic errors from bias evolution and photo-z shapes in our calibration procedure.« less

Cross-correlation redshift calibration without spectroscopic calibration samples in DES Science Verification Data

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

Davis, C.; Rozo, E.; Roodman, A.

Galaxy cross-correlations with high-fidelity redshift samples hold the potential to precisely calibrate systematic photometric redshift uncertainties arising from the unavailability of complete and representative training and validation samples of galaxies. However, application of this technique in the Dark Energy Survey (DES) is hampered by the relatively low number density, small area, and modest redshift overlap between photometric and spectroscopic samples. We propose instead using photometric catalogs with reliable photometric redshifts for photo-z calibration via cross-correlations. We verify the viability of our proposal using redMaPPer clusters from the Sloan Digital Sky Survey (SDSS) to successfully recover the redshift distribution of SDSS spectroscopic galaxies. We demonstrate how to combine photo-z with cross-correlation data to calibrate photometric redshift biases while marginalizing over possible clustering bias evolution in either the calibration or unknown photometric samples. We apply our method to DES Science Verification (DES SV) data in order to constrain the photometric redshift distribution of a galaxy sample selected for weak lensing studies, constraining the mean of the tomographic redshift distributions to a statistical uncertainty ofmore » $$\\Delta z \\sim \\pm 0.01$$. We forecast that our proposal can in principle control photometric redshift uncertainties in DES weak lensing experiments at a level near the intrinsic statistical noise of the experiment over the range of redshifts where redMaPPer clusters are available. Here, our results provide strong motivation to launch a program to fully characterize the systematic errors from bias evolution and photo-z shapes in our calibration procedure.« less

Remote excitation fluorescence correlation spectroscopy using silver nanowires

NASA Astrophysics Data System (ADS)

Su, Liang; Yuan, Haifeng; Lu, Gang; Hofkens, Johan; Roeffaers, Maarten; Uji-i, Hiroshi

2014-11-01

Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Applications of fluorescence spectroscopy to problems of food safety: detection of fecal contamination and of the presence of central nervous system tissue and diagnosis of neurological disease

NASA Astrophysics Data System (ADS)

Adhikary, Ramkrishna; Bose, Sayantan; Casey, Thomas A.; Gapsch, Al; Rasmussen, Mark A.; Petrich, Jacob W.

2010-02-01

Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.

Planetary Surface Analysis Using Fast Laser Spectroscopic Techniques: Combined Microscopic Raman, LIBS, and Fluorescence Spectroscopy

NASA Astrophysics Data System (ADS)

Blacksberg, J.; Rossman, G. R.; Maruyama, Y.; Charbon, E.

2011-12-01

In situ exploration of planetary surfaces has to date required multiple techniques that, when used together, yield important information about their formation histories and evolution. We present a time-resolved laser spectroscopic technique that could potentially collect complementary sets of data providing information on mineral structure, composition, and hydration state. Using a picosecond-scale pulsed laser and a fast time-resolved detector we can simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence emissions that are separated in time due to the unique decay times of each process. The use of a laser with high rep rate (40 KHz) and low pulse energy (1 μJ/pulse) allows us to rapidly collect high signal to noise Raman spectra while minimizing sample damage. Increasing the pulse energy by about an order of magnitude creates a microscopic plasma near the surface and enables the collection of LIBS spectra at an unusually high rep rate and low pulse energy. Simultaneously, broader fluorescence peaks can be detected with lifetimes varying from nanosecond to microsecond. We will present Raman, LIBS, and fluorescence spectra obtained on natural mineral samples such as sulfates, clays, pyroxenes and carbonates that are of interest for Mars mineralogy. We demonstrate this technique using a photocathode-based streak camera detector as well as a newly-developed solid state Single Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. We will discuss the impact of system design and detector choice on science return of a potential planetary surface mission, with a specific focus on size, weight, power, and complexity. The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA).

Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications

PubMed Central

Olson, Eben; Torres, Richard; Levene, Michael J.

2013-01-01

We describe an optical system which reduces the cost and complexity of fluorescence correlation spectroscopy (FCS), intended to increase the suitability of the technique for clinical use. Integration of the focusing optics and sample chamber into a plastic component produces a design which is simple to align and operate. We validate the system by measurements on fluorescent dye, and compare the results to a commercial instrument. In addition, we demonstrate its application to measurements of concentration and multimerization of the clinically relevant protein von Willebrand factor (vWF) in human plasma. PMID:23847733

Photophysical properties of fluorescently-labeled peptoids.

PubMed

Rudat, Birgit; Birtalan, Esther; Vollrath, Sidonie B L; Fritz, Daniel; Kölmel, Dominik K; Nieger, Martin; Schepers, Ute; Müllen, Klaus; Eisler, Hans-Jürgen; Lemmer, Uli; Bräse, Stefan

2011-09-01

Fluorescently-labeled biomolecules are often utilized in biochemical or cellular experiments without further detailed spectroscopical characterization. This report is intended to narrow this gap and therefore presents the photophysical investigation of a library of 17 fluorescently-labeled molecules, namely peptoid transporters. First, one peptoid structure is labeled with seven different fluorophores and the spectroscopical properties are examined. Absorption and fluorescence maxima are almost identical for free dyes and conjugated dyes, suggesting free choice of a spectrally suitable fluorophore for different applications. Otherwise, extinction coefficients and quantum yields, and therefore the brightness of all seven dyes are strongly influenced. For the fluorophores, e.g. rhodamine B, the extent of this influence depends on the peptoid itself. This is shown by comparing different structures in the second part of this report. Especially the side chain functionalities influence the brightness. And finally, peptoids having two identical fluorescent labels are presented, which show decreased quantum yields. Possible reasons for the observed photophysical properties are discussed. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Spectroscopic Evidence of Anthropogenic Compounds Extraction from Polymers by Fluorescent Dissolved Organic Matter in Natural Water

NASA Astrophysics Data System (ADS)

Miranda, M.; Trojzuck, A.; Voss, D.; Gassmann, S.; Zielinski, O.

2016-04-01

FDOM is one of the most important carriers of anthropogenic compounds in natural waters. It can combine with environmental contaminants and polymers to form diverse chemical structures. To this end, here a microfluidic chip was designed for the analysis of these changes in fluorescent dissolved organic matter (FDOM) fingerprints due to thermal treatment and varying time intervals of exposure. Excitation Emission Matrix Spectroscopy (EEMS) approach was utilized to detect and identify the inherent compounds in sampled FDOM. Strong direct correlations were founded, Spearman rank correlation values (ρ = 0.85 at α = 0.1, n = 4) and linear correlation R2 = 0.8359 were noted between thermal treatment pattern 2 and fluorescence intensity of samples. Materials, acrylic based glue and cyclic olefin copolymer (COC) polymer, used to design the microfluidic sensor were determined to possess unique spectral features in the ultraviolet to green spectrum using EEMS. The study therefore provides an insight on methods to identify contaminants in natural waters. This underlines the potential of optical sensors providing measurements at fast intervals, enabling environmental monitoring.

Fluorometric detection of nitroaromatics by fluorescent lead complexes: A spectroscopic assessment of detection mechanism

NASA Astrophysics Data System (ADS)

Chattopadhyay, Tanmay; Chatterjee, Sourav; Majumder, Ishani; Ghosh, Soumen; Yoon, Sangee; Sim, Eunji

2018-04-01

Three Schiff base ligands such as 2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-2-hydroxymethyl-propane-1,3-diol (HL1), 2-[(2-Hydroxy-benzylidene)-amino]-2-hydroxymethyl-propane-1,3-diol (HL2), 2-[(3,5-Dichloro-2-hydroxy-benzylidene)-amino]-2-hydroxymethyl-propane-1,3-diol (HL3) have been synthesized by condensation of aldehydes (such as 3,5-Dichloro-2-hydroxy benzaldehyde, 2-Hydroxy-benzaldehyde, and 2-Hydroxy-3-methoxy-benzaldehyde) with Tris-(hydroxymethyl)amino methane and characterized by IR, UV-vis and 1H NMR spectroscopy. Then all these three ligands have been used to prepare Pb(II) complexes by reaction with lead(II) acetate tri-hydrate in methanol. In view of analytical and spectral (IR, UV-vis and Mass) studies, it has been concluded that, except HL2, other two ligands form 1:1 metal complexes (1 and 3) with lead. Between two complexes, complex 3 is highly fluorescent and this property has been used to identify the pollutant nitroaromatics. Finally, the quenching mechanism has been established by means of spectroscopic investigation.

Multiprobe Spectroscopic Inverstigation of Molecular-level Behavior within Aqueous 1-Butyl-3-methylimidazolium Tetrafluoroborate

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

Sarkar, Abhra; Ali, Maroof; Baker, Gary A

2009-01-01

In this work, an array of molecular-level solvent featuressincluding solute-solvent/solvent-solvent interactions, dipolarity, heterogeneity, dynamics, probe accessibility, and diffusionswere investigated across the entire composition of ambient mixtures containing the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, [bmim][BF4], and pH 7.0 phosphate buffer, based on results assembled for nine different molecular probes utilized in a range of spectroscopic modes. These studies uncovered interesting and unusual solvatochromic probe behavior within this benchmark mixture. Solvatochromic absorbance probessa watersoluble betaine dye (betaine dye 33), N,N-diethyl-4-nitroaniline, and 4-nitroanilineswere employed to determine ET (a blend of dipolarity/polarizability and hydrogen bond donor contributions) and the Kamlet-Taft indices * (dipolarity/polarizability), R (hydrogenmore » bond donor acidity), and (hydrogen bond acceptor basicity) characterizing the [bmim][BF4] + phosphate buffer system. These parameters each showed a marked deviation from ideality, suggesting selective solvation of the individual probe solutes by [bmim][BF4]. Similar conclusions were derived from the responses of the fluorescent polarity-sensitive probes pyrene and pyrene-1-carboxaldehyde. Importantly, the fluorescent microfluidity probe 1,3-bis(1-pyrenyl)propane senses a microviscosity within the mixture that significantly exceeds expectations derived from simple interpolation of the behavior in the neat solvents. On the basis of results from this probe, a correlation between microviscosity and bulk viscosity was established; pronounced solvent-solvent hydrogen-bonding interactions were implicit in this behavior. The greatest deviation from ideal additive behavior for the probes studied herein was consistently observed to occur in the buffer-rich regime. Nitromethane-based fluorescence quenching of pyrene within the [bmim][BF4] + phosphate buffer system showed unusual compliance with

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.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery

PubMed Central

Jermyn, Michael; Gosselin, Yoann; Valdes, Pablo A.; Sibai, Mira; Kolste, Kolbein; Mercier, Jeanne; Angulo, Leticia; Roberts, David W.; Paulsen, Keith D.; Petrecca, Kevin; Daigle, Olivier; Wilson, Brian C.; Leblond, Frederic

2015-01-01

In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-of-magnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival. PMID:26713218

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

PubMed Central

Kinjo, Masataka

2018-01-01

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. PMID:29570669

Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yilmaz, Hasan

2016-03-01

Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Novel Multidimensional Cross-Correlation Data Comparison Techniques for Spectroscopic Discernment in a Volumetrically Sensitive, Moderating Type Neutron Spectrometer

NASA Astrophysics Data System (ADS)

Hoshor, Cory; Young, Stephan; Rogers, Brent; Currie, James; Oakes, Thomas; Scott, Paul; Miller, William; Caruso, Anthony

2014-03-01

A novel application of the Pearson Cross-Correlation to neutron spectral discernment in a moderating type neutron spectrometer is introduced. This cross-correlation analysis will be applied to spectral response data collected through both MCNP simulation and empirical measurement by the volumetrically sensitive spectrometer for comparison in 1, 2, and 3 spatial dimensions. The spectroscopic analysis methods discussed will be demonstrated to discern various common spectral and monoenergetic neutron sources.

Scanning fluorescence correlation spectroscopy comes full circle.

PubMed

Gunther, German; Jameson, David M; Aguilar, Joao; Sánchez, Susana A

2018-02-07

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

Spectroscopic identification of rare earth elements in phosphate glass

NASA Astrophysics Data System (ADS)

Devangad, Praveen; Tamboli, Maktum; Muhammed Shameem, K. M.; Nayak, Rajesh; Patil, Ajeetkumar; Unnikrishnan, V. K.; Santhosh, C.; Kumar, G. A.

2018-01-01

In this work, rare earth-doped phosphate glasses were synthesized and characterized using three different spectroscopic techniques. The absorption spectra of the prepared praseodymium (Pr) and samarium (Sm) doped glasses, recorded by a UV-VIS-NIR spectrophotometer, show the characteristic absorption bands of these elements. To confirm this inference, laser-induced fluorescence spectra of Pr and Sm were obtained at a laser excitation of 442 nm. Their emission bands are reported here. The elemental analysis of these samples was carried out using a laser-induced breakdown spectroscopy (LIBS) system. Characteristic emission lines of Pr and Sm have been identified and reported by the recorded LIBS spectra of glass samples. Results prove that using these three complimentary spectroscopic techniques (absorption, fluorescence and LIBS), we can meaningfully characterize rare earth-doped glass samples.

Detection of rheumatoid arthritis by evaluation of normalized variances of fluorescence time correlation functions

NASA Astrophysics Data System (ADS)

Dziekan, Thomas; Weissbach, Carmen; Voigt, Jan; Ebert, Bernd; MacDonald, Rainer; Bahner, Malte L.; Mahler, Marianne; Schirner, Michael; Berliner, Michael; Berliner, Birgitt; Osel, Jens; Osel, Ilka

2011-07-01

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

A dual spectroscopic fluorescence probe based on carbon dots for detection of 2,4,6-trinitrophenol/Fe (III) ion by fluorescence and frequency doubling scattering spectra and its analytical applications.

PubMed

Xu, Jinxia; Bai, Zhangjun; Zu, Fanlin; Yan, Fanyong; Wei, Junfu; Zhang, Saihui; Luo, Yunmei

2018-07-05

A convenient, highly sensitive and reliable assay for 2,4,6‑trinitrophenol (TNP) and Fe (III) ion (Fe 3+ ) in the dual spectroscopic manner is developed based on novel carbon dots (CDs). The CDs with highly blue emitting fluorescent were easily prepared via the one-step potassium hydroxide-assisted reflux method from dextrin. The as-synthesized CDs exhibited the high crystalline quality, the excellent fluorescence characteristics with a high quantum yield of ~13.1%, and the narrow size distribution with an average diameter of 6.3±0.5nm. Fluorescence and frequency doubling scattering (FDS) spectra of CDs show the unique changes in the presence of TNP/Fe 3+ by different mechanism. The fluorescence of CDs decreased apparently in the presence of TNP via electron-transfer. Thus, after the experimental conditions were optimized, the linear range for detection TNP is 0-50μM, the detection limit was 19.1nM. With the addition of Fe 3+ , the FDS of CDs appeared to be highly sensitive with a quick response to Fe 3+ as a result of the change concentration of the scattering particle. The emission peak for FDS at 450nm was enhanced under the excitation wavelength at 900nm. The fluorescence response changes linearly with Fe 3+ concentration in the range of 8-40μM, the detection limits were determined to be 44.1nM. The applications of CDs were extended for the detection of TNP, Fe 3+ in real water samples with a high recovery. The results reported here may become the potential tools for the fast response of TNP and Fe 3+ in the analysis of environmental pollutants. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorescence spectroscopic detection of virus-induced atherosclerosis

NASA Astrophysics Data System (ADS)

Yan, Wei-dong; Perk, Masis; Nation, Patric N.; Power, Robert F.; Liu, Liying; Jiang, Xiuyan; Lucas, Alexandra

1994-07-01

Laser-induced fluorescence (LF) has been developed as a diagnostic tool for the detection of atherosclerosis. We have examined the use of LF for the identification of accelerated atherosclerotic plaque growth induced by Marek's Disease Virus (MDV) infection in White Leghorn rooster chicks (R) as well as plaque regression after treatment. Twenty-eight newborn R were infected with 12,000 cfu of MDV. Twelve parallel control R had saline injection. LF spectra were recorded from the arteries in vitro with a CeramOptec laser angioplasty catheter during 308 nm XeCl excimer laser excitation. Significant differences were detected at 440 to 475, 525, 550, 600, and 650 nm in MDV-R (p<0.05). In a subsequent study, 60 R were infected with 5,000 cfu of MDV, and were then treated with either Pravastatin (PRV) or placebo at 3 months post infection. These PRV-R were followed for 6 months to detect changes in atherosclerotic plaque development. PRV reduced intimal proliferation produced by MDV infection on histological examination (PRV-R 128.0+/- 44.0 micrometers , placebo-R 412.2+/- 91.5 micrometers , pequals0.007). MDV infected, PRV treated R were examined for LF changes that correlated with decreased atherosclerosis. There was an associated significant increase in LF intensity in PRV-R at 405 to 425 nm (p<0.001). In conclusion, LF can detect intimal proliferation in virus- induced atherosclerosis and atherosclerotic plaque regression after PRV therapy.

Advancements of two dimensional correlation spectroscopy in protein researches

NASA Astrophysics Data System (ADS)

Tao, Yanchun; Wu, Yuqing; Zhang, Liping

2018-05-01

The developments of two-dimensional correlation spectroscopy (2DCOS) applications in protein studies are discussed, especially for the past two decades. The powerful utilities of 2DCOS combined with various analytical techniques in protein studies are summarized. The emphasis is on the vibration spectroscopic techniques including IR, NIR, Raman and optical activity (ROA), as well as vibration circular dichroism (VCD) and fluorescence spectroscopy. In addition, some new developments, such as hetero-spectral 2DCOS, moving-window correlation, and model based correlation, are also reviewed for their utility in the investigation of the secondary structure, denaturation, folding and unfolding changes of protein. Finally, the new possibility and challenges of 2DCOS in protein research are highlighted as well.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

PubMed

Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

2017-07-16

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Molecular insight into the inclusion of the dietary plant flavonol fisetin and its chromophore within a chemically modified γ-cyclodextrin: Multi-spectroscopic, molecular docking and solubility studies.

PubMed

Pahari, Biswapathik; Chakraborty, Sandipan; Sengupta, Pradeep K

2018-09-15

We explored the encapsulation of dietary plant flavonols fisetin and its chromophore 3-hydroxyflavone, within 2-hydroxypropyl-γ-cyclodextrin (HPγ-CDx) nano-cavity in aqueous solution using multi-spectroscopic approaches and molecular docking. Upon addition of HPγ-CDx, dramatic changes occur in the intrinsic 'two color' fluorescence behavior of the fluorophores. This is manifested by significant increase in the steady state fluorescence intensities, anisotropies, average fluorescence lifetimes and rotational correlation times. Furthermore, in the CDx environment, intrinsically achiral flavonols exhibit prominent induced circular dichroism bands. These findings indicate that the flavonol molecules spontaneously enter the relatively hydrophobic, chiral environment of the HPγ-CDx nano-cavities. Molecular docking computations corroborate the spectroscopic findings, and predict selectivity in orientation of the encapsulated flavonols. HPγ-CDx inclusion increases the aqueous solubility of individual flavonols ∼100-1000 times. The present study demonstrates that the hydroxypropyl substituent in γ-CDx controls the inclusion mode of the flavonols, leading to their enhanced solubilization and altered spectral signatures. Copyright © 2018 Elsevier Ltd. All rights reserved.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Fluorescence decay time imaging using an imaging photon detector with a radio frequency photon correlation system

NASA Astrophysics Data System (ADS)

Morgan, Christopher G.; Mitchell, A. C.; Murray, J. G.

1990-05-01

An imaging photon detector has been modified to incorporate fast timing electronics coupled to a custom built photon correlator interfaced to a RISC computer. Using excitation with intensity- muodulated light, fluorescence images can be readily obtained where contrast is determined by the decay time of emission, rather than by intensity. This technology is readily extended to multifrequency phase/demodulation fluorescence imaging or to differential polarised phase fluorometry. The potential use of the correlator for confocal imaging with a laser scanner is also briefly discussed.

Two-dimensional correlation spectroscopic analysis on the interaction between humic acids and aluminum coagulant.

PubMed

Jin, Pengkang; Song, Jina; Wang, Xiaochang C; Jin, Xin

2018-02-01

In this study, two-dimensional correlation spectroscopy integrated with synchronous fluorescence and infrared absorption spectroscopy was employed to investigate the interaction between humic acids and aluminum coagulant at slightly acidic and neutral pH. Higher fluorescence quenching was produced for fulvic-like and humic-like fractions at pH5. At pH5, the humic-like fractions originating from the carboxylic acid, carboxyl and polysaccharide compounds were bound to aluminum first, followed by the fulvic-like fractions originating from the carboxyl and polysaccharide compounds. This finding also demonstrated that the activated functional groups of HA were involved in forming the Al-HA complex, which was accompanied by the removal of other groups by co-precipitation. Meanwhile, at pH7, almost no fluorescence quenching occurred, and surface complexation was observed to occur, in which the activated functional groups were absorbed on the amorphous Al(OH) 3 . Two-dimensional FT-IR correlation spectroscopy indicated the sequence of HA structural change during coagulation with aluminum, with IR bands affected in the order of COOH>COO - >NH deformation of amide II>aliphatic hydroxyl COH at pH5, and COO - >aliphatic hydroxyl COH at pH7. This study provides a promising pathway for analysis and insight into the priority of functional groups in the interaction between organic matters and metal coagulants. Copyright © 2017. Published by Elsevier B.V.

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

Multivariate optical element platform for compressed detection of fluorescence markers

NASA Astrophysics Data System (ADS)

Priore, Ryan J.; Swanstrom, Joseph A.

2014-05-01

The success of a commercial fluorescent diagnostic assay is dependent on the selection of a fluorescent biomarker; due to the broad nature of fluorescence biomarker emission profiles, only a small number of fluorescence biomarkers may be discriminated from each other as a function of excitation source. Multivariate Optical Elements (MOEs) are thin-film devices that encode a broad band, spectroscopic pattern allowing a simple broadband detector to generate a highly sensitive and specific detection for a target analyte. MOEs have historically been matched 1:1 to a discrete analyte or class prediction; however, MOE filter sets are capable of sensing projections of the original sparse spectroscopic space enabling a small set of MOEs to discriminate a multitude of target analytes. This optical regression can offer real-time measurements with relatively high signal-to-noise ratios that realize the advantages of multiplexed detection and pattern recognition in a simple optical instrument. The specificity advantage of MOE-based sensors allows fluorescent biomarkers that were once incapable of discrimination from one another via optical band pass filters to be employed in a common assay panel. A simplified MOE-based sensor may ultimately reduce the requirement for highly trained operators as well as move certain life science applications like disease prognostication from the laboratory to the point of care. This presentation will summarize the design and fabrication of compressed detection MOE filter sets for detecting multiple fluorescent biomarkers simultaneously with strong spectroscopic interference as well as comparing the detection performance of the MOE sensor with traditional optical band pass filter methodologies.

Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

2007-11-01

Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

Computational and spectroscopic data correlation study of N,N'-bisarylmalonamides (Part II).

PubMed

Arsovski, Violeta M; Božić, Bojan Đ; Mirković, Jelena M; Vitnik, Vesna D; Vitnik, Željko J; Petrović, Slobodan D; Ušćumlić, Gordana S; Mijin, Dušan Ž

2015-09-01

To complement a previous UV study, we present a quantitative evaluation of substituent effects on spectroscopic data ((1)H and (13)C NMR chemical shifts as well as FT-IR absorption frequency) applied to N,N'-bisarylmalonamides, using simple and extended Hammett equations as well as the Swain-Lupton equation. Furthermore, the DFT CAM-B3LYP/6-311+G(d,p) method was applied to study the impact of different solvents on the geometry of the molecules and their spectral data. Additionally, experimental data are correlated with theoretical results; excellent linear dependence was obtained. The overall results presented in this paper show that N,N'-bisarylmalonamides are prominent candidates for model molecules.

Spectroscopic characterization of Venus at the single molecule level.

PubMed

David, Charlotte C; Dedecker, Peter; De Cremer, Gert; Verstraeten, Natalie; Kint, Cyrielle; Michiels, Jan; Hofkens, Johan

2012-02-01

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments. This journal is © The Royal Society of Chemistry and Owner Societies 2012

Determining Serpin Conformational Distributions with Single Molecule Fluorescence

PubMed Central

Mushero, Nicole; Gershenson, Anne

2012-01-01

Conformational plasticity is key to inhibitory serpin function, and this plasticity gives serpins relatively easy access to alternative, dysfunctional conformations. Thus, a given serpin population may contain both functional and dysfunctional proteins. Single molecule fluorescence (SMF), with its ability to interrogate one fluorescently labeled protein at a time, is a powerful method for elucidating conformational distributions and monitoring how these distributions change over time. SMF and related methods have been particularly valuable for characterizing serpin polymerization. Fluorescence correlation spectroscopy experiments have revealed a second lag phase during in vitro α1-antitrypsin polymerization associated with the formation of smaller oligomers that then condense to form longer polymers [Purkayastha, P., Klemke, J. W., Lavender, S., Oyola, R., Cooperman, B. S., and Gai, F. (2005). Alpha 1-antitrypsin polymerization: A fluorescence correlation spectroscopic study. Biochemistry 44, 2642–2649.]. SMF studies of in vitro neuroserpin polymerization have confirmed that a monomeric intermediate is required for polymer formation while providing a test of proposed polymerization mechanisms [Chiou, A., Hägglöf, P., Orte, A., Chen, A. Y., Dunne, P. D., Belorgey, D., Karlsson-Li, S., Lomas, D., and Klenerman, D. (2009)]. Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence. Biophys. J. 97, 2306–2315.]. SMF has also been used to monitor protease–serpin interactions. Single pair Förster resonance energy transfer studies of covalent protease–serpin complexes suggest that the extent of protease structural disruption in the complex is protease dependent [Liu, L., Mushero, N., Hedstrom, L., and Gershenson, A. (2006). Conformational distributions of protease-serpin complexes: A partially translocated complex. Biochemistry 45, 10865–10872.]. SMF techniques are still evolving and the combination of

Remote temperature measurements in femto-liter volumes using dual-focus-Fluorescence Correlation Spectroscopy.

PubMed

Müller, Claus B; Weiss, Kerstin; Loman, Anastasia; Enderlein, Jörg; Richtering, Walter

2009-05-07

Remote temperature measurements in microfluidic devices with micrometer spatial resolution are important for many applications in biology, biochemistry and chemistry. The most popular methods use the temperature-dependent fluorescence lifetime of Rhodamine B, or the temperature-dependent size of thermosensitive materials such as microgel particles. Here, we use the recently developed method of dual-focus fluorescence correlation spectroscopy (2fFCS) for measuring the absolute diffusion coefficient of small fluorescent molecules at nanomolar concentrations and show how these data can be used for remote temperature measurements on a micrometer scale. We perform comparative temperature measurements using all three methods and show that the accuracy of 2fFCS is comparable or even better than that achievable with Rhodamine B fluorescence lifetime measurements. The temperature dependent microgel swelling leads to an enhanced accuracy within a narrow temperature range around the volume phase transition temperature, but requires the availability of specific microgels, whereas 2fFCS is applicable under very general conditions.

Shedding light on the photostability of two intermolecular charge-transfer complexes between highly fluorescent bis-1,8-naphthalimide dyes and some π-acceptors: A spectroscopic study in solution and solid states

NASA Astrophysics Data System (ADS)

Refat, Moamen S.; Ismail, Lamia A.; Adam, Abdel Majid A.

2015-01-01

Given the great importance of the various uses of 1,8-naphthalimides in the trends of biology, medicine and industry, the current study focused on extending the scope of these dyes by introducing some of their charge-transfer (CT) complexes. For this purpose, two highly fluorescent bis-1,8-naphthalimide dyes and their complexes with some π-acceptors have been synthesized and characterized spectroscopically. The π-acceptors include picric acid (PA), chloranilic acid (CLA), tetracyanoquinodimethane (TCNQ) and dichlorodicyanobenzoquinone (DDQ). The molecular structure, spectroscopic and fluorescence properties as well as the binding modes were deduced from IR, UV-vis and 1H NMR spectral studies. The binding ratio of complexation was determined to be 1:1 according to the elemental analyses and photometric titrations. It has been found that the order of acceptance ability for the different acceptors is TCNQ > DDQ > CLA > PA. The photostability of 1,8-naphthalimide dye as a donor and its charge-transfer complex doped in polymethyl methacrylate/PMMA were exposed to UV-Vis radiation and the change in the absorption spectra was achieved at different times during irradiation period.

An investigation of nonsimultaneous laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Fletcher, D. G.

1993-01-01

An alternative to simultaneous, two-line laser-induced fluorescence for thermodynamic property measurement is presented. This spectroscopic approach is similar to multiple-overheat hot-wire anemometry and is based on laser excitation of different fluorescence transitions for separate, sequential wind tunnel runs. Both fluctuating and mean thermodynamic property measurements seem to be achievable with this method without exciting the transitions during the same laser pulse.

A simple and highly sensitive spectroscopic fluorescence-detection system for multi-channel plastic-microchip electrophoresis based on side-entry laser-beam zigzag irradiation.

PubMed

Anazawa, Takashi; Uchiho, Yuichi; Yokoi, Takahide; Chalkidis, George; Yamazaki, Motohiro

2017-06-27

A five-color fluorescence-detection system for eight-channel plastic-microchip electrophoresis was developed. In the eight channels (with effective electrophoretic lengths of 10 cm), single-stranded DNA fragments were separated (with single-base resolution up to 300 bases within 10 min), and seventeen-loci STR genotyping for forensic human identification was successfully demonstrated. In the system, a side-entry laser beam is passed through the eight channels (eight A channels), with alternately arrayed seven sacrificial channels (seven B channels), by a technique called "side-entry laser-beam zigzag irradiation." Laser-induced fluorescence from the eight A channels and Raman-scattered light from the seven B channels are then simultaneously, uniformly, and spectroscopically detected, in the direction perpendicular to the channel array plane, through a transmission grating and a CCD camera. The system is therefore simple and highly sensitive. Because the microchip is fabricated by plastic-injection molding, it is inexpensive and disposable and thus suitable for actual use in various fields.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Biophysical influence of coumarin 35 on bovine serum albumin: Spectroscopic study

NASA Astrophysics Data System (ADS)

Bayraktutan, Tuğba; Onganer, Yavuz

2017-01-01

The binding mechanism and protein-fluorescence probe interactions between bovine serum albumin (BSA) and coumarin 35 (C35) was investigated by using UV-Vis absorption and fluorescence spectroscopies since they remain major research topics in biophysics. The spectroscopic data indicated that a fluorescence quenching process for BSA-C35 system was occurred. The fluorescence quenching processes were analyzed using Stern-Volmer method. In this regard, Stern-Volmer quenching constants (KSV) and binding constants were calculated at different temperatures. The distance r between BSA (donor) and C35 (acceptor) was determined by exploiting fluorescence resonance energy transfer (FRET) method. Synchronous fluorescence spectra were also studied to observe information about conformational changes. Moreover, thermodynamics parameters were calculated for better understanding of interactions and conformational changes of the system.

Electrically induced microflows probed by fluorescence correlation spectroscopy.

PubMed

Ybert, C; Nadal, F; Salomé, R; Argoul, F; Bourdieu, L

2005-03-01

We report on the experimental characterisation of electrically induced flows at the micrometer scale through Fluorescence Correlation Spectroscopy (FCS) measurements. We stress the potential of FCS as a useful characterisation technique in microfluidics devices for transport properties cartography. The experimental results obtained in a model situation are in agreement with previous calculations (F. Nadal, F. Argoul, P. Kestener, B. Pouligny, C. Ybert, A. Ajdari, Eur. Phys. J. E 9, 387 (2002)) predicting the structure and electric-field dependency of the induced flow. Additionally, the present study evidences a complex behaviour of the probe nanobeads under electric field whose precise understanding might prove relevant for situations where nano-objects interact with an external electric field.

Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy

PubMed Central

Arkhipov, Anton; Hüve, Jana; Kahms, Martin; Peters, Reiner; Schulten, Klaus

2007-01-01

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved. PMID:17704168

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

PubMed Central

Hunt, Marguerite E.; Scherrer, Michael P.; Ferrari, Frank D.; Matz, Mikhail V.

2010-01-01

Background Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. Results Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. Conclusions The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit. PMID:20644720

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

NASA Astrophysics Data System (ADS)

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-03-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers.

PubMed

Haring, Martijn T; Liv, Nalan; Zonnevylle, A Christiaan; Narvaez, Angela C; Voortman, Lenard M; Kruit, Pieter; Hoogenboom, Jacob P

2017-03-02

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

PubMed Central

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-01-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample. PMID:28252673

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells

PubMed Central

Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-01-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments. PMID:18842624

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

PubMed

Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-11-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

Calibration and validation of projection lithography in chemically amplified resist systems using fluorescence imaging

NASA Astrophysics Data System (ADS)

Mason, Michael D.; Ray, Krishanu; Feke, Gilbert D.; Grober, Robert D.; Pohlers, Gerd; Cameron, James F.

2003-05-01

Coumarin 6 (C6), a pH sensitive fluorescent molecule were doped into commercial resist systems to demonstrate a cost-effective fluorescence microscopy technique for detecting latent photoacid images in exposed chemically amplified resist films. The fluorescenec image contrast is optimized by carefully selecting optical filters to match the spectroscopic properties of C6 in the resist matrices. We demonstrate the potential of this technique for two sepcific non-invasive applications. First, a fast, conventient, fluorescence technique is demonstrated for determination of quantum yeidsl of photo-acid generation. Since the Ka of C6 in the 193nm resist system lies wihtin the range of acid concentrations that can be photogenerated, we have used this technique to evaluate the acid generation efficiency of various photo-acid generators (PAGs). The technique is based on doping the resist formulations containing the candidate PAGs with C6, coating one wafer per PAG, patterning the wafer with a dose ramp and spectroscopically imaging the wafers. The fluorescence of each pattern in the dose ramp is measured as a single image and analyzed with the optical titration model. Second, a nondestructive in-line diagnostic technique is developed for the focus calibration and validation of a projection lithography system. Our experimental results show excellent correlation between the fluorescence images and scanning electron microscope analysis of developed features. This technique has successfully been applied in both deep UV resists e.g., Shipley UVIIHS resist and 193 nm resists e.g., Shipley Vema-type resist. This method of focus calibration has also been extended to samples with feature sizes below the diffraction limit where the pitch between adjacent features is on the order of 300 nm. Image capture, data analysis, and focus latitude verification are all computer controlled from a single hardware/software platform. Typical focus calibration curves can be obtained within several

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

PubMed

Stortz, Martin; Angiolini, Juan; Mocskos, Esteban; Wolosiuk, Alejandro; Pecci, Adali; Levi, Valeria

2018-05-01

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus. Copyright © 2017 Elsevier Inc. All rights reserved.

Analyzing Baryon Acoustic Oscillations in Sparse Spectroscopic Samples via Cross-Correlation with Dense Photometry

NASA Astrophysics Data System (ADS)

Patej, Anna; Eisenstein, Daniel J.

2018-04-01

We develop a formalism for measuring the cosmological distance scale from baryon acoustic oscillations (BAO) using the cross-correlation of a sparse redshift survey with a denser photometric sample. This reduces the shot noise that would otherwise affect the auto-correlation of the sparse spectroscopic map. As a proof of principle, we make the first on-sky application of this method to a sparse sample defined as the z > 0.6 tail of the Sloan Digital Sky Survey's (SDSS) BOSS/CMASS sample of galaxies and a dense photometric sample from SDSS DR9. We find a 2.8σ preference for the BAO peak in the cross-correlation at an effective z = 0.64, from which we measure the angular diameter distance DM(z = 0.64) = (2418 ± 73 Mpc)(rs/rs, fid). Accordingly, we expect that using this method to combine sparse spectroscopy with the deep, high quality imaging that is just now becoming available will enable higher precision BAO measurements than possible with the spectroscopy alone.

PyCorrFit-generic data evaluation for fluorescence correlation spectroscopy.

PubMed

Müller, Paul; Schwille, Petra; Weidemann, Thomas

2014-09-01

We present a graphical user interface (PyCorrFit) for the fitting of theoretical model functions to experimental data obtained by fluorescence correlation spectroscopy (FCS). The program supports many data file formats and features a set of tools specialized in FCS data evaluation. The Python source code is freely available for download from the PyCorrFit web page at http://pycorrfit.craban.de. We offer binaries for Ubuntu Linux, Mac OS X and Microsoft Windows. © The Author 2014. Published by Oxford University Press.

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

PubMed

Sutter, Marc; Oliveira, Sabrina; Sanders, Niek N; Lucas, Bart; van Hoek, Arie; Hink, Mark A; Visser, Antonie J W G; De Smedt, Stefaan C; Hennink, Wim E; Jiskoot, Wim

2007-03-01

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Fluorescence correlation spectroscopy to measure the metabolism of high-density lipoprotein

NASA Astrophysics Data System (ADS)

Deitrick, Russell; Gibson, Emily; Razzaghi, Hamid

2009-10-01

High-density lipoprotein (HDL), referred to as the ``good cholesterol'', carries free cholesterol to the liver to be filtered from the bloodstream and is important to our understanding of atherosclerosis. HDL is metabolized in part by the enzyme Endothelial Lipase (EL). With this project we will use fluorescence correlation spectroscopy (FCS) to study the metabolism of HDL by EL comparing wild type with different genetic mutations. FCS is an advanced microscopy technique in which we record fluctuations in the fluorescence of dye-labeled molecules (in this case, HDL labeled with Nile Red) as they freely diffuse through a small focal volume. This data can be analyzed mathematically using the cross-correlation function, from which we can ultimately ascertain much information. In our case, we are interested in the diffusion coefficient which, via the Stokes-Einstein relation for a sphere, we can determine the size of HDL as it undergoes the process of metabolism. Preliminary results seem to indicate that the metabolic process occurs very quickly, that the final size of HDL depends primarily on the concentration of EL, and that the wild and mutant variants of EL have a similar effectiveness. In following experiments, we hope to investigate these relationships further.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

PubMed Central

Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

2013-01-01

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology. PMID:23457228

Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy.

PubMed Central

Hess, Samuel T; Webb, Watt W

2002-01-01

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 micros to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as approximately 80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (approximately 4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS. PMID:12324447

Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Foo, Yong Hwee; Naredi-Rainer, Nikolaus; Lamb, Don C.; Ahmed, Sohail; Wohland, Thorsten

2012-01-01

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (Kds) of biomolecules. The determination of a Kd depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ∼0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the Kd. PMID:22404940

Insights into accelerated liposomal release of topotecan in plasma monitored by a non-invasive fluorescence spectroscopic method

PubMed Central

Fugit, Kyle D.; Jyoti, Amar; Upreti, Meenakshi; Anderson, Bradley D.

2014-01-01

A non-invasive fluorescence method was developed to monitor liposomal release kinetics of the anticancer agent topotecan (TPT) in physiological fluids and subsequently used to explore the cause of accelerated release in plasma. Analyses of fluorescence excitation spectra confirmed that unencapsulated TPT exhibits a red shift in its spectrum as pH is increased. This property was used to monitor TPT release from actively loaded liposomal formulations having a low intravesicular pH. Mathematical release models were developed to extract reliable rate constants for TPT release in aqueous solutions monitored by fluorescence and release kinetics obtained by HPLC. Using the fluorescence method, accelerated TPT release was observed in plasma as previously reported in the literature. Simulations to estimate the intravesicular pH were conducted to demonstrate that accelerated release correlated with alterations in the low intravesicular pH. This was attributed to the presence of ammonia in plasma samples rather than proteins and other plasma components generally believed to alter release kinetics in physiological samples. These findings shed light on the critical role that ammonia may play in contributing to the preclinical/clinical variability and performance seen with actively-loaded liposomal formulations of TPT and other weakly-basic anticancer agents. PMID:25456833

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.

Quenching of p-Cyanophenylalanine Fluorescence by Various Anions.

PubMed

Pazos, Ileana M; Roesch, Rachel M; Gai, Feng

2013-03-20

To expand the spectroscopic utility of the non-natural amino acid p -cyanophenylalanine (Phe CN ), we examine the quenching efficiencies of a series of commonly encountered anions toward its fluorescence. We find that iodide exhibits an unusually large Stern-Volmer quenching constant, making it a convenient choice in Phe CN fluorescence quenching studies. Indeed, using the villin headpiece subdomain as a testbed we demonstrate that iodide quenching of Phe CN fluorescence offers a convenient means to reveal protein conformational heterogeneity. Furthermore, we show that the amino group of Phe CN strongly quenches its fluorescence, suggesting that Phe CN could be used as a local pH sensor.

Spectroscopic Chemical Analysis Methods and Apparatus

NASA Technical Reports Server (NTRS)

Hug, William F. (Inventor); Lane, Arthur L. (Inventor); Bhartia, Rohit (Inventor); Reid, Ray D. (Inventor)

2017-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted along with photoluminescence spectroscopy (i.e. fluorescence and/or phosphorescence spectroscopy) to provide high levels of sensitivity and specificity in the same instrument.

Spectroscopic Chemical Analysis Methods and Apparatus

NASA Technical Reports Server (NTRS)

Hug, William F. (Inventor); Lane, Arthur L. (Inventor); Reid, Ray D. (Inventor); Bhartia, Rohit (Inventor)

2018-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted along with photoluminescence spectroscopy (i.e. fluorescence and/or phosphorescence spectroscopy) to provide high levels of sensitivity and specificity in the same instrument.

Studies on the binding behavior of prodigiosin with bovine hemoglobin by multi-spectroscopic techniques

NASA Astrophysics Data System (ADS)

Tang, Jing; Yang, Chao; Zhou, Lin; Ma, Fei; Liu, Shuchao; Wei, Shaohua; Zhou, Jiahong; Zhou, Yanhuai

2012-10-01

In this article, the interaction mechanism of prodigiosin (PG) with bovine hemoglobin (BHb) is studied in detail using various spectroscopic technologies. UV-vis absorption and fluorescence spectra demonstrate the interaction process. The Stern-Volmer plot and the time-resolved fluorescence study suggest the quenching mechanism of fluorescence of BHb by PG is a static quenching procedure, and the hydrophobic interactions play a major role in binding of PG to BHb. Furthermore, synchronous fluorescence studies, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra reveal that the conformation of BHb is changed after conjugation with PG.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

A correlative study of Papanicolaou smear, fluorescent antibody, and culture for the diagnosis of Chlamydia trachomatis.

PubMed

Spence, M R; Barbacci, M; Kappus, E; Quinn, T

1986-11-01

A prospective study of 300 patients undergoing therapeutic termination of pregnancy was conducted. A Papanicolaou smear was obtained and a clinical evaluation of the cervix was made. Specimens from the cervix were examined by both direct fluorescent antibody and culture techniques for the presence of Chlamydia trachomatis. The presence of inflammation on Papanicolaou smear could be correlated with C trachomatis isolation. Papanicolaou smear findings consistent with C trachomatis lacked both sensitivity and specificity when compared with direct fluorescent antibody and/or culture techniques. A correlation was found between the clinical diagnosis of cervicitis and C trachomatis. This interrelationship was absent when the component findings of cervicitis (ectopy, friability, and purulent mucus) were examined independently.

Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

NASA Astrophysics Data System (ADS)

Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

2016-03-01

Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

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

Del Razo, Mauricio; Pan, Wenxiao; Qian, Hong

2014-05-30

The currently existing theory of fluorescence correlation spectroscopy (FCS) is based on the linear fluctuation theory originally developed by Einstein, Onsager, Lax, and others as a phenomenological approach to equilibrium fluctuations in bulk solutions. For mesoscopic reaction-diffusion systems with nonlinear chemical reactions among a small number of molecules, a situation often encountered in single-cell biochemistry, it is expected that FCS time correlation functions of a reaction-diffusion system can deviate from the classic results of Elson and Magde [Biopolymers (1974) 13:1-27]. We first discuss this nonlinear effect for reaction systems without diffusion. For nonlinear stochastic reaction-diffusion systems there are no closedmore » solutions; therefore, stochastic Monte-Carlo simulations are carried out. We show that the deviation is small for a simple bimolecular reaction; the most significant deviations occur when the number of molecules is small and of the same order. Extending Delbrück-Gillespie’s theory for stochastic nonlinear reactions with rapidly stirring to reaction-diffusion systems provides a mesoscopic model for chemical and biochemical reactions at nanometric and mesoscopic level such as a single biological cell.« less

Oral cancer detection based on fluorescence polarization of blood plasma at excitation wavelength 405 nm

NASA Astrophysics Data System (ADS)

Pachaiappan, Rekha; Prakasarao, Aruna; Manoharan, Yuvaraj; Dornadula, Koteeswaran; Singaravelu, Ganesan

2017-02-01

During metabolism the metabolites such as hormones, proteins and enzymes were released in to the blood stream by the cells. These metabolites reflect any change that occurs due to any disturbances in normal metabolic function of the human system. This was well observed with the altered spectral signatures observed with fluorescence spectroscopic technique. Previously many have reported on the significance of native fluorescence spectroscopic method in the diagnosis of cancer. As fluorescence spectroscopy is sensitive and simple, it has complementary techniques such as excitation-emission matrix, synchronous and polarization. The fluorescence polarization measurement provides details about any association or binding reactions and denaturing effects that occurs due to change in the micro environment of cells and tissues. In this study, we have made an attempt in the diagnosis of oral cancer at 405 nm excitation using fluorescence polarization measurement. The fluorescence anisotropic values calculated from polarized fluorescence spectral data of normal and oral cancer subjects yielded a good accuracy when analyzed with linear discriminant analysis based artificial neural network. The results will be discussed in detail.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

PubMed

Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

2016-04-01

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Laser excited atomic fluorescence spectrometry as a powerful tool for analytical applications and spectroscopic studies

NASA Astrophysics Data System (ADS)

Gornushkin, Igor B.

1997-12-01

Laser-excited atomic fluorescence spectrometry (LEAFS) with a novel diffusive tube electrothermal atomizer (ETA) has been used for the study of atomization and diffusion processes and for the direct trace analysis of complex matrices. A novel ETA was a graphite tube sealed by two graphite electrodes. A sample was introduced into the tube and the furnace assembly was heated. The vaporized sample diffused through the hot graphite and the atomic fraction of the vapor was excited by a tunable dye laser above the tube. Temporal behavior of atomic fluorescence of Cu, Ag, and Ni atoms, diffused through the furnace tube, was studied at different temperatures; the values for activation energies and diffusion coefficients were derived on the basis of the diffusion/vaporization kinetic model. The femtogram/nanogram concentrations of silver were determined in coastal Atlantic water and soil samples. Use of the new ETA resulted in significant reduction of matrix interferences, ultra-low limits of detection, good accuracy and precision. LEAFS coupled with laser ablation (LA) was studied in terms of its analytical and spectroscopic potential. Low concentrations of lead (0.15 ppm-750 ppm) in metallic matrices (copper, brass, steel, and zinc) were measured in a low pressure argon atmosphere. No matrix effect was observed, providing a universal calibration curve for all samples. A limit of detection of 22 ppb (0.5 fg) was achieved. Also, the lifetime of the metastable 6p21D level of lead was measured and found to be in good agreement with the literature data. A simple open-air LA-LEAFS system was used for the determination of cobalt in solid matrices (graphite, soil, and steel). The fluorescence of cobalt was excited from a level which was already populated in the ablation plasma and was monitored at the Stokes-shifted wavelength. Detection limits in the ppb to ppm range and linearity over four orders of magnitude were achieved. The resonance shadowgraph technique has been developed

Fluorescence spectroscopic and molecular docking studies of the binding interaction between the new anaplastic lymphoma kinase inhibitor crizotinib and bovine serum albumin

NASA Astrophysics Data System (ADS)

Abdelhameed, Ali S.; Alanazi, Amer M.; Bakheit, Ahmed H.; Darwish, Hany W.; Ghabbour, Hazem A.; Darwish, Ibrahim A.

2017-01-01

Binding of the recently introduced anti-cancer drug, crizotinib (CRB) with the bovine serum albumin (BSA) was comprehensively studied with the aid of fluorescence and UV-Vis spectroscopic as well as molecular docking techniques. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the CRB and BSA with binding constants of 104 L mol- 1. BSA conformational changes were investigated using three dimensional (3D) and synchronous fluorescence measurements. Moreover, the results of site marker competitive experiments and molecular docking, it could be deduced that CRB was inserted into the subdomain IIA (site I) of BSA yielding a more stabilized system. This was further confirmed with the molecular docking results which revealed that CRB is located in the active site residues Try149, Glu152, Ser191, Arg194, Arg198, Trp213, Arg217, Arg256, His287, Ala290, Glu291, Ser343, Asp450 within a radius of 6 Å. Combining the molecular docking studies and the computed thermodynamic parameters, it can be inferred that hydrophobic and electrostatic interactions are the major binding forces involved in formation of the CRB-BSA complex.

Analysing baryon acoustic oscillations in sparse spectroscopic samples via cross-correlation with dense photometry

NASA Astrophysics Data System (ADS)

Patej, A.; Eisenstein, D. J.

2018-07-01

We develop a formalism for measuring the cosmological distance scale from baryon acoustic oscillations (BAO) using the cross-correlation of a sparse redshift survey with a denser photometric sample. This reduces the shot noise that would otherwise affect the autocorrelation of the sparse spectroscopic map. As a proof of principle, we make the first on-sky application of this method to a sparse sample defined as the z > 0.6 tail of the Sloan Digital Sky Survey's (SDSS) BOSS/CMASS sample of galaxies and a dense photometric sample from SDSS DR9. We find a 2.8σ preference for the BAO peak in the cross-correlation at an effective z = 0.64, from which we measure the angular diameter distance DM(z = 0.64) = (2418 ± 73 Mpc)(rs/rs, fid). Accordingly, we expect that using this method to combine sparse spectroscopy with the deep, high-quality imaging that is just now becoming available will enable higher precision BAO measurements than possible with the spectroscopy alone.

[Laser Induced Fluorescence Spectroscopic Analysis of Aromatics from One Ring to Four Rings].

PubMed

Zhang, Peng; Liu, Hai-feng; Yue, Zong-yu; Chen, Bei-ling; Yao, Ming-fa

2015-06-01

In order to distinguish small aromatics preferably, a Nd : YAG Laser was used to supply an excitation laser, which was adjusted to 0.085 J x cm(-2) at 266 nm. Benzene, toluene, naphthalene, phenanthrene, anthracene, pyrene and chrysene were used as the representative of different rings aromatics. The fluorescence emission spectra were researched for each aromatic hydrocarbon and mixtures by Laser induced fluorescence (LIF). Results showed that the rings number determined the fluorescence emission spectra, and the structure with same rings number did not affect the emission fluorescence spectrum ranges. This was due to the fact that the absorption efficiency difference at 266 nm resulted in that the fluorescence intensities of each aromatic hydrocarbon with same rings number were different and the fluorescence intensities difference were more apparently with aromatic ring number increasing. When the absorption efficiency was similar at 266 nm and the concentrations of each aromatic hydrocarbon were same, the fluorescence intensities were increased with aromatic ring number increasing. With aromatic ring number increasing, the fluorescence spectrum and emission peak wavelength were all red-shifted from ultraviolet to visible and the fluorescence spectrum range was also wider as the absorption efficiency was similar. The fluorescence emission spectra from one to four rings could be discriminated in the following wavelengths, 275 to 320 nm, 320 to 375 nm, 375 to 425 nm, 425 to 556 nm, respectively. It can be used for distinguish the type of the polycyclic aromatic hydrocarbons (PAHs) as it exists in single type. As PAHs are usually exist in a variety of different rings number at the same time, the results for each aromatic hydrocarbon may not apply to the aromatic hydrocarbon mixtures. For the aromatic hydrocarbon mixtures, results showed that the one- or two-ring PAHs in mixtures could not be detected by fluorescence as three- or four-ring PAHs existed in mixture

New look at the Badger-Bauer rule: Correlations of spectroscopic IR and NMR parameters with hydrogen bond energy and geometry. FHF complexes

NASA Astrophysics Data System (ADS)

Tupikina, E. Yu.; Denisov, G. S.; Melikova, S. M.; Kucherov, S. Yu.; Tolstoy, P. M.

2018-07-01

In this work correlation dependencies between hydrogen bond energy ΔE for complexes with Fsbnd H⋯F hydrogen bond and their spectroscopic characteristics of the IR and NMR spectra are presented. We considered 26 complexes in a wide hydrogen bond energy range 0.2-47 kcal/mol. For each complex we calculated complexation energy (MP2/6-311++G(d,p)), IR spectroscopic parameters (FH stretching frequency ν, FH stretching frequency in local mode approximation νLM at MP2/6-311++G(d,p) level) and NMR parameters (chemical shift of hydrogen δH and fluorine nuclei δF, Nuclear Independent Chemical Shielding and spin-spin coupling constants 1JFH, 1hJH...F, 2hJFF at B3LYP/pcSseg-2 level). It was shown that changes of parameters upon complexation, i.e. changes of the stretching frequency in local mode approximation ΔνLM, change of the proton chemical shift ΔδH and change of the absolute value of spin-spin coupling constant 1JFH could be used for estimation of corresponding hydrogen bond strength. Furthermore, we build correlation dependencies between abovementioned spectroscopic characteristics and geometric ones, such as the asymmetry of bridging proton position q1 = 0.5·(rFH - rH…F).

Interaction of sodium benzoate with trypsin by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software.

Application of UV and Fluorescence Indices for Assessing the Performance of Ozonation Process: Towards Smart Water Treatment

NASA Astrophysics Data System (ADS)

Li, Wen-Tao; Abbt-Braun, Gudrun; Dodd, Michael; Korshin, Gregory; Li, Ai-Min

2017-04-01

The UV absorbance and fluorescence indices were comprehensively studied as surrogate indicators for assessing the degradation of dissolved organic matter (DOM), the formation of bromate and biodegradable dissolved organic carbon (BDOC) and the elimination of trace organic contaminants (TOrCs) during the ozonation of surface water and wastewater effluent. Spectroscopic monitoring was carried out using benchtop UV/Vis and fluorescence spectrophotometers and a newly developed miniature LED UV/fluorescence sensor capable of rapidly measuring UVA280 and protein-like and humic-like fluorescence. With the increase of O3/DOC mass ratio, the plots of BDOC formation were characterized three phases of initial lag, transition slope and final plateau. With the decrease of UV absorbance and fluorescence, BDOC concentrations initially increased slowly and then rose more noticeably. Inflection points in plots of BDOC versus changes of spectroscopic indicators were close to 35-45% loss of UVA254 or UVA280 and 75-85% loss of humic-like fluorescence. According to the data from size exclusion chromatography (SEC) with organic carbon detection and 2D synchronous correlation analyses, DOM fractions assigned to operationally defined large biopolymers (apparent molecular weight, AMW>20 kDa) and medium AMW humic substances (AMW 5.5-20 kDa) were transformed into medium-size building blocks (AMW 3-5.5 kDa) and other smaller AMW species (AMW<3 kDa) associated with BDOC at increasing O3/DOC ratios. Appreciable bromate formation was observed only after the values of UVA254, UVA280 and humic-like fluorescence in O3-treated samples were decreased by 45-55%, 50-60% and 86-92% relative to their respective initial levels. No significant differences in plots of bromate concentrations versus decreases of humic-like fluorescence were observed for surface water and wastewater effluent samples. This was in contrast with the plots of bromate concentration versus UVA254 and UVA280 which exhibited

Spectroscopic studies on the interaction of bovine serum albumin with surfactants and apigenin

NASA Astrophysics Data System (ADS)

Zhao, Xu-Na; Liu, Yi; Niu, Li-Yuan; Zhao, Chen-Ping

The binding of apigenin (Ap) to bovine serum albumin (BSA) has been studied using the methods of fluorescence spectroscopy and UV-vis absorption spectroscopy. The spectroscopic analysis of the quenching mechanism indicates that the quenching constants are inversely correlated with the temperatures and the quenching process could result from a static interaction. The type of interaction force was discussed and the binding site of Ap was in site I (subdomain IIA) of BSA. The thermodynamic parameters ΔH and ΔS are -42.02 kJ mol-1 and -48.31 J mol-1 K-1, respectively and the negative ΔG implying that the binding interaction was spontaneous. The distance r between BSA and Ap was calculated according to Förster's theory and the value is 3.44 nm. The synchronous and three-dimensional fluorescence spectra show that the binding of Ap to BSA could lead to the changes in the conformation and microenvironment of BSA. At the same time, the effects of ionic surfactants on the interaction of Ap and BSA have also been investigated.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics.

PubMed

Wei, Liping; Yan, Wenrong; Ho, Derek

2017-12-04

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics

PubMed Central

Yan, Wenrong; Ho, Derek

2017-01-01

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices. PMID:29207568

Principal component and spatial correlation analysis of spectroscopic-imaging data in scanning probe microscopy.

PubMed

Jesse, Stephen; Kalinin, Sergei V

2009-02-25

An approach for the analysis of multi-dimensional, spectroscopic-imaging data based on principal component analysis (PCA) is explored. PCA selects and ranks relevant response components based on variance within the data. It is shown that for examples with small relative variations between spectra, the first few PCA components closely coincide with results obtained using model fitting, and this is achieved at rates approximately four orders of magnitude faster. For cases with strong response variations, PCA allows an effective approach to rapidly process, de-noise, and compress data. The prospects for PCA combined with correlation function analysis of component maps as a universal tool for data analysis and representation in microscopy are discussed.

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

NASA Astrophysics Data System (ADS)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

Correlation Between Structural, Spectroscopic, and Reactivity Properties Within a Series of Structurally Analogous Metastable Manganese(III)-Alkylperoxo Complexes

PubMed Central

Coggins, Michael K.; Martin-Diaconescu, Vlad; DeBeer, Serena; Kovacs, Julie A.

2013-01-01

Manganese–peroxos are proposed as key intermediates in a number of important biochemical and synthetic transformations. Our understanding of the structural, spectroscopic, and reactivity properties of these metastable species is limited, however, and correlations between these properties have yet to be established experimentally. Herein we report the crystallographic structures of a series of structurally related metastable Mn(III)–OOR compounds, and examine their spectroscopic and reactivity properties. The four reported Mn(III)–OOR compounds extend the number of known end-on Mn(III)–(η1-peroxos) to six. The ligand backbone is shown to alter the metal–ligand distances and modulate the electronic properties key to bonding and activation of the peroxo. The mechanism of thermal decay of these metastable species is examined via variable-temperature kinetics. Strong correlations between structural (O–O and Mn⋯Npy,quin distances), spectroscopic (E(πv*(O–O) → Mn CT band), νO–O), and kinetic (ΔH‡ and ΔS‡) parameters for these complexes provide compelling evidence for rate-limiting O–O bond cleavage. Products identified in the final reaction mixtures of Mn(III)–OOR decay are consistent with homolytic O–O bond scission. The N-heterocyclic amines and ligand backbone (Et vs Pr) are found to modulate structural and reactivity properties, and O–O bond activation is shown, both experimentally and theoretically, to track with metal ion Lewis acidity. The peroxo O–O bond is shown to gradually become more activated as the N-heterocyclic amines move closer to the metal ion causing a decrease in π-donation from the peroxo πv*(O–O) orbital. The reported work represents one of very few examples of experimentally verified relationships between structure and function. PMID:23432090

Correlation between structural, spectroscopic, and reactivity properties within a series of structurally analogous metastable manganese(III)-alkylperoxo complexes.

PubMed

Coggins, Michael K; Martin-Diaconescu, Vlad; DeBeer, Serena; Kovacs, Julie A

2013-03-20

Manganese-peroxos are proposed as key intermediates in a number of important biochemical and synthetic transformations. Our understanding of the structural, spectroscopic, and reactivity properties of these metastable species is limited, however, and correlations between these properties have yet to be established experimentally. Herein we report the crystallographic structures of a series of structurally related metastable Mn(III)-OOR compounds, and examine their spectroscopic and reactivity properties. The four reported Mn(III)-OOR compounds extend the number of known end-on Mn(III)-(η(1)-peroxos) to six. The ligand backbone is shown to alter the metal-ligand distances and modulate the electronic properties key to bonding and activation of the peroxo. The mechanism of thermal decay of these metastable species is examined via variable-temperature kinetics. Strong correlations between structural (O-O and Mn···N(py,quin) distances), spectroscopic (E(πv*(O-O) → Mn CT band), ν(O-O)), and kinetic (ΔH(‡) and ΔS(‡)) parameters for these complexes provide compelling evidence for rate-limiting O-O bond cleavage. Products identified in the final reaction mixtures of Mn(III)-OOR decay are consistent with homolytic O-O bond scission. The N-heterocyclic amines and ligand backbone (Et vs Pr) are found to modulate structural and reactivity properties, and O-O bond activation is shown, both experimentally and theoretically, to track with metal ion Lewis acidity. The peroxo O-O bond is shown to gradually become more activated as the N-heterocyclic amines move closer to the metal ion causing a decrease in π-donation from the peroxo πv*(O-O) orbital. The reported work represents one of very few examples of experimentally verified relationships between structure and function.

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

PubMed Central

Ridgeway, William K.; Millar, David P.; Williamson, James R.

2012-01-01

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods. PMID:22869699

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Synthesis, spectroscopic, fluorescence properties and biological evaluation of novel Pd(II) and Cd(II) complexes of NOON tetradentate Schiff bases.

PubMed

Ali, Omyma A M

2014-01-01

The solid complexes of Pd(II) and Cd(II) with N,N/bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H2L(1)), and N,N/bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H2L(2)) have been synthesized and characterized by several techniques using elemental analysis (CHN), FT-IR, (1)H NMR, UV-Vis spectra and thermal analysis. Elemental analysis data proved 1:1 stoichiometry for the reported complexes while spectroscopic data indicated square planar and octahedral geometries for Pd(II) and Cd(II) complexes, respectively. The prepared ligands, Pd(II) and Cd(II) complexes exhibited intraligand (π-π(∗)) fluorescence and can potentially serve as photoactive materials. Thermal behavior of the complexes was studied and kinetic parameters were determined by Coats-Redfern method. Both the ligands and their complexes have been screened for antimicrobial activities. Copyright © 2013 Elsevier B.V. All rights reserved.

Polyproline and the “spectroscopic ruler” revisited with single-molecule fluorescence

PubMed Central

Schuler, Benjamin; Lipman, Everett A.; Steinbach, Peter J.; Kumke, Michael; Eaton, William A.

2005-01-01

To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances. PMID:15699337

Interaction of sodium benzoate with trypsin by spectroscopic techniques.

PubMed

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software. Copyright © 2011 Elsevier B.V. All rights reserved.

Fluorescence and Raman Spectroscopy of Doped Nanodiamonds

NASA Astrophysics Data System (ADS)

Kudryavtsev, O. S.; Khomich, A. A.; Sedov, V. S.; Ekimov, E. A.; Vlasov, I. I.

2018-05-01

Raman and fluorescence spectroscopic techniques were used to study doped nanodiamonds synthesized at high pressure and high temperature (HPHT technique) and by chemical vapor deposition from the gas phase (CVD technique). For the CVD diamonds, a hundred-fold increase in fluorescence intensity of the silicon-vacancy centers normalized to the volume of the probe material was observed with an increase in synthesized diamond particle diameter from 150 to 300 nm. Graphitization temperature upon heating in the air significantly lower than for detonation nanodiamonds was found for the boron-doped HPHT nanodiamonds.

Fluorescence Lifetime Study of Cyclodextrin Complexes of Substituted Naphthalenes.

DTIC Science & Technology

1987-08-15

Spectroscopy iip 17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse If necessary and identify by block number) FIELD GROUP SUB-GROUP fluorescence lifetime...measurements cyclodextrins spectroscopic techniques 19. TRACT (Continue on revere if necsary and identify by block number

Fluorescence spectroscopic study on the interaction of resveratrol with lipoxygenase

NASA Astrophysics Data System (ADS)

Pinto, María del Carmen; Duque, Antonio Luis; Macías, Pedro

2010-09-01

The interaction of lipoxygenase with (E)-resveratrol was investigated by fluorescence spectroscopy. The data obtained revealed that the quenching of intrinsic fluorescence of lipoxygenase is produced by the formation of a complex lipoxygenase-(E)-resveratrol. From the value obtained for the binding constant, according to the Stern-Volmer modified equation, was deduced the existence of static quenching mechanism and, as consequence, the existence of a strong interaction between (E)-resveratrol and lipoxygenase. The values obtained for the thermodynamic parameter Δ H (-3.58 kJ mol -1) and Δ S (87.97 J mol -1K -1) suggested the participation of hydrophobic interactions and hydrogen bonds in the stabilization of the complex ligand-protein. From the static quenching we determined that only exist one independent binding site. Based on the Förster energy transfer theory, the distance between the acceptor ((E)-resveratrol) and the donor (Trp residues of lipoxygenase) was calculated to be 3.42 nm. Finally, based on the information obtained from the evaluation of synchronous and three-dimensional fluorescence spectroscopy, we deduced that the interaction of (E)-resveratrol with lipoxygenase produces micro-environmental and conformational alterations of protein in the binding region.

An integrated platform for surface forces measurements and fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Ashis; Zhao, Jiang; Bae, Sung Chul; Granick, Steve

2003-06-01

We describe an apparatus to measure the diffusion of dilute fluorophores in molecularly thin liquid films within a surface forces apparatus (SFA). The design is a significant modification of the traditional SFA in that it allows one to combine nanorheology with the single-molecule sensitive technique of fluorescence correlation spectroscopy. The primary enabling idea was to place a miniaturized SFA onto the stage of an optical microscope equipped with a long working distance objective and illuminated by a femtosecond laser. A secondary enabling idea was that the silver coating on the backside of mica, normally used in the traditional SFA design for interferometric measurements of the film thickness, was replaced by multilayer dielectric coatings that allowed simultaneous interferometry and fluorescence measurements in different regions of the optical spectrum. To illustrate the utility of this instrument, we contrast the translational diffusion of rhodamine dye molecules (in the solvent, 1,2-propane diol), in the unconfined bulk state and confined between mica sheets to the thickness 2.5 nm. The diffusion coefficient is found to decrease by 2 orders of magnitude under confinement.

In situ fluorescence spectroscopy correlates ionomer degradation to reactive oxygen species generation in an operating fuel cell.

PubMed

Prabhakaran, Venkateshkumar; Arges, Christopher G; Ramani, Vijay

2013-11-21

The rate of generation of reactive oxygen species (ROS) within the polymer electrolyte membrane (PEM) of an operating proton exchange member fuel cell (PEMFC) was monitored using in situ fluorescence spectroscopy. A modified barrier layer was introduced between the PEM and the electrocatalyst layer to eliminate metal-dye interactions and fluorescence resonance energy transfer (FRET) effects during measurements. Standard fuel cell operating parameters (temperature, relative humidity, and electrode potential) were systematically varied to evaluate their influence on the rate of ROS generation during PEMFC operation. Independently, the macroscopic rate of PEM degradation was measured by monitoring the fluoride ion emission rate (FER) in the effluent stream at each operating condition. The ROS generation reaction rate constant (estimated from the in situ fluorescence experiments) correlated perfectly with the measured FER across all conditions, demonstrating unequivocally for the first time that a direct correlation exists between in situ ROS generation and PEM macroscopic degradation. The activation energy for ROS generation within the PEM was estimated to be 12.5 kJ mol(-1).

The intrinsic fluorescence of FAD and its application in analytical chemistry: a review

NASA Astrophysics Data System (ADS)

Galbán, Javier; Sanz-Vicente, Isabel; Navarro, Jesús; de Marcos, Susana

2016-12-01

This review (with 106 references) mainly deals with the analytical applications of flavin-adenine dinucleotide (FAD) fluorescence. In the first section, the spectroscopic properties of this compound are reviewed at the light of his different acid-base, oxidation and structural forms; the chemical and spectroscopic properties of flavin mononucleotide (FMN) and other flavins will be also briefly discussed. The second section discusses how the properties of FAD fluorescence changes in flavoenzymes (FvEs), again considering the different chemical and structural forms; the glucose oxidase (GOx) and the choline oxidase (ChOx) cases will be commented. Since almost certainly the most reported analytical application of FAD fluorescence is as an auto-indicator in enzymatic methods catalysed by FvE oxidoreductases, it is important to know how the concentrations of the different forms of FAD changes along the reaction and, consequently, the fluorescence and the analytical signals. An approach to do this will be presented in section 3. The fourth part of the paper compiles the analytical applications which have been reported until now based in these fluorescence properties. Finally, some suggestions about tentative future research are also given.

The intrinsic fluorescence of FAD and its application in analytical chemistry: a review.

PubMed

Galbán, Javier; Sanz-Vicente, Isabel; Navarro, Jesús; de Marcos, Susana

2016-12-19

This review (with 106 references) mainly deals with the analytical applications of flavin-adenine dinucleotide (FAD) fluorescence. In the first section, the spectroscopic properties of this compound are reviewed at the light of his different acid-base, oxidation and structural forms; the chemical and spectroscopic properties of flavin mononucleotide (FMN) and other flavins will be also briefly discussed. The second section discusses how the properties of FAD fluorescence changes in flavoenzymes (FvEs), again considering the different chemical and structural forms; the glucose oxidase (GOx) and the choline oxidase (ChOx) cases will be commented. Since almost certainly the most reported analytical application of FAD fluorescence is as an auto-indicator in enzymatic methods catalysed by FvE oxidoreductases, it is important to know how the concentrations of the different forms of FAD changes along the reaction and, consequently, the fluorescence and the analytical signals. An approach to do this will be presented in section 3. The fourth part of the paper compiles the analytical applications which have been reported until now based in these fluorescence properties. Finally, some suggestions about tentative future research are also given.

Correlation of conformational heterogeneity of the tryptophyl side chain and time-resolved fluorescence intensity decay kinetics

NASA Astrophysics Data System (ADS)

Laws, William R.; Ross, J. B. Alexander

1992-04-01

The time-resolved fluorescence properties of a tryptophan residue should be useful for probing protein structure, function, and dynamics. To date, however, the non-single exponential fluorescence intensity decay kinetics for numerous peptides and proteins having a single tryptophan residue have not been adequately explained. Many possibilities have been considered and include: (1) contributions from the 1La and 1Lb states of indole; (2) excited-state hydrogen exchange; and (3) environmental heterogeneity from (chi) 1 and (chi) 2 rotamers. In addition, it has been suggested that generally many factors contribute to the decay and a distribution of probabilities may be more appropriate. Two recent results support multiple species due to conformational heterogeneity as the major contributor to complex kinetics. First, a rotationally constrained tryptophan analogue has fluorescence intensity decay kinetics that can be described by the sum of two exponentials with amplitudes comparable to the relative populations of the two rotational isomers. Second, the multiple exponentials observed for tyrosine-containing model compounds and peptides correlate with the (chi) 1 rotamer populations independently determined by 1H NMR. We now report similar correlations between rotamer populations and fluorescence intensity decay kinetics for a tryptophan analogue of oxytocin. It appears for this compound that either (chi) 2 rotations do not appreciably alter the indole environment, (chi) 2 rotations are rapid enough to average the observed dependence, or only one of two possible (chi) 2 populations is associated with each (chi) 1 rotamer.

Fluorescence and Spectral Imaging

PubMed Central

DaCosta, Ralph S.; Wilson, Brian C.; Marcon, Norman E.

2007-01-01

Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots). This is an area of great promise, but still in its infancy, and preclinical studies are currently under way. PMID:18167619

Interventional fluorescence spectroscopy: preliminary results to detect tumor margins during glioma resection with two fluorescence spectra of PpIX

NASA Astrophysics Data System (ADS)

Alston, L. M.; Guyotat, J.; Mahieu-Williame, L.; Hebert, M.; Kantapareddy, P.; Meyronet, D.; Rousseau, D.; Montcel, B.

2017-07-01

We show the feasibility of using an intraoperative spectroscopic device to identify tumors margins during glioma resection. The collected fluorescence spectra is fitted with two reference spectra of PpIX and the contribution of each spectrum enables to overcome the sensitivity of current techniques by seeing tumor margins and low grade gliomas.

Synthesis and characterization of photoswitchable fluorescent silica nanoparticles.

PubMed

Fölling, Jonas; Polyakova, Svetlana; Belov, Vladimir; van Blaaderen, Alfons; Bossi, Mariano L; Hell, Stefan W

2008-01-01

We have designed and synthesized a new functional (amino reactive) highly efficient fluorescent molecular switch (FMS) with a photochromic diarylethene and a rhodamine fluorescent dye. The reactive group in this FMS -N-hydroxysuccinimide ester- allows selective labeling of amino containing molecules or other materials. In ethanolic solutions, the compound displays a large fluorescent quantum yield of 52 % and a large fluorescence modulation ratio (94 %) between two states that may be interconverted with red and near-UV light. Silica nanoparticles incorporating the new FMS were prepared and characterized, and their spectroscopic and switching properties were also studied. The dye retained its properties after the incorporation into the silica, thereby allowing light-induced reversible high modulation of the fluorescence signal of a single particle for up to 60 cycles, before undergoing irreversible photobleaching. Some applications of these particles in fluorescence microscopy are also demonstrated. In particular, subdiffraction images of nanoparticles were obtained, in the focal plane of a confocal microscope.

Combined fluorescence-Raman spectroscopy measurements with an optical fiber probe for the diagnosis of melanocytic lesions

NASA Astrophysics Data System (ADS)

Cosci, Alessandro; Cicchi, Riccardo; Rossari, Susanna; De Giorgi, Vincenzo; Massi, Daniela; Pavone, Francesco S.

2012-02-01

We have designed and developed an optical fiber-probe for spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multidimensional approach. Concerning fluorescence spectroscopy, the excitation is provided by two laser diodes, one emitting in the UV (378 nm) and the other emitting in the visible (445 nm). These two lasers are used to selectively excite fluorescence from NADH and FAD, which are among the brightest endogenous fluorophores in human tissues. For Raman and NIR spectroscopy, the excitation is provided by a third laser diode with 785 nm excitation wavelength. Laser light is delivered to the tissue through the central optical fiber of a fiber bundle. The surrounding 48 fibers of the bundle are used for collecting fluorescence and Raman and for delivering light to the spectrograph. Fluorescence and Raman spectra are acquired on a cooled CCD camera. The instrument has been tested on fresh human skin biopsies clinically diagnosed as malignant melanoma, melanocytic nevus, or healthy skin, finding an optimal correlation with the subsequent histological exam. In some cases our examination was not in agreement with the clinical observation, but it was with the histological exam, demonstrating that the system can potentially contribute to improve clinical diagnostic capabilities and hence reduce the number of unnecessary biopsies.

Spectroscopic chemical analysis methods and apparatus

NASA Technical Reports Server (NTRS)

Hug, William F. (Inventor); Reid, Ray D. (Inventor)

2009-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

Spectroscopic chemical analysis methods and apparatus

NASA Technical Reports Server (NTRS)

Hug, William F. (Inventor); Reid, Ray D. (Inventor); Bhartia, Rohit (Inventor)

2013-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted along with photoluminescence spectroscopy (i.e. fluorescence and/or phosphorescence spectroscopy) to provide high levels of sensitivity and specificity in the same instrument.

Spectroscopic chemical analysis methods and apparatus

NASA Technical Reports Server (NTRS)

Reid, Ray D. (Inventor); Hug, William F. (Inventor)

2010-01-01

Spectroscopic chemical analysis methods and apparatus are disclosed which employ deep ultraviolet (e.g. in the 200 nm to 300 nm spectral range) electron beam pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor light emitting devices, and hollow cathode metal ion lasers to perform non-contact, non-invasive detection of unknown chemical analytes. These deep ultraviolet sources enable dramatic size, weight and power consumption reductions of chemical analysis instruments. Chemical analysis instruments employed in some embodiments include capillary and gel plane electrophoresis, capillary electrochromatography, high performance liquid chromatography, flow cytometry, flow cells for liquids and aerosols, and surface detection instruments. In some embodiments, Raman spectroscopic detection methods and apparatus use ultra-narrow-band angle tuning filters, acousto-optic tuning filters, and temperature tuned filters to enable ultra-miniature analyzers for chemical identification. In some embodiments Raman analysis is conducted simultaneously with native fluorescence spectroscopy to provide high levels of sensitivity and specificity in the same instrument.

ALA-induced fluorescence in the canine oral cavity.

PubMed

Vaidyanathan, Vijay; Wiggs, Robert; Stohl, Josh; Baxi, Mehul

2006-06-01

We examined whether 5-aminolevulinic acid (ALA) could enhance the spectroscopic contrast between normal and diseased oral tissues, without prolonged photosensitivity. ALA is a promising photosensitizing agent. Adose of 25 mg/kg of ALA was administered intravenously to five dogs with gingivitis and three dogs with oral cancer, respectively. Fluorescence was recorded from the diseased sites in the oral cavity in addition to normal sites. ALA-induced proto-porphyrin IX fluorescence at all gingivitis sites reached a peak in 2-3 h and returned to baseline in 24 h. Fluorescence from the gingivitis site was observed earlier and was higher than the fluorescence from the normal site. For dogs with cancer, fluorescence from the cancerous sites occurred earlier in time compared to gingivitis sites and was comparatively higher in intensity. The fluorescence from the diseased sites was found to be higher than the normal site. Clinical and fluorescence data suggest that a dose of 25 mg/kg may be satisfactory for diagnostic purposes and would have minimal side effects.

Growth and spectroscopic properties of Sm3+:KY(WO4)2 crystal

NASA Astrophysics Data System (ADS)

Demesh, M. P.; Dernovich, O. P.; Gusakova, N. V.; Yasukevich, A. S.; Kornienko, A. A.; Dunina, E. B.; Fomicheva, L. A.; Pavlyuk, A. A.; Kuleshov, N. V.

2018-01-01

A Sm3+:KY(WO4)2 crystal was grown by the modified Czochralski technique. Polarized absorption and fluorescence spectra, as well as a fluorescence decay curve, were recorded at room temperature. Radiative properties such as emission probabilities, branching ratios and radiative lifetimes were investigated within the framework of the Judd-Ofelt theory as well as the theory of f-f transition intensities which takes into account the influence of the excited configurations. Emission cross section spectra were determined. 4G5/2 fluorescence decay was analyzed within the framework of the Inokuti-Hirayama model. The spectroscopic properties of Sm:KYW crystal were compared with those of other Sm3+-doped materials.

Fluorescence correlation spectroscopy directly monitors coalescence during nanoparticle preparation.

PubMed

Schaeffel, David; Staff, Roland Hinrich; Butt, Hans-Juergen; Landfester, Katharina; Crespy, Daniel; Koynov, Kaloian

2012-11-14

Dual color fluorescence cross-correlation spectroscopy (DC FCCS) experiments were conducted to study the coalescence and aggregation during the formation of nanoparticles. To assess the generality of the method, three completely different processes were selected to prepare the nanoparticles. Polymeric nanoparticles were formed either by solvent evaporation from emulsion nanodroplets of polymer solutions or by miniemulsion polymerization. Inorganic nanocapsules were formed by polycondensation of alkoxysilanes at the interface of nanodroplets. In all cases, DC FCCS provided fast and unambiguous information about the occurrence of coalescence and thus a deeper insight into the mechanism of nanoparticle formation. In particular, it was found that coalescence played a minor role for the emulsion-solvent evaporation process and the miniemulsion polymerization, whereas substantial coalescence was detected during the formation of the inorganic nanocapsules. These findings demonstrate that DC FCCS is a powerful tool for monitoring nanoparticles genesis.

Spectroscopic studies on the interaction of fluorescein and safranine T in PC liposomes

NASA Astrophysics Data System (ADS)

Bozkurt, Ebru; Bayraktutan, Tuğba; Acar, Murat; Toprak, Mahmut

2013-01-01

In this study, the fluorescence quenching of fluorescein by safranine T in liposome media had been investigated systematically by fluorescence spectroscopy, UV-vis absorption spectroscopy and fluorescence decay lifetime measurements. The spectroscopic data were analyzed using a Stern-Volmer equation to determine the quenching process. The experimental results showed that the intrinsic fluorescence of fluorescein was strongly quenched by safranine T, and that the quenching mechanism was considered as static quenching by forming a ground-complex. The Stern-Volmer quenching constant Ksv, and the bimolecular quenching constant Kq were estimated. The distances between the donor (fluorescein) and the acceptor (safranine T) were calculated according to the Förster non-radiation energy transfer theory. In addition, the partition coefficient of the safranine T (Kp) in the L-egg lecithin phosphatidylcholine liposomes was also calculated by utilizing the fluorescence quenching.

Imaging fluorescence-correlation spectroscopy for measuring fast surface diffusion at liquid/solid interfaces.

PubMed

Cooper, Justin T; Harris, Joel M

2014-08-05

The development of techniques to probe interfacial molecular transport is important for understanding and optimizing surface-based analytical methods including surface-enhanced spectroscopies, biological assays, and chemical separations. Single-molecule-fluorescence imaging and tracking has been used to measure lateral diffusion rates of fluorescent molecules at surfaces, but the technique is limited to the study of slower diffusion, where molecules must remain relatively stationary during acquisition of an image in order to build up sufficient intensity in a spot to detect and localize the molecule. Although faster time resolution can be achieved by fluorescence-correlation spectroscopy (FCS), where intensity fluctuations in a small spot are related to the motions of molecules on the surface, long-lived adsorption events arising from surface inhomogeneity can overwhelm the correlation measurement and mask the surface diffusion of the moving population. Here, we exploit a combination of these two techniques, imaging-FCS, for measurement of fast interfacial transport at a model chromatographic surface. This is accomplished by rapid imaging of the surface using an electron-multiplied-charged-coupled-device (CCD) camera, while limiting the acquisition to a small area on the camera to allow fast framing rates. The total intensity from the sampled region is autocorrelated to determine surface diffusion rates of molecules with millisecond time resolution. The technique allows electronic control over the acquisition region, which can be used to avoid strong adsorption sites and thus minimize their contribution to the measured autocorrelation decay and to vary the acquisition area to resolve surface diffusion from adsorption and desorption kinetics. As proof of concept, imaging-FCS was used to measure surface diffusion rates, interfacial populations, and adsorption-desorption rates of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiI) on planar C18- and C1

Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

NASA Astrophysics Data System (ADS)

Swift, Kerry M.; Matayoshi, Edmund D.

2008-02-01

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

Co-treatment of fruit and vegetable waste in sludge digesters: Chemical and spectroscopic investigation by fluorescence and Fourier transform infrared spectroscopy.

PubMed

Provenzano, Maria Rosaria; Cavallo, Ornella; Malerba, Anna Daniela; Di Maria, Francesco; Cucina, Mirko; Massaccesi, Luisa; Gigliotti, Giovanni

2016-04-01

In a previous work co-digestion of food waste and sewage sludge was performed in a pilot apparatus reproducing operating conditions of an existing full scale digester and processing waste mixed sludge (WMS) and fruit and vegetable waste (FVW) at different organic loading rates. An analysis of the relationship among bio-methane generation, process stability and digestate phytotoxicity was conducted. In this paper we considered humification parameters and spectroscopic analysis. Humification parameters indicated a higher not humified fraction (NH) and a lower degree of humification (DH) of FVW with respect to WMS (NH=19.22 and 5.10%; DH=36.65 and 61.94% for FVW and WMS, respectively) associated with their different chemical compositions and with the stabilization process previously undergone by sludge. FVW additions seemed to be favourable from an agronomical point of view since a lower percentage of organic carbon was lost. Fourier transform infrared spectra suggested consumption of aliphatics associated with rising in bio-methane generation followed by accumulation of aliphatics and carboxylic acids when the biogas production dropped. The trend of peaks ratios can be used as an indicator of the process efficiency. Fluorescence intensity of peak B associated with tryptophan-like substances and peak D associated with humic-like substances observed on tridimensional Excitation Emission Matrix maps increased up to sample corresponding to the highest rate of biogas production. Overall spectroscopic results provided evidence of different chemical pathways of anaerobic digestion associated with increasing amount of FVW which led to different levels of biogas production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy.

PubMed

Kaur, Gurpreet; Costa, Mauro W; Nefzger, Christian M; Silva, Juan; Fierro-González, Juan Carlos; Polo, Jose M; Bell, Toby D M; Plachta, Nicolas

2013-01-01

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin.

PubMed

de Paula Campos, Carolina; de Paula D'Almeida, Camila; Nogueira, Marcelo Saito; Moriyama, Lilian Tan; Pratavieira, Sebastião; Kurachi, Cristina

2017-12-01

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Intensity and amplitude correlations in the fluorescence from atoms with interacting Rydberg states

NASA Astrophysics Data System (ADS)

Xu, Qing; Mølmer, Klaus

2015-09-01

We explore the fluorescence signals from a pair of atoms driven towards Rydberg states on a three-level ladder transition. The dipole-dipole interactions between Rydberg excited atoms significantly distort the dark state and electromagnetically induced transparency behavior observed with independent atoms and, thus, their steady-state light emission. We calculate and analyze the temporal correlations between intensities and amplitudes of the signals emitted by the atoms and explain their origin in the atomic Rydberg state interactions.

Spectroscopic study of N2(b1Πu, ν = 8) by atmospheric-pressure resonant-enhanced multiphoton ionization and fluorescence detection.

PubMed

Adams, Steven F; Williamson, James M

2013-12-19

A spectroscopic analysis of the strongly perturbed N2(b(1)Πu, ν = 8) state has been conducted, accounting for b(1)Πu(ν = 8) ← X (1)Σg(+)(ν = 0) transitions, for the first time, up to J' = 20. A novel laser spectroscopy technique, using a combination of resonant-enhanced multiphoton ionization and fluorescence detection at atmospheric pressure, avoids the severe effects of perturbation reported in past extreme vacuum ultraviolet absorption experiments that produced weak and unusable spectra for the ν = 8 level. The R, Q, and P branches of the three-photon absorption transition b(1)Πu(ν = 8) ← X(1)Σg(+)(ν = 0) were fit, allowing rotational term energy assignment up to J' = 20 and molecular constants to be determined. Evidence of the previously suspected perturbation in b(1)Πu(ν = 8) is clear in this data, with significant Λ-type doubling at higher J' along with an anomalous negative value determined for the centrifugal distortion coefficient.

Spectroscopic investigation of inner filter effect by magnolol solutions

NASA Astrophysics Data System (ADS)

Li, Hongmei; YuzhuHu

2007-12-01

Spectroscopy is useful tool for aggregation studies on fluorephores. One of the major problems with this technique is that the inner filter effect becomes unavoidable since the samples are used at high concentration. In this work, our investigation on magnolol spectroscopic properties shows that the inner filter effect (IFE) of fluorescence plays a critical role in the spectra of magnolol. The strong dependence of the fluorescence parameters on the concentration accounts for the apparent experimental evidence of magnolol aggregation at high concentrations. There are some questions despite the aggregation model based on fluorescent aggregates seems to describe the behavior of the system. The mathematical correction on the emission intensities shows the linear fluorescence-concentration relationship. Furthermore, we propose a mathematic model of excitation spectrum based on the primary IFE (absorption of light of excitation wavelength), which provide a correct explanation of the unusual spectral shift and spectral narrowing in the excitation spectra of magnolol at high concentrations. The shapes of spectra are completely independent on magnolol aggregation and are due only to experimental artifacts, i.e. IFE.

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores.

PubMed

Yao, H; Hruska, Z; Kincaid, R; Brown, R; Cleveland, T; Bhatnagar, D

2010-05-01

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r(2), was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1-20, 20-100, and >or=100 ng g(-1) (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to

Protochlorophyll complexes with similar steady-state fluorescence characteristics can differ in fluorescence lifetimes. A model study in Triton X-100.

PubMed

Myśliwa-Kurdziel, Beata; Solymosi, Katalin; Kruk, Jerzy; Böddi, Béla; Strzałka, Kazimierz

2007-03-01

The steady-state and time-resolved fluorescence characteristics of protochlorophyll (Pchl) dissolved in neat Triton X-100 and in Triton X-100 micelles were investigated, and the fluorescence lifetimes of different Pchl spectral forms were studied. Varying the concentration of Pchl or diluting the micellar solutions either with a buffer or with a micellar solution, 631-634, 645-655, 680-692 and above 700 nm emitting Pchl complexes were prepared, the ratios of which varied from one another. The fluorescence decay of the 631-634 nm emitting (monomeric) form had a mono-exponential character with a 5.4-ns fluorescence lifetime. The long-wavelength Pchl complexes (aggregates) had two fluorescence lifetime values within a range of 1.4-3.9 ns and 0.15-0.84 ns, which showed high variability in different environments. Depending on the conditions, either mono- or double-exponential fluorescence decay was found for a fluorescence band at 680-685 nm. These data show that despite their very similar steady-state fluorescence properties, Pchl complexes can differ in fluorescence lifetimes, which may reflect different molecular structures, intrinsic geometries or different molecular interactions. This underlines the importance of complex spectroscopic analysis for a precise description of native and artificial chlorophyllous pigment forms.

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight.

PubMed

Durgannavar, Amar K; Patgar, Manjanath B; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

2016-05-01

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K(A), are 7.159 × 10(3), 9.398 × 10(3) and 16.101 × 10(3)  L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.

Quantitative fluorescence correlation spectroscopy on DNA in living cells

NASA Astrophysics Data System (ADS)

Hodges, Cameron; Kafle, Rudra P.; Meiners, Jens-Christian

2017-02-01

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

Detection of Organic Matter in Greenland Ice Cores by Deep-UV Fluorescence

NASA Astrophysics Data System (ADS)

Willis, M.; Malaska, M.; Wanger, G.; Bhartia, R.; Eshelman, E.; Abbey, W.; Priscu, J. C.

2017-12-01

The Greenland Ice Sheet is an Earthly analog for icy ocean worlds in the outer Solar System. Future missions to such worlds including Europa, Enceladus, and Titan may potentially include spectroscopic instrumentation to examine the surface/subsurface. The primary goal of our research is to test deep UV/Raman systems for in the situ detection and localization of organics in ice. As part of this effort we used a deep-UV fluorescence instrument able to detect naturally fluorescent biological materials such as aromatic molecules found in proteins and whole cells. We correlated these data with more traditional downstream analyses of organic material in natural ices. Supraglacial ice cores (2-4 m) were collected from several sites on the southwest outlet of the Greenland Ice Sheet using a 14-cm fluid-free mechanical coring system. Repeat spectral mapping data were initially collected longitudinally on uncut core sections. Cores were then cut into 2 cm thick sections along the longitudinal axis, slowly melted and analyzed for total organic carbon (TOC), total dissolved nitrogen (TDN), and bacterial density. These data reveal a spatial correlation between organic matter concentration, cell density, and the deep UV fluorescence maps. Our results provide a profile of the organics embedded within the ice from the top surface into the glacial subsurface, and the TOC:TDN data from the clean interior of the cores are indicative of a biological origin. This work provides a background dataset for future work to characterize organic carbon in the Greenland Ice Sheet and validation of novel instrumentation for in situ data collection on icy bodies.

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.

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

Fluorescence Spectroscopy Applied to Monitoring Biodiesel Degradation: Correlation with Acid Value and UV Absorption Analyses.

PubMed

Vasconcelos, Maydla Dos Santos; Passos, Wilson Espíndola; Lescanos, Caroline Honaiser; Pires de Oliveira, Ivan; Trindade, Magno Aparecido Gonçalves; Caires, Anderson Rodrigues Lima; Muzzi, Rozanna Marques

2018-01-01

The techniques used to monitor the quality of the biodiesel are intensely discussed in the literature, partly because of the different oil sources and their intrinsic physicochemical characteristics. This study aimed to monitor the thermal degradation of the fatty acid methyl esters of Sesamum indicum L. and Raphanus sativus L. biodiesels (SILB and RSLB, resp.). The results showed that both biodiesels present a high content of unsaturated fatty acids, ∼84% (SILB) and ∼90% (RSLB). The SILB had a high content of polyunsaturated linoleic fatty acid (18  :  2), about 49%, and the oleic monounsaturated (18  :  1), ∼34%. On the other hand, RSLB presented a considerable content of linolenic fatty acid (18  :  3), ∼11%. The biodiesel samples were thermal degraded at 110°C for 48 hours, and acid value, UV absorption, and fluorescence spectroscopy analysis were carried out. The results revealed that both absorption and fluorescence presented a correlation with acid value as a function of degradation time by monitoring absorptions at 232 and 270 nm as well as the emission at 424 nm. Although the obtained correlation is not completely linear, a direct correlation was observed in both cases, revealing that both properties can be potentially used for monitoring the biodiesel degradation.

Individual bioaerosol particle discrimination by multi-photon excited fluorescence.

PubMed

Kiselev, Denis; Bonacina, Luigi; Wolf, Jean-Pierre

2011-11-21

Femtosecond laser induced multi-photon excited fluorescence (MPEF) from individual airborne particles is tested for the first time for discriminating bioaerosols. The fluorescence spectra, analysed in 32 channels, exhibit a composite character originating from simultaneous two-photon and three-photon excitation at 790 nm. Simulants of bacteria aggregates (clusters of dyed polystyrene microspheres) and different pollen particles (Ragweed, Pecan, Mulberry) are clearly discriminated by their MPEF spectra. This demonstration experiment opens the way to more sophisticated spectroscopic schemes like pump-probe and coherent control. © 2011 Optical Society of America

Fluorescence Correlation Spectroscopy to Study Diffusion of Polymer Chains within Layered Hydrogen-Bonded Polymer Films

NASA Astrophysics Data System (ADS)

Pristinski, Denis; Kharlampieva, Evguenia; Sukhishvili, Svetlana

2002-03-01

Fluorescence Correlation Spectroscopy (FCS) has been used to probe molecular motions within polymer multilayers formed by hydrogen-bonding sequential self-assembly. Polyethylene glycol (PEG) molecules were end-labeled with the fluorescent tags, and self-assembled with polymethacrylic acid (PMAA) using layer-by-layer deposition. We have found that molecules included in the top adsorbed layer have significant mobility at the millisecond time scale, probably due to translational diffusion. However, their dynamics deviate from classical Brownian motion with a single diffusion time. Possible reasons for the deviation are discussed. We found that motions were significantly slowed with increasing depth within the PEG/PMAA multilayer. This phenomena occured in a narrow pH range around 4.0 in which intermolecular interactions were relatively weak.

Dynamic MR perfusion and proton MR spectroscopic imaging in Sturge-Weber syndrome: correlation with neurological symptoms.

PubMed

Lin, Doris D M; Barker, Peter B; Hatfield, Laura A; Comi, Anne M

2006-08-01

To investigate physiological alterations in Sturge-Weber syndrome (SWS) using MR perfusion imaging (PWI) and proton spectroscopic imaging (MRSI), and their association with neurological status. Six consecutive patients with a clinically established diagnosis of SWS underwent MRI using a 1.5 Tesla scanner. The protocol consisted of conventional anatomic scans, dynamic PWI, and multislice MRSI. A pediatric neurologist evaluated the neurological scores, and the imaging results were correlated with neurological scores using nonparametric correlation analysis. Two patients had classic neuroimaging findings of unilateral cerebral atrophy with corresponding leptomeningeal enhancement and hypoperfusion (prolonged mean transit time). Two patients had bilateral disease, and two had normal symmetric perfusion. Among clinical measures, the highest correlation was between hemiparesis index and hypoperfused tissue volume (Spearman's correlation coefficient, rho = 0.943, P < 0.05). There was also a trend of correlation, although not statistically significant (P = 0.06), between the hemiparesis score and the NAA/Cr ratio in the mid to posterior centrum semiovale, lateral gray matter (GM), and splenium. In SWS, PWI indicates cerebral hypoperfusion predominantly due to impaired venous drainage, with only the most severely affected regions in some patients also showing arterial perfusion deficiency. The extent and severity of the perfusion abnormality and neuronal loss/dysfunction reflect the severity of neurological symptoms and disability, and the highest correlation is found with the degree of hemiparesis. These parameters may be useful as quantitative measures of disease burden; however, further studies in larger number of patients (and with a more homogeneous age range) are required to confirm the preliminary findings reported here.

Fluorescence correlation spectroscopy, Raster image correlation spectroscopy and Number & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)

PubMed Central

Moens, Pierre D.J.; Gratton, Enrico; Salvemini, Iyrri L.

2010-01-01

Fluorescence correlation spectroscopy (FCS) was developed in 1972 by Magde, Elson and Webb (Magde et al., 1972). Photon counting detectors and avalanche photodiodes have become standards in FCS to the point that there is a widespread belief that these detectors are essential to perform FCS experiments, despite the fact that FCS was developed using analog detectors. Spatial and temporal intensity fluctuation correlations using analog detection on a commercial Olympus Fluoview 300 microscope has been reported by Brown et al. (2008). However, each analog instrument has its own idiosyncrasies that need to be understood before using the instrument for FCS. In this work we explore the capabilities of the Nikon C1, a low cost confocal microscope, to obtain single point FCS, Raster-scan Image Correlation Spectroscopy (RICS) and Number & Brightness data both in solution and incorporated into the membrane of Giant Unilamellar Vesicles (GUVs). We show that it is possible to obtain dynamic information about fluorescent molecules from single point FCS, RICS and Number & Brightness using the Nikon C1. We highlighted the fact that care should be taken in selecting the acquisition parameters in order to avoid possible artifacts due to the detector noise. However, due to relatively large errors in determining the distribution of digital levels for a given microscope setting, the system is probably only adequate for determining relative brightness within the same image. PMID:20734406

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

PubMed

Zhou, Kai-Li; Pan, Dong-Qi; Lou, Yan-Yue; Shi, Jie-Hua

2018-04-16

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, K b , value was found to lie between 2.69 × 10 3 and 9.55 × 10 3  M -1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH 0 ) and entropy change (ΔS 0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction. Copyright © 2018 John Wiley & Sons, Ltd.

Spectroscopic studies on some fluorescent mixed-ligand titanium(IV) complexes.

PubMed

Baranwal, Balram Prasad; Singh, Alok Kumar; Varma, Anand

2011-12-15

A novel route to synthesize some titanium(IV) complexes containing acetylacetone, straight chain carboxylic acid and hydroxycarboxylic acid ligands has been investigated. Complexes with the general formula [Ti(acac)Cl(2-n)(OOCR*)(n)(OOCC(15)H(31))] (where Hacac=acetylacetone, R*COOH=hydroxycarboxylic acids and n=1 or 2) have been isolated and characterized. Molecular weight determinations indicated mononuclear nature of the complexes. LMCT bands were observed in the electronic spectra. Infrared spectra suggested bidentate nature of the ligands. Fluorescent behaviour of the complexes was noticed on the basis of fluorescence spectra. Powder XRD indicated them to be semi-crystalline having the crystallite size in 136-185 nm range. Transmission electron microscopy (TEM) indicated spherical particles of ~ 200 nm diameter. On the basis of physico-chemical studies, it is suggested that titanium is having coordination number 7 or 8 in these complexes. Copyright © 2011 Elsevier B.V. All rights reserved.

Second Harmonic Correlation Spectroscopy: Theory and Principles for Determining Surface Binding Kinetics.

PubMed

Sly, Krystal L; Conboy, John C

2017-06-01

A novel application of second harmonic correlation spectroscopy (SHCS) for the direct determination of molecular adsorption and desorption kinetics to a surface is discussed in detail. The surface-specific nature of second harmonic generation (SHG) provides an efficient means to determine the kinetic rates of adsorption and desorption of molecular species to an interface without interference from bulk diffusion, which is a significant limitation of fluorescence correlation spectroscopy (FCS). The underlying principles of SHCS for the determination of surface binding kinetics are presented, including the role of optical coherence and optical heterodyne mixing. These properties of SHCS are extremely advantageous and lead to an increase in the signal-to-noise (S/N) of the correlation data, increasing the sensitivity of the technique. The influence of experimental parameters, including the uniformity of the TEM00 laser beam, the overall photon flux, and collection time are also discussed, and are shown to significantly affect the S/N of the correlation data. Second harmonic correlation spectroscopy is a powerful, surface-specific, and label-free alternative to other correlation spectroscopic methods for examining surface binding kinetics.

Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

DOE PAGES

Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; ...

2014-08-14

Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase themore » concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.« less

Novel functionalized fluorescent polymeric nanoparticles for immobilization of biomolecules

NASA Astrophysics Data System (ADS)

Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Abid, C. K. V. Zainul; Singh, Harpal

2013-07-01

Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable, monodisperse, spherical nano sized particles exhibiting high luminescence properties. Particles with 1% SLS (S1) showed good dispersion stability and fluorescence intensity and were chosen as ideal candidates for further immobilization studies. Steady state fluorescence studies showed 10 times higher fluorescence intensity of S1 nanoparticles than that of pyrene solution in solvent-toluene at the same concentration. Environmental factors such as pH, ionic strength and time were found to have no effect on fluorescence intensity of FPNPs. Surface β-di-ketone groups were utilized for the covalent immobilization of enzyme conjugated antibodies without any activation or pre-treatment of nanoparticles.Novel, size controlled fluorescent polymeric nanoparticles (FPNP) were synthesized having acetoacetoxy functionality on the surface for immobilization of biomolecules which can be utilized as biomarkers and labels in fluoroimmunoassays. Core-shell nanoparticles of poly(styrene, St-methyl methacrylate, MMA-acetoacetoxy ethyl methacrylate, AAEM), stabilized by various concentrations of surfactant, sodium lauryl sulphate (SLS), were obtained by facile miniemulsion co-polymerization encapsulated with pyrene molecules in their hydrophobic core. Analytical, spectroscopic and imaging characterization techniques revealed the formation of stable

An improved cyan fluorescent protein variant useful for FRET.

PubMed

Rizzo, Mark A; Springer, Gerald H; Granada, Butch; Piston, David W

2004-04-01

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Biochemical activity of a fluorescent dye rhodamine 6G: Molecular modeling, electrochemical, spectroscopic and thermodynamic studies.

PubMed

Al Masum, Abdulla; Chakraborty, Maharudra; Ghosh, Soumen; Laha, Dipranjan; Karmakar, Parimal; Islam, Md Maidul; Mukhopadhyay, Subrata

2016-11-01

Interaction of CT DNA with Rhodamine 6G (R6G) has been studied using molecular docking, electrochemical, spectroscopic and thermodynamic methods. From the study, it was illustrated that Rhodamine 6G binds to the minor groove of CT DNA. The binding was cooperative in nature. Circular voltametric study showed significant change in peak current and peak potential due to complexation. All the studies showed that the binding constant was in the order of 10 6 M -1 . Circular dichroic spectra showed significant conformational change on binding and DNA unwind during binding. Thermodynamic study showed that binding was favored by negative enthalpy and positive entropy change. From thermodynamic study it was also observed that several positive and negative free energies played significant role during binding and the unfavorable conformational free energy change was overcame by highly negative hydrophobic and salt dependent free energy changes. The experimental results were further validated using molecular docking study and the effect of structure on binding has been studied theoretically. From docking study it was found that the hydrophobic interaction and hydrogen bonds played a significant role during binding. The dye was absorbed by cell and this phenomenon was studied using fluorescent microscope. Cell survivability test showed that the dye active against Human Breast Cancer cells MDA-MB 468. ROS study showed that the activity is due to the production of reactive oxygen. Copyright © 2016 Elsevier B.V. All rights reserved.

Spectroscopic methods for the photodiagnosis of nonmelanoma skin cancer.

PubMed

Drakaki, Eleni; Vergou, Theognosia; Dessinioti, Clio; Stratigos, Alexander J; Salavastru, Carmen; Antoniou, Christina

2013-06-01

The importance of dermatological noninvasive imaging techniques has increased over the last decades, aiming at diagnosing nonmelanoma skin cancer (NMSC). Technological progress has led to the development of various analytical tools, enabling the in vivo/in vitro examination of lesional human skin with the aim to increase diagnostic accuracy and decrease morbidity and mortality. The structure of the skin layers, their chemical composition, and the distribution of their compounds permits the noninvasive photodiagnosis of skin diseases, such as skin cancers, especially for early stages of malignant tumors. An important role in the dermatological diagnosis and disease monitoring has been shown for promising spectroscopic and imaging techniques, such as fluorescence, diffuse reflectance, Raman and near-infrared spectroscopy, optical coherence tomography, and confocal laser-scanning microscopy. We review the use of these spectroscopic techniques as noninvasive tools for the photodiagnosis of NMSC.

Spectroscopic methods for the photodiagnosis of nonmelanoma skin cancer

NASA Astrophysics Data System (ADS)

Drakaki, Eleni; Vergou, Theognosia; Dessinioti, Clio; Stratigos, Alexander J.; Salavastru, Carmen; Antoniou, Christina

2013-06-01

The importance of dermatological noninvasive imaging techniques has increased over the last decades, aiming at diagnosing nonmelanoma skin cancer (NMSC). Technological progress has led to the development of various analytical tools, enabling the in vivo/in vitro examination of lesional human skin with the aim to increase diagnostic accuracy and decrease morbidity and mortality. The structure of the skin layers, their chemical composition, and the distribution of their compounds permits the noninvasive photodiagnosis of skin diseases, such as skin cancers, especially for early stages of malignant tumors. An important role in the dermatological diagnosis and disease monitoring has been shown for promising spectroscopic and imaging techniques, such as fluorescence, diffuse reflectance, Raman and near-infrared spectroscopy, optical coherence tomography, and confocal laser-scanning microscopy. We review the use of these spectroscopic techniques as noninvasive tools for the photodiagnosis of NMSC.

One-Pot Synthesis, Spectroscopic and Physicochemical Studies of Quinoline Based Blue Emitting Donor-Acceptor Chromophores with Their Biological Application.

PubMed

Asiri, Abdullah M; Khan, Salman A; Al-Thaqafya, Saad H

2015-09-01

Blue emitting cyano substituted isoquinoline dyes were synthesized by one-pot multicomponent reactions (MCRs) of aldehydes, malononitrile, 6-methoxy-1,2,3,4-tetrahydro-naphthalin-1-one and ammonium acetate. Results obtained from spectroscopic (FT-IR, (1)H-NMR, (13)C-NMR, EI-MS) and elemental analysis of synthesized compounds was in good agreement with their chemical structures. UV-vis and fluorescence spectroscopy measurements proved that all compounds are good absorbent and fluorescent. Fluorescence polarity study demonstrated that these compounds were sensitive to the polarity of the microenvironment provided by different solvents. In addition, spectroscopic and physicochemical parameters, including electronic absorption, excitation coefficient, stokes shift, oscillator strength, transition dipole moment and fluorescence quantum yield were investigated in order to explore the analytical potential of synthesized compounds. The anti-bacterial activity of these compounds were first studied in vitro by the disk diffusion assay against two Gram-positive and two Gram-negative bacteria then the minimum inhibitory concentration (MIC) was determined with the reference of standard drug chloramphenicol. The results displayed that compound 3 was better inhibitors of both types of the bacteria (Gram-positive and Gram-negative) than chloramphenicol. Graphical Abstract ᅟ.

Comparison of laser spectroscopic PNC method with laser integral fluorescence in optical caries diagnostics

NASA Astrophysics Data System (ADS)

Masychev, Victor I.

2001-05-01

In this research we represent the results of approbation of two methods of optical caries diagnostics: PNC-spectral diagnostics and caries detection by laser integral fluorescence. The research was conducted in a dental clinic. PNC-method analyzes parameters of probing laser radiation and PNC-spectrums of stimulated secondary radiations: backscattering and endogenous fluorescence of caries- involved bacteria. Ia-Ne laser ((lambda) equals632.8 nm, 1-2 mW) was used as a source of probing (stimulated) radiation. For registration of signals, received from intact and pathological teeth PDA-detector was applied. PNC-spectrums were processed by special algorithms, and were displayed on PC monitor. The method of laser integral fluorescence was used for comparison. In this case integral power of fluorescence of human teeth was measured. As a source of probing (stimulated) radiation diode lasers ((lambda) equals655 nm, 0.1 mW and 630 nm, 1 mW) and Ia-Na laser were applied. For registration of signals Si-photodetector was used. Integral power was shown in a digital indicator. Advantages and disadvantages of these methods are described in this research. It is disclosed that the method of laser integral power of fluorescence has the following characteristics: simplicity of construction and schema-technical decisions. However the method of PNC-spectral diagnostics are characterized by considerably more sensitivity in diagnostics of initial caries and capability to differentiate pathologies of various stages (for example, calculus/initial caries). Estimation of spectral characteristics of PNC-signals allows eliminating a number of drawbacks, which are character for detection by method of laser integral fluorescence (for instance, detection of fluorescent fillings, plagues, calculus, discolorations generally, amalgam, gold fillings as if it were caries).

Polarization properties of fluorescent BSA protected Au25 nanoclusters.

PubMed

Raut, Sangram; Chib, Rahul; Rich, Ryan; Shumilov, Dmytro; Gryczynski, Zygmunt; Gryczynski, Ignacy

2013-04-21

BSA protected gold nanoclusters (Au25) are attracting a great deal of attention due to their unique spectroscopic properties and possible use in biophysical applications. Although there are reports on synthetic strategies, spectroscopy and applications, little is known about their polarization behavior. In this study, we synthesized the BSA protected Au25 nanoclusters and studied their steady state and time resolved fluorescence properties including polarization behavior in different solvents: glycerol, propylene glycol and water. We demonstrated that the nanocluster absorption spectrum can be separated from the extinction spectrum by subtraction of Rayleigh scattering. The nanocluster absorption spectrum is well approximated by three Gaussian components. By a comparison of the emissions from BSA Au25 clusters and rhodamine B in water, we estimated the quantum yield of nanoclusters to be higher than 0.06. The fluorescence lifetime of BSA Au25 clusters is long and heterogeneous with an average value of 1.84 μs. In glycerol at -20 °C the anisotropy is high, reaching a value of 0.35. However, the excitation anisotropy strongly depends on the excitation wavelengths indicating a significant overlap of the different transition moments. The anisotropy decay in water reveals a correlation time below 0.2 μs. In propylene glycol the measured correlation time is longer and the initial anisotropy depends on the excitation wavelength. BSA Au25 clusters, due to long lifetime and high polarization, can potentially be used in studying large macromolecules such as protein complexes with large molecular weight.

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Fluorescence Spectroscopy for the Monitoring of Food Processes.

PubMed

Ahmad, Muhammad Haseeb; Sahar, Amna; Hitzmann, Bernd

Different analytical techniques have been used to examine the complexity of food samples. Among them, fluorescence spectroscopy cannot be ignored in developing rapid and non-invasive analytical methodologies. It is one of the most sensitive spectroscopic approaches employed in identification, classification, authentication, quantification, and optimization of different parameters during food handling, processing, and storage and uses different chemometric tools. Chemometrics helps to retrieve useful information from spectral data utilized in the characterization of food samples. This contribution discusses in detail the potential of fluorescence spectroscopy of different foods, such as dairy, meat, fish, eggs, edible oil, cereals, fruit, vegetables, etc., for qualitative and quantitative analysis with different chemometric approaches.

Spectral dependence of irreversible light-induced fluorescence quenching: Chlorophyll forms with maximal emission at 700-702 and 705-710nm as spectroscopic markers of conformational changes in the core complex.

PubMed

Nematov, Sherzod; Casazza, Anna Paola; Remelli, William; Khuvondikov, Vakhobjon; Santabarbara, Stefano

2017-07-01

The spectral dependence of the irreversible non-photochemical fluorescence quenching associated with photoinhibition in vitro has been comparatively investigated in thylakoid membranes, PSII enriched particles and PSII core complexes isolated from spinach. The analysis of the fluorescence emission spectra of dark-adapted and quenched samples as a function of the detection temperature in the 280-80K interval, indicates that Chlorophyll spectral forms having maximal emission in the 700-702nm and 705-710nm ranges gain relative intensity in concomitance with the establishment of irreversible light-induced quenching, acting thereby as spectroscopic markers. The relative enhancement of the 700-702nm and 705-710nm forms emission could be due either to an increase of their stoichiometric abundance or to their intrinsically low fluorescence quantum yields. These two factors, that can also coexist, need to be promoted by light-induced alterations in chromophore-protein as well as chromophore-chromophore interactions. The bands centred at about 701 and 706nm are also observed in the PSII core complex, suggesting their, at least partial, localisation in proximity to the reaction centre, and the occurrence of light-induced conformational changes in the core subunits. Copyright © 2017 Elsevier B.V. All rights reserved.

Stochastic approach to data analysis in fluorescence correlation spectroscopy.

PubMed

Rao, Ramachandra; Langoju, Rajesh; Gösch, Michael; Rigler, Per; Serov, Alexandre; Lasser, Theo

2006-09-21

Fluorescence correlation spectroscopy (FCS) has emerged as a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In FCS, the experimental data is conventionally fit using standard local search techniques, for example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these categories of algorithms is the sound knowledge of the behavior of fit parameters and in most cases good initial guesses for accurate fitting, otherwise leading to fitting artifacts. For known fit models and with user experience about the behavior of fit parameters, these local search algorithms work extremely well. However, for heterogeneous systems or where automated data analysis is a prerequisite, there is a need to apply a procedure, which treats FCS data fitting as a black box and generates reliable fit parameters with accuracy for the chosen model in hand. We present a computational approach to analyze FCS data by means of a stochastic algorithm for global search called PGSL, an acronym for Probabilistic Global Search Lausanne. This algorithm does not require any initial guesses and does the fitting in terms of searching for solutions by global sampling. It is flexible as well as computationally faster at the same time for multiparameter evaluations. We present the performance study of PGSL for two-component with triplet fits. The statistical study and the goodness of fit criterion for PGSL are also presented. The robustness of PGSL on noisy experimental data for parameter estimation is also verified. We further extend the scope of PGSL by a hybrid analysis wherein the output of PGSL is fed as initial guesses to ML. Reliability studies show that PGSL and the hybrid combination of both perform better than ML for various thresholds of the mean-squared error (MSE).

Excited state complex formation between methyl glyoxal and some aromatic bio-molecules: a fluorescence quenching study

NASA Astrophysics Data System (ADS)

Banerjee, D.; Mandal, A.; Mukherjee, S.

2003-01-01

Fluorescence quenching of some important aromatic bio-molecules (ABM) such as 3-aminophthalhydrazide (luminol), tryptophan (Try), phenylalanine and tyrosine (Tyr) by methyl glyoxal (MG) has been studied employing different spectroscopic techniques. The interaction of MG with ABM in the excited state has been analysed using Stern-Volmer (S-V) mechanism. In the case of MG-luminol system time correlated single photon counting (TCSPC) technique has also been applied to explain the S-V mechanism. The bimolecular rate constants obtained are found to be higher than the rate constant for diffusion controlled process. A plausible explanation of the quenching mechanism has been discussed on the basis of hydrogen bonding, charge transfer and energy transfer interaction between the colliding species.

Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein.

PubMed

van Thor, Jasper J; Georgiev, Georgi Y; Towrie, Michael; Sage, J Timothy

2005-09-30

Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO2. Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 angstroms of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 angstroms resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm(-1) (+) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence.

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy.

PubMed

Staaf, Elina; Bagawath-Singh, Sunitha; Johansson, Sofia

2017-02-01

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy.

PubMed

Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

2016-05-01

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K(a)) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.

Spectroscopic Study on the Interaction of 4-dimethylaminochalcones with Phospholipids

NASA Astrophysics Data System (ADS)

Tomečková, V.; Revická, M.; Sassen, A.; Veliká, B.; Stupák, M.; Perjési, P.

2014-11-01

The ultraviolet-visible and fluorescence spectroscopic properties of 4'-dimethylaminochalcone ( 1a) and its cyclic analogs 2a-4a have been studied in the presence of phospholipid vesicles (i.e., egg yolk lecithin and dipalmitoylpho sphatidylcholine), bovine serum albumin (BSA), and lipoprotein particles (i.e., bovine serum albumin plus egg yolk lecithin). The spectral results showed that compounds 1a-4a formed hydrophobic interactions with the phospholipids, lipoproteins, and BSA at the polar/nonpolar interface. Compounds 3a and 4a exhibited the strongest hydrophobic interactions of all of the compounds tested towards the phospholipids. Compound 2a gave the best fluorescent fluorophore indicating interactions with the lipids, lipoproteins, and proteins. Fluorescent microscopic imaging of breast cancer cells treated with compounds 1a-4a revealed that they could be used to stain all of the cellular components and destroy the nuclear structure. Compounds 1a-4a were found to be concentrated predominantly on the surfaces of the liposomes and lipoproteins.

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.

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA

NASA Astrophysics Data System (ADS)

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-01

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (Kb) between TMG and DNA was 2.27 × 104 M- 1, that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH < 0 and ΔS < 0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking.

Monitoring biological aerosols using UV fluorescence

NASA Astrophysics Data System (ADS)

Eversole, Jay D.; Roselle, Dominick; Seaver, Mark E.

1999-01-01

An apparatus has been designed and constructed to continuously monitor the number density, size, and fluorescent emission of ambient aerosol particles. The application of fluorescence to biological particles suspended in the atmosphere requires laser excitation in the UV spectral region. In this study, a Nd:YAG laser is quadrupled to provide a 266 nm wavelength to excite emission from single micrometer-sized particles in air. Fluorescent emission is used to continuously identify aerosol particles of biological origin. For calibration, biological samples of Bacillus subtilis spores and vegetative cells, Esherichia coli, Bacillus thuringiensis and Erwinia herbicola vegetative cells were prepared as suspensions in water and nebulized to produce aerosols. Detection of single aerosol particles, provides elastic scattering response as well as fluorescent emission in two spectral bands simultaneously. Our efforts have focuses on empirical characterization of the emission and scattering characteristics of various bacterial samples to determine the feasibility of optical discrimination between different cell types. Preliminary spectroscopic evidence suggest that different samples can be distinguished as separate bio-aerosol groups. In addition to controlled sample results, we will also discuss the most recent result on the effectiveness of detection outdoor releases and variations in environmental backgrounds.

Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

NASA Astrophysics Data System (ADS)

Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

2014-11-01

Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.

PubMed

Jutila, Arimatti; Zhu, Keng; Tuominen, Esa K J; Kinnunen, Paavo K J

2004-11-01

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.

Studying flow close to an interface by total internal reflection fluorescence cross-correlation spectroscopy: Quantitative data analysis

NASA Astrophysics Data System (ADS)

Schmitz, R.; Yordanov, S.; Butt, H. J.; Koynov, K.; Dünweg, B.

2011-12-01

Total internal reflection fluorescence cross-correlation spectroscopy (TIR-FCCS) has recently [S. Yordanov , Optics ExpressOPEXFF1094-408710.1364/OE.17.021149 17, 21149 (2009)] been established as an experimental method to probe hydrodynamic flows near surfaces, on length scales of tens of nanometers. Its main advantage is that fluorescence occurs only for tracer particles close to the surface, thus resulting in high sensitivity. However, the measured correlation functions provide only rather indirect information about the flow parameters of interest, such as the shear rate and the slip length. In the present paper, we show how to combine detailed and fairly realistic theoretical modeling of the phenomena by Brownian dynamics simulations with accurate measurements of the correlation functions, in order to establish a quantitative method to retrieve the flow properties from the experiments. First, Brownian dynamics is used to sample highly accurate correlation functions for a fixed set of model parameters. Second, these parameters are varied systematically by means of an importance-sampling Monte Carlo procedure in order to fit the experiments. This provides the optimum parameter values together with their statistical error bars. The approach is well suited for massively parallel computers, which allows us to do the data analysis within moderate computing times. The method is applied to flow near a hydrophilic surface, where the slip length is observed to be smaller than 10nm, and, within the limitations of the experiments and the model, indistinguishable from zero.

Pilot Assessment of the Repeatability of Indocyanine Green Fluorescence Imaging and Correlation with Traditional Foot Perfusion Assessments.

PubMed

Venermo, M; Settembre, N; Albäck, A; Vikatmaa, P; Aho, P-S; Lepäntalo, M; Inoue, Y; Terasaki, H

2016-10-01

Ankle brachial index (ABI), toe pressures (TP), and transcutaneous oxygen pressure (TcPO 2 ) are traditionally used in the assessment of critical limb ischemia (CLI). Indocyanine green (ICG) fluorescence imaging can be used to evaluate local circulation in the foot and to evaluate the severity of ischemia. This prospective study analyzed the suitability of a fluorescence imaging system (photodynamic eye [PDE]) in CLI. Forty-one patients with CLI were included. Of the patients, 66% had diabetes and there was an ischemic tissue lesion in 70% of the limbs. ABI, toe pressures, TcPO 2 and ICG-fluorescence imaging (ICG-FI) were measured in each leg. To study the repeatability of the ICG-FI, each patient underwent the study twice. After the procedure, foot circulation was measured using a time-intensity curve, where T1/2 (the time needed to achieve half of the maximum fluorescence intensity) and PDE10 (increase of the intensity during the first 10 s) were determined. A time-intensity curve was plotted using the same areas as for the TcPO 2 probes (n=123). The mean ABI was 0.43, TP 21 mmHg, TcPO 2 23 mmHg, T1/2 38 s, and PDE10 19 AU. Time-intensity curves were repeatable. In a Bland-Altman scatter plot, the 95% limits of agreement of PDE10 was 9.9 AU and the corresponding value of T1/2 was 14 s. Correlation between ABI and TP was significant (R=.73, p<.001), and it was weaker in diabetic patients (R=.47, p=.048) compared with non-diabetic patients (R=.89, p=.002). Correlations between ABI and TcPO 2 and TP and TcPO 2 were weak (R=.37, p=.05 and R=.43, p=.037, respectively). Correlation between TcPO 2 and PDE10 was strong in diabetic patients (R=.70, p=.003). According to this pilot study, ICG-FI with PDE can be used in the assessment of blood supply in the ischemic foot. Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

In vivo Diagnosis of Cervical Intraepithelial Neoplasia Using 337-nm- Excited Laser-Induced Fluorescence

NASA Astrophysics Data System (ADS)

Ramanujam, N.; Mitchell, M. F.; Mahadevan, A.; Warren, S.; Thomsen, S.; Silva, E.; Richards-Kortum, R.

1994-10-01

Laser-induced fluorescence at 337-nm excitation was used in vivo to differentiate neoplastic [cervical intraepithelial neoplasia (CIN)], nonneoplastic abnormal (inflammation and human papilloma viral infection), and normal cervical tissues. A colposcope (low-magnification microscope used to view the cervix with reflected light) was used to identify 66 normal and 49 abnormal (5 inflammation, 21 human papilloma virus infection, and 23 CIN) sites on the cervix in 28 patients. These sites were then interrogated spectroscopically. A two-stage algorithm was developed to diagnose CIN. The first stage differentiated histologically abnormal tissues from colposcopically normal tissues with a sensitivity, specificity, and positive predictive value of 92%, 90%, and 88%, respectively. The second stage differentiated preneoplastic and neoplastic tissues from nonneoplastic abnormal tissues with a sensitivity, specificity, and positive predictive value of 87%, 73%, and 74%, respectively. Spectroscopic differences were consistent with a decrease in the absolute contribution of collagen fluorescence, an increase in the absolute contribution of oxyhemoglobin attenuation, and an increase in the relative contribution of reduced nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence as tissue progresses from normal to abnormal in the same patient. These results suggest that in vivo fluorescence spectroscopy of the cervix can be used to diagnose CIN at colposcopy.

Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.

PubMed

Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad

2008-06-01

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

Multi-spectroscopic investigation on the complexation of tetracycline with dissolved organic matter derived from algae and macrophyte.

PubMed

Bai, Leilei; Zhao, Zhen; Wang, Chunliu; Wang, Changhui; Liu, Xin; Jiang, Helong

2017-11-01

Interactions of antibiotics with algae-derived dissolved organic matter (ADOM) and macrophyte-derived dissolved organic matter (MDOM) are of vital importance to the transport and ecotoxicity of antibiotics in eutrophic freshwater lakes. Multi-spectroscopic techniques were used to investigate the complexation of tetracycline (TTC) with ADOM and MDOM collected from Lake Taihu (China). The 3 fluorescent components, tyrosine-, tryptophan-, and humic-like component, were identified by excitation emission matrix spectra with parallel factor analysis. Their fluorescence was quenched at different degree by TTC titration through static quenching. The complexation of TTC induced conformational changes in DOM fractions. Synchronous fluorescence spectra combined with two dimensional correlation spectroscopy further suggested that the formation of TTC-DOM complexes occurred on the sequential order of tryptophan-like→tyrosine-like→humic-like component. The effective quenching constants of tryptophan- and tyrosine-like component were similar, higher than those of humic-like component. The strong binding ability and abundant content of protein-like substances indicated their prominent role in the TTC-DOM complexation. Fourier transform infrared spectroscopy further revealed that the heterogeneous functional groups, including amide I and II, aromatics, and aliphatics, were responsible for the complexation. These results highlight the significant impact of the overgrowth of algae and macrophyte on the environmental behavior of antibiotics in waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Laser-induced fluorescence ratios of Cajanus cajan L. under the stress of cadmium and its correlation with pigment content and pigment ratios.

PubMed

Maurya, Renu; Gopal, R

2008-04-01

Laser-induced fluorescence spectra were used to characterize the effect of cadmium on the pigment status of the leaves of Cajanus cajan L. Laser-induced fluorescence spectra of untreated as well as cadmium treated (0.01 mM, 0.10 mM, and 1.00 mM) Cajanus cajan L. were recorded using the 355 nm line of a Nd:YAG laser as the excitation source and a monochromator with an intensified charge-coupled device as a detector in the region 400-800 nm. The fluorescence intensity ratios (FIR) of control as well as treated Cajanus cajan L. have been calculated by evaluating curve fitted parameters using a Gaussian spectral function. In addition, some growth parameters, such as photosynthetic pigment content, were also measured. The 355 nm line of the laser-light-excited leaves not only showed a fluorescence emission in the red spectral region (650-800 nm), but also in the blue-green region (400-570 nm). The chlorophyll FIR F690/F740 strongly correlated with the photosynthetic pigment content (total chlorophyll and carotenoids) and its ratio. Consequently, a correlation was also seen between the ratio of the blue-green fluorescence F470/F540 and the photosynthetic pigment content. The results indicated that the plants treated with 0.01 mM of cadmium exhibited better growth, while higher concentrations of cadmium were hazardous for Cajanus cajan L.

Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

NASA Astrophysics Data System (ADS)

Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

2017-03-01

Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

Fluorescence Correlation Spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mRNA-complexes.

PubMed

Zhang, Heyang; De Smedt, Stefaan C; Remaut, Katrien

2018-05-10

Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine ® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production. The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, ...), but should release

Spectroscopic detection of chemotherapeutics and antioxidants

NASA Astrophysics Data System (ADS)

Latka, Ines; Grüner, Roman; Matthäus, Christian; Dietzek, Benjamin; Werncke, W.; Lademann, Jürgen; Popp, Jürgen

2012-06-01

The hand-foot-syndrome presents a severe dermal side-effect of chemotherapeutic cancer treatment. The cause of this side-effect is the elimination of systemically administered chemotherapeutics with the sweat. Transported to the skin surface, the drugs subsequently penetrate into the skin in the manner of topically applied substances. Upon accumulation of the chemotherapeutics in the skin the drugs destroy cells and tissue - in the same way as they are supposed to act in cancer cells. Aiming at the development of strategies to illuminate the molecular mechanism underlying the handfoot- syndrome (and, in a second step, strategies to prevent this severe side-effect), it might be important to evaluate the concentration and distribution of chemotherapeutics and antioxidants in the human skin. The latter can be estimated by the carotenoid concentration, as carotenoids serve as marker substances for the dermal antioxidative status.Following the objectives outlined above, this contribution presents a spectroscopic study aiming at the detection and quantification of carotenoids and selected chemotherapeutics in human skin. To this end, spontaneous Raman scattering and coherent anti-Stokes Raman scattering (CARS) microspectroscopy are combined with two-photon excited fluorescence. While the latter technique is Please verify that (1) all pages are present, (2) all figures are correct, (3) all fonts and special characters are correct, and (4) all text and figures fit within the red margin lines shown on this review document. Complete formatting information is available at http://SPIE.org/manuscripts Return to your MySPIE To Do List at http://myspie.org and approve or disapprove this submission. Your manuscript will not be published without this approval.restricted to the detection of fluorescent chemotherapeutics, e.g., doxorubicin, the vibrational spectroscopic techniques can - in principle - be applied to any type of analyte molecules. Furthermore, we will present the

Interaction of Lysozyme with Rhodamine B: A combined analysis of spectroscopic & molecular docking.

PubMed

Millan, Sabera; Satish, Lakkoji; Kesh, Sandeep; Chaudhary, Yatendra S; Sahoo, Harekrushna

2016-09-01

The interaction of Rhodamine B (RB) with Lysozyme (Lys) was investigated by different optical spectroscopic techniques such as absorption, fluorescence, and circular-dichroism (CD), along with molecular docking studies. The fluorescence results (including steady-state and time-resolved mode) revealed that the addition of RB effectively causes strong quenching of intrinsic fluorescence in Lysozyme and mostly, by the static quenching mechanism. Different binding and thermodynamic parameters were calculated at different temperatures and the binding constant value was found to be 2963.54Lmol(-1) at 25°C. The average distance (r0) was found to be 3.31nm according to Förster's theory of non-radiative energy transfer between Lysozyme and RB. The conformational change in Lysozyme during interaction with RB was confirmed from absorbance, synchronous fluorescence, and circular dichroism measurements. Finally, molecular docking studies were done to confirm that the dye binds with Lysozyme. Copyright © 2016 Elsevier B.V. All rights reserved.

Photoacoustic spectroscopic imaging of intra-tumor heterogeneity and molecular identification

NASA Astrophysics Data System (ADS)

Stantz, Keith M.; Liu, Bo; Cao, Minsong; Reinecke, Dan; Miller, Kathy; Kruger, Robert

2006-02-01

Purpose. To evaluate photoacoustic spectroscopy as a potential imaging modality capable of measuring intra-tumor heterogeneity and spectral features associated with hemoglobin and the molecular probe indocyanine green (ICG). Material and Methods. Immune deficient mice were injected with wildtype and VEGF enhanced MCF-7 breast cancer cells or SKOV3x ovarian cancer cells, which were allowed to grow to a size of 6-12 mm in diameter. Two mice were imaged alive and after euthanasia for (oxy/deoxy)-hemoglobin content. A 0.4 mL volume of 1 μg/mL concentration of ICG was injected into the tail veins of two mice prior to imaging using the photoacoustic computed tomography (PCT) spectrometer (Optosonics, Inc., Indianapolis, IN 46202) scanner. Mouse images were acquired for wavelengths spanning 700-920 nm, after which the major organs were excised, and similarly imaged. A histological study was performed by sectioning the organ and optically imaging the fluorescence distribution. Results. Calibration of PCT-spectroscopy with different samples of oxygenated blood reproduced a hemoglobin dissociation curve consistent with empirical formula with an average error of 5.6%. In vivo PCT determination of SaO II levels within the tumor vascular was measurably tracked, and spatially correlated to the periphery of the tumor. Statistical and systematic errors associated with hypoxia were estimated to be 10 and 13%, respectively. Measured ICG concentrations determined by contrast-differential PCT images in excised organs (tumor, liver) were approximately 0.8 μg/mL, consistent with fluorescent histological results. Also, the difference in the ratio of ICG concentration in the gall bladder-to-vasculature between the mice was consistent with excretion times between the two mice. Conclusion. PCT spectroscopic imaging has shown to be a noninvasive modality capable of imaging intra-tumor heterogeneity of (oxy/deoxy)-hemoglobin and ICG in vivo, with an estimated error in SaO II at 17% and in

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy.

PubMed

Butte, Pramod V; Fang, Qiyin; Jo, Javier A; Yong, William H; Pikul, Brian K; Black, Keith L; Marcu, Laura

2010-01-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Butte, Pramod V.; Fang, Qiyin; Jo, Javier A.; Yong, William H.; Pikul, Brian K.; Black, Keith L.; Marcu, Laura

2010-03-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Binding of fluorescent acridine dyes acridine orange and 9-aminoacridine to hemoglobin: Elucidation of their molecular recognition by spectroscopy, calorimetry and molecular modeling techniques.

PubMed

Chatterjee, Sabyasachi; Kumar, Gopinatha Suresh

2016-06-01

The molecular interaction between hemoglobin (HHb), the major human heme protein, and the acridine dyes acridine orange (AO) and 9-aminoacridine (9AA) was studied by various spectroscopic, calorimetric and molecular modeling techniques. The dyes formed stable ground state complex with HHb as revealed from spectroscopic data. Temperature dependent fluorescence data showed the strength of the dye-protein complexation to be inversely proportional to temperature and the fluorescence quenching was static in nature. The binding-induced conformational change in the protein was investigated using circular dichroism, synchronous fluorescence, 3D fluorescence and FTIR spectroscopy results. Circular dichroism data also quantified the α-helicity change in hemoglobin due to the binding of acridine dyes. Calorimetric studies revealed the binding to be endothermic in nature for both AO and 9AA, though the latter had higher affinity, and this was also observed from spectroscopic data. The binding of both dyes was entropy driven. pH dependent fluorescence studies revealed the existence of electrostatic interaction between the protein and dye molecules. Molecular modeling studies specified the binding site and the non-covalent interactions involved in the association. Overall, the results revealed that a small change in the acridine chromophore leads to remarkable alteration in the structural and thermodynamic aspects of binding to HHb. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Spectroscopic study of fluorescent probes based on G-quadruplex oligonucleotides labeled with ethynylpyrenyldeoxyuridine.

PubMed

Switalska, Angelika; Kierzek, Ryszard; Dembska, Anna; Juskowiak, Bernard

2017-12-01

The design, synthesis, and spectral properties of four pyrene labeled oligonucleotide probes with G-quadruplex structure (Tel22-Tpy, Tel22-Upy, Tel22-6Upy, Tel22-18Upy) based on the 22-mer human telomeric sequence (Tel22) have been reported. Pyrene labels in the form of ethynylpyrenyldeoxyuridine have been inserted efficiently into oligodeoxynucleotides probes using phosphoramidite chemistry. The probes exhibited abilities to fold into G-quadruplex structures and to bind metal cations (Na + and K + ). Folding properties of probes and their spectral behavior were examined by recording the UV-vis, fluorescence, and CD spectra as well as by analyzing melting profiles. Fluorescence characteristics and G-quadruplex folding of probes were also studied at the interface of cationic dioctadecyldimethylammonium bromide (DODAB) monolayer. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. Copyright © 2017 Elsevier B.V. All rights reserved.

Optical caries diagnostics: comparison of laser spectroscopic PNC method with method of laser integral fluorescence

NASA Astrophysics Data System (ADS)

Masychev, Victor I.

2000-11-01

In this research we present the results of approbation of two methods of optical caries diagnostics: PNC-spectral diagnostics and caries detection by laser integral fluorescence. The research was conducted in a dental clinic. PNC-method analyses parameters of probing laser radiation and PNC-spectrums of stimulated secondary radiations: backscattering and endogenous fluorescence of caries-involved bacterias. He-Ne-laser ((lambda) =632,8 nm, 1-2mW) was used as a source of probing (stimulated) radiation. For registration of signals, received from intact and pathological teeth PDA-detector was applied. PNC-spectrums were processed by special algorithms, and were displayed on PC monitor. The method of laser integral fluorescence was used for comparison. In this case integral power of fluorescence of human teeth was measured. As a source of probing (stimulated) radiation diode lasers ((lambda) =655 nm, 0.1 mW and 630nm, 1mW) and He-Ne laser were applied. For registration of signals Si-photodetector was used. Integral power was shown in a digital indicator. Advantages and disadvantages of these methods are described in this research. It is disclosed that the method of laser integral power of fluorescence has the following characteristics: simplicity of construction and schema-technical decisions. However the method of PNC-spectral diagnostics are characterized by considerably more sensitivity in diagnostics of initial caries and capability to differentiate pathologies of various stages (for example, calculus/initial caries). Estimation of spectral characteristics of PNC-signals allows eliminating a number of drawbacks, which are character for detection by method of laser integral fluorescence (for instance, detection of fluorescent fillings, plagues, calculus, discolorations generally, amalgam, gold fillings as if it were caries.

Analysis of single-molecule fluorescence spectroscopic data with a Markov-modulated Poisson process.

PubMed

Jäger, Mark; Kiel, Alexander; Herten, Dirk-Peter; Hamprecht, Fred A

2009-10-05

We present a photon-by-photon analysis framework for the evaluation of data from single-molecule fluorescence spectroscopy (SMFS) experiments using a Markov-modulated Poisson process (MMPP). A MMPP combines a discrete (and hidden) Markov process with an additional Poisson process reflecting the observation of individual photons. The algorithmic framework is used to automatically analyze the dynamics of the complex formation and dissociation of Cu2+ ions with the bidentate ligand 2,2'-bipyridine-4,4'dicarboxylic acid in aqueous media. The process of association and dissociation of Cu2+ ions is monitored with SMFS. The dcbpy-DNA conjugate can exist in two or more distinct states which influence the photon emission rates. The advantage of a photon-by-photon analysis is that no information is lost in preprocessing steps. Different model complexities are investigated in order to best describe the recorded data and to determine transition rates on a photon-by-photon basis. The main strength of the method is that it allows to detect intermittent phenomena which are masked by binning and that are difficult to find using correlation techniques when they are short-lived.

Fluorescence spectroscopy reveals accompanying occurrence of ammonium with fulvic acid-like organic matter in a fluvio-lacustrine aquifer of Jianhan Plain.

PubMed

Huang, Shuangbing; Wang, Yanxin; Ma, Teng; Wang, Yanyan; Zhao, Long

2016-05-01

This study is the first to investigate the simultaneous presence of NH4 (+) and fluorescent organic matter components (FOCs) from a fluvio-lacustrine aquifer in Central Jianghan Plain. Sediment, groundwater, and surface water samples were collected for the sediment organic matter extraction, 3D fluorescence spectroscopy characterization, and/or hydrochemical analysis. NH4 (+) and dissolved organic carbon was ubiquitous in the groundwater. The fluorescence spectroscopy revealed good relationships between NH4 (+) and fulvic acid-like components (FALCs) in the groundwater and sediment-extracted organic matter (SEOM) solutions. NH4 (+) also exhibited significant positive correlation with protein-like component (PLC) (p < 0.001), with the stronger in the SEOM solutions than that in groundwater. Comparisons of spectroscopic indices [e.g., humification index (HIX), biological index (BIX), spectra slope (S275-295), and specific UV absorbance (SUVA254)] between the groundwater and SEOM solutions revealed more labile properties of SEOM. This result indicates that the decreasing NH4 (+)-FOCs correlations of groundwater relative to sediments may be attributed to microbial degradation. Factor analysis identifies important factors that cause NH4 (+) occurrence in the groundwater. The accompanying increase of FALC (C1) and NH4-N with the mole concentration of the normalized HCO3 (-)/(Ca(2+)+Mg(2+)) and [H(+)] suggests that couple effects of various biodegradations simultaneously occur in the aquifer, promoting the occurrence of NH4-DOMs.

Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

PubMed

Di Rienzo, Carmine; Cardarelli, Francesco; Di Luca, Mariagrazia; Beltram, Fabio; Gratton, Enrico

2016-08-23

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Metallic-nanoparticles-enhanced fluorescence from individual micron-sized aerosol particles on-the-fly.

PubMed

Sivaprakasam, Vasanthi; Hart, Matthew B; Jain, Vaibhav; Eversole, Jay D

2014-08-11

Fluorescence spectra from individual aerosol particles that were either coated or embedded with metallic nanoparticles (MNPs) was acquired on-the-fly using 266 nm and 355 nm excitation. Using aqueous suspensions of MNPs with either polystyrene latex (PSL) spheres or dissolved proteins (tryptophan or ovalbumin), we generated PSL spheres coated with MNPs, or protein clusters embedded with MNPs as aerosols. Both enhanced and quenched fluorescence intensities were observed as a function of MNP concentration. Optimizing MNP material, size and spacing should yield enhanced sensitivity for specific aerosol materials that could be exploited to improve detection limits of single-particle, on-the-fly fluorescence or Raman based spectroscopic sensors.

Entangled-photon coincidence fluorescence imaging

PubMed Central

Scarcelli, Giuliano; Yun, Seok H.

2009-01-01

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations. PMID:18825257

X-ray fluorescence tomographic system design and image reconstruction.

PubMed

Cong, Wenxiang; Shen, Haiou; Cao, Guohua; Liu, Hong; Wang, Ge

2013-01-01

In this paper, we presented a new design of x-ray fluorescence CT imaging system. For detecting fuorescence signals of gold nanoparticles in-vivo, multiple spectroscopic detectors are arranged and rotated orthogonal to an excited region of interest so that a localized scan can be acquired with a maximized efficiency. Excitation filtration was employed to minimize the effects of low-energy x-rays and background scattering for lowering radiation dose to the object. Numerical simulations showed that the radiation dose is less than 300 mGy/second for a complete 30 views tomographic scan; and the sensitivity of 3D fluorescence signal detection is up to 0.2% contrast concentrations of nanoparticles. The x-ray fluorescence computed tomography is an important molecular imaging tool. It can be used directly in samall animal research. It has great translational potential for future clinical applications.

Nine New Fluorescent Probes

NASA Astrophysics Data System (ADS)

Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

1989-06-01

Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA.

PubMed

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-05

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (K b ) between TMG and DNA was 2.27×10 4 M -1 , that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH<0 and ΔS<0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of hepatocarcinoma in rats by integration of the fluorescence spectrum: Experimental model

NASA Astrophysics Data System (ADS)

Marcassa, J. C.; Ferreira, J.; Zucoloto, S.; Castro E Silva, O., Jr.; Marcassa, L. G.; Bagnato, V. S.

2006-05-01

The incorporation of spectroscopic techniques into diagnostic procedures may greatly improve the chances for precise diagnostics. One promising technique is fluorescence spectroscopy, which has recently been used to detect many different types of diseases. In this work, we use laser-induced tissue fluorescence to detect hepatocarcinoma in rats using excitation light at wavelengths of 443 and 532 nm. Hepatocarcinoma was induced chemically in Wistar rats. The collected fluorescence spectrum ranges from the excitation wavelength up to 850 nm. A mathematical procedure carried out on the spectrum determines a figure of merit value, which allows the detection of hepatocarcinoma. The figure of merit involves a procedure which evaluates the ratio between the backscattered excitation wavelength and the broad emission fluorescence band. We demonstrate that a normalization allowed by integration of the fluorescence spectra is a simple operation that may allow the detection of hepatocarcinoma.

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.

Confined detection volume of fluorescence correlation spectroscopy by bare fiber probes.

PubMed

Lu, Guowei; Lei, Franck H; Angiboust, Jean-François; Manfait, Michel

2010-04-01

A fiber-tip-based near-field fluorescence correlation spectroscopy (FCS) has been developed for confining the detection volume to sub-diffraction-limited dimensions. This near-field FCS is based on near-field illumination by coupling a scanning near-field optical microscope (SNOM) to a conventional confocal FCS. Single-molecule FCS analysis at 100 nM Rhodamine 6G has been achieved by using bare chemically etched, tapered fiber tips. The detection volume under control of the SNOM system has been reduced over one order of magnitude compared to that of the conventional confocal FCS. Related factors influencing the near-field FCS performance are investigated and discussed in detail. In this proof-of-principle study, the preliminary experimental results suggest that the fiber-tip-based near-field FCS might be a good alternative to realize localized analysis at the single-molecule level.

Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes

PubMed Central

Macháň, Radek; Hof, Martin

2010-01-01

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support. PMID:20386647

Photophysics of α-furil at room temperature and 77 K: Spectroscopic and quantum chemical studies

NASA Astrophysics Data System (ADS)

Kundu, Pronab; Chattopadhyay, Nitin

2016-06-01

Steady state and time resolved spectroscopic measurements have been exploited to assign the emissions from different conformations of α-furil (2, 2'-furil) in solution phase at room temperature as well as cryogen (liquid nitrogen, LN2) frozen matrices of ethanol and methylcyclohexane. Room temperature studies reveal a single fluorescence from the trans-planar conformer of the fluorophore or two fluorescence bands coming from the trans-planar and the relaxed skew forms depending on excitation at the nπ∗ or the ππ∗ absorption band, respectively. Together with the fluorescence bands, the LN2 studies in both the solvents unambiguously ascertain two phosphorescence emissions with lifetimes 5 ± 0.3 ms (trans-planar triplet) and 81 ± 3 ms (relaxed skew triplet). Quantum chemical calculations have been performed using density functional theory at CAM-B3LYP/6-311++G∗∗ level to prop up the spectroscopic surveillance. The simulated potential energy curves (PECs) illustrate that α-furil is capable of giving two emissions from each of the S1 and the T1 states - one corresponding to the trans-planar and the other to the relaxed skew conformation. Contrary to the other 1,2-dicarbonyl molecular systems like benzil and α-naphthil, α-furil does not exhibit any fluorescence from its second excited singlet (S2) state. This is ascribed to the proximity of the minimum of the PEC of the S2 state and the hill-top of the PEC of the S1 state.

Optical diagnostic of breast cancer using Raman, polarimetric and fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Anwar, Shahzad; Firdous, Shamaraz; Rehman, Aziz-ul; Nawaz, Muhammed

2015-04-01

We presented the optical diagnostic of normal and cancerous human breast tissues using Raman, polarimetric and fluorescence spectroscopic techniques. Breast cancer is the second leading cause of cancer death among women worldwide. Optical diagnostics of cancer offered early intervention and the greatest chance of cure. Spectroscopic data were collected from freshly excised surgical specimens of normal tissues with Raman bands at 800, 1171 and 1530 cm-1 arising mainly by lipids, nucleic acids, proteins, carbohydrates and amino acids. For breast cancer, Raman bands are observed at 1070, 1211, 1495, 1583 and 1650 cm-1. Results demonstrate that the spectra of normal tissue are dominated by lipids and amino acids. Polarization decomposition of the Mueller matrix and confocal microscopic fluorescence provides detailed description of cancerous tissue and distinguishes between the normal and malignant one. Based on these findings, we successfully differentiate normal and malignant breast tissues at an early stage of disease. There is a need to develop a new tool for noninvasive, real-time diagnosis of tissue abnormalities and a test procedure for detecting breast cancer at an early stage.

Spectroscopic methods for the study of energetic characteristics of the normal and photoproduct forms of 3-hydroxyflavones.

PubMed

Ushakou, Dzmitryi V; Tomin, Vladimir I

2018-06-07

We report spectroscopic properties of 3-hydroxyflavone (3-HF) and 4'-N,N-dimethylamino-3-hydroxyflavone (DMA3HF) in acetonitrile and ethyl acetate at different temperatures in the range from 10 °C to about 67 °C. These compounds are characterized by excited-state intramolecular proton transfer (ESIPT) which leads to occurrence of two forms of these molecules. For this reason their fluorescence spectra have two bands which correspond to emission of normal and photoproduct (tautomer) forms. The correlation between ratio of integrated intensity of these two bands and inverse absolute temperature (the Arrhenius plot) have been applied to estimate energetic properties, such as difference between energy levels of excited states as well ground states for normal and tautomer forms for each molecule. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel heterocyclic thiosemicarbazones derivatives as colorimetric and "turn on" fluorescent sensors for fluoride anion sensing employing hydrogen bonding.

PubMed

Ashok Kumar, S L; Saravana Kumar, M; Sreeja, P B; Sreekanth, A

2013-09-01

Two novel heterocyclic thiosemicarbazone derivatives have been synthesized, and characterized, by means of spectroscopic and single crystal X-ray diffraction methods. Their chromophoric-fluorogenic response towards anions in competing solvent dimethyl sulfoxide (DMSO) was studied. The receptor shows selective recognition towards fluoride anion. The binding affinity of the receptors with fluoride anion was calculated using UV-visible and fluorescence spectroscopic techniques. Copyright © 2013 Elsevier B.V. All rights reserved.

Designing an anion-functionalized fluorescent ionic liquid as an efficient and reversible turn-off sensor for detecting SO2.

PubMed

Che, Siying; Dao, Rina; Zhang, Weidong; Lv, Xiaoyu; Li, Haoran; Wang, Congmin

2017-03-30

A novel anion-functionalized fluorescent ionic liquid was designed and prepared, which was capable of capturing sulphur dioxide with high capacity and could also be used as a good colorimetric and fluorescent SO 2 sensor. Compared to conventional fluorescent sensors, this fluorescent ionic liquid did not undergo aggregation-caused quenching or aggregation-induced emission, and the fluorescence was quenched when exposed to SO 2 , and the fluorescence would quench when exposed to SO 2 . The experimental absorption, spectroscopic investigation, and quantum chemical calculations indicated that the quenching of the fluorescence originated from SO 2 physical absorption, not chemical absorption. Furthermore, this fluorescent ionic liquid exhibited high selectivity, good quantification, and excellent reversibility for SO 2 detection, and showed potential for an excellent liquid sensor.

Design and fabrication of SiO2/TiO2 and MgO/TiO2 based high selective optical filters for diffuse reflectance and fluorescence signals extraction

PubMed Central

Pimenta, S.; Cardoso, S.; Miranda, A.; De Beule, P.; Castanheira, E.M.S.; Minas, G.

2015-01-01

This paper presents the design, optimization and fabrication of 16 MgO/TiO2 and SiO2/TiO2 based high selective narrow bandpass optical filters. Their performance to extract diffuse reflectance and fluorescence signals from gastrointestinal tissue phantoms was successfully evaluated. The obtained results prove their feasibility to correctly extract those spectroscopic signals, through a Spearman’s rank correlation test (Spearman’s correlation coefficient higher than 0.981) performed between the original spectra and the ones obtained using those 16 fabricated optical filters. These results are an important step for the implementation of a miniaturized, low-cost and minimal invasive microsystem that could help in the detection of gastrointestinal dysplasia. PMID:26309769

Design and fabrication of SiO2/TiO2 and MgO/TiO2 based high selective optical filters for diffuse reflectance and fluorescence signals extraction.

PubMed

Pimenta, S; Cardoso, S; Miranda, A; De Beule, P; Castanheira, E M S; Minas, G

2015-08-01

This paper presents the design, optimization and fabrication of 16 MgO/TiO2 and SiO2/TiO2 based high selective narrow bandpass optical filters. Their performance to extract diffuse reflectance and fluorescence signals from gastrointestinal tissue phantoms was successfully evaluated. The obtained results prove their feasibility to correctly extract those spectroscopic signals, through a Spearman's rank correlation test (Spearman's correlation coefficient higher than 0.981) performed between the original spectra and the ones obtained using those 16 fabricated optical filters. These results are an important step for the implementation of a miniaturized, low-cost and minimal invasive microsystem that could help in the detection of gastrointestinal dysplasia.

Liquid nitrogen-assisted synthesis of fluorescent carbon dots from Blueberry and their performance in Fe3+ detection

NASA Astrophysics Data System (ADS)

Aslandaş, Ayşe Merve; Balcı, Neslihan; Arık, Mustafa; Şakiroğlu, Halis; Onganer, Yavuz; Meral, Kadem

2015-11-01

Fluorescent carbon dots (C-dots) were synthesized by a facile method containing liquid N2 treatment and centrifuge processes. The photophysical properties of the C-dots in an aqueous solution were examined at various conditions such as concentration, temperature, pH and excitation wavelength by using UV-vis absorption, fluorescence and time-resolved fluorescence spectroscopies. The C-dots emitted a broad fluorescence between approximately 350-550 nm and their fluorescence was tuned by changing excitation wavelength. The as-prepared C-dots were applied to Fe3+ detection from aqueous solution. Spectroscopic data revealed that the as-prepared C-dots were used to detect Fe3+ in the range of 12.5 μM to 100 μM as a fluorescence sensor.

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red

NASA Astrophysics Data System (ADS)

Halder, Animesh; Saha, Baishakhi; Maity, Pabitra; Kumar, Gopinatha Suresh; Sinha, Deepak Kumar; Karmakar, Sanat

2018-02-01

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by 10 nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Spectroscopic and theoretical investigation of conformational changes of proteins by synthesized pyrimidine derivative and its sensitivity towards FRET application

NASA Astrophysics Data System (ADS)

Ghosh, Swadesh; Singharoy, Dipti; Bhattacharya, Subhash Chandra

2018-04-01

Interest in synthesizing and characterizing (IR, NMR and HRMS spectroscopic methods) a pyrimidine based Schiff-base ligand, 2-(2-(Anthracen-9-ylmethylene) hydrazinyl)-4,6-dimethyl pyrimidine (ANHP) has been developed for its application to ascertain the conformational change of protein and sensitivity towards fluorescence resonance energy transfer (FRET) process. Location of ANHP in bovine serum albumin (BSA) and human serum albumin (HSA) proteins environment has been determined using different spectroscopic techniques. Weakly fluorescent ANHP have shown greater protein induced fluorescence enhancement (PIFE) in case of HSA than BSA, though in both cases energy transfer efficiency are almost same but difference in binding constant values encourages us to find the location of ANHP within the complex protein environment. From the FRET parameter and α-helicity change, it has been found that ANHP bound with Trp-214 of HSA and surface Trp-134 of BSA. Conformational changes of proteins have been observed more for HSA than BSA in presence of ANHP, which has confirmed the location of ANHP in both the protein environments. Coupled with experimental studies, molecular docking analysis has also been done to explain the locations and distance dependent FRET process of ANHP in both proteins.

Spectroscopic study of intermolecular complexes between FAD and some β-carboline derivatives

NASA Astrophysics Data System (ADS)

Codoñer, Armando; Monzó, Isidro S.; Tomás, Francisco; Valero, Rosa

The formation of molecular complexes between flavine adenine dinucleotide (FAD) and some β-carboline derivatives [antidepressant drugs that have a pronounced inhibition of monoamine oxidase (MAO)] has been studied by using electronic absorption and fluorescence spectroscopic methods. Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures. The influence of substituents in the β-carboline molecule on the stability of the complexes formed was also investigated.

Depth-resolved fluorescence of human ectocervical tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-04-01

The depth-resolved autofluorescence of normal and dysplastic human ectocervical tissue within 120um depth were investigated utilizing a portable confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of all ectocervical tissue samples, strong keratin fluorescence with the spectral characteristics similar to collagen was observed, which created serious interference in seeking the correlation between tissue fluorescence and tissue pathology. While from the underlying non-keratinizing epithelial layer, the measured NADH fluorescence induced by 355nm excitation and FAD fluorescence induced by 457nm excitation were strongly correlated to the tissue pathology. The ratios between NADH over FAD fluorescence increased statistically in the CIN epithelial relative to the normal and HPV epithelia, which indicated increased metabolic activity in precancerous tissue. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes.

PubMed

Freiberg, Arvi; Rätsep, Margus; Timpmann, Kõu

2012-08-01

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2011. Published by Elsevier B.V.

Electrochemical and spectroscopic study on the interaction between isoprenaline and DNA using multivariate curve resolution-alternating least squares.

PubMed

Ni, Yongnian; Wei, Min; Kokot, Serge

2011-11-01

Interaction of isoprenaline (ISO) with calf-thymus DNA was studied by spectroscopic and electrochemical methods. The behavior of ISO was investigated at a glassy carbon electrode (GCE) by cyclic voltammetry (CV) and differential pulse stripping voltammetry (DPSV); ISO was oxidized and an irreversible oxidation peak was observed. The binding constant K and the stoichiometric coefficient m of ISO with DNA were evaluated. Also, with the addition of DNA, hyperchromicity of the UV-vis absorption spectra of ISO was noted, while the fluorescence intensity decreased significantly. Multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics method was applied to resolve the combined spectroscopic data matrix, which was obtained by the UV-vis and fluorescence methods. Pure spectra of ISO, DNA and ISO-DNA complex, and their concentration profiles were then successfully obtained. The results indicated that the ISO molecule intercalated into the base-pairs of DNA, and the complex of ISO-DNA was formed. Copyright © 2011 Elsevier B.V. All rights reserved.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

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.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

PubMed

Tsuganezawa, Keiko; Shinohara, Yoshiyasu; Ogawa, Naoko; Tsuboi, Shun; Okada, Norihisa; Mori, Masumi; Yokoyama, Shigeyuki; Noda, Nobuo N; Inagaki, Fuyuhiko; Ohsumi, Yoshinori; Tanaka, Akiko

2013-10-01

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Spectroscopic and structural study of the newly synthesized heteroligand complex of copper with creatinine and urea.

PubMed

Gangopadhyay, Debraj; Singh, Sachin Kumar; Sharma, Poornima; Mishra, Hirdyesh; Unnikrishnan, V K; Singh, Bachcha; Singh, Ranjan K

2016-02-05

Study of copper complex of creatinine and urea is very important in life science and medicine. In this paper, spectroscopic and structural study of a newly synthesized heteroligand complex of copper with creatinine and urea has been discussed. Structural studies have been carried out using DFT calculations and spectroscopic analyses were carried out by FT-IR, Raman, UV-vis absorption and fluorescence techniques. The copper complex of creatinine and the heteroligand complex were found to have much increased water solubility as compared to pure creatinine. The analysis of FT-IR and Raman spectra helps to understand the coordination properties of the two ligands and to determine the probable structure of the heteroligand complex. The LIBS spectra of the heteroligand complex reveal that the complex is free from other metal impurities. UV-visible absorption spectra and the fluorescence emission spectra of the aqueous solution of Cu-Crn-urea heteroligand complex at different solute concentrations have been analyzed and the complex is found to be rigid and stable in its monomeric form at very low concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescent Sensing of Fluoride in Cellular System

PubMed Central

Jiao, Yang; Zhu, Baocun; Chen, Jihua; Duan, Xiaohong

2015-01-01

Fluoride ions have the important roles in a lot of physiological activities related with biological and medical system, such as water fluoridation, caries treatment, and bone disease treatment. Great efforts have been made to develop new methods and strategies for F- detection in the past decades. Traditional methods for the detection of F- including ion chromatography, ion-selective electrodes, and spectroscopic techniques have the limitations in the biomedicine research. The fluorescent probes for F- are very promising that overcome some drawbacks of traditional fluoride detection methods. These probes exhibit high selectivity, high sensitivity as well as quick response to the detection of fluoride anions. The review commences with a brief description of photophysical mechanisms for fluorescent probes for fluoride, including photo induced electron transfer (PET), intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), and excited-state intramolecular proton transfer (ESIPT). Followed by a discussion about common dyes for fluorescent fluoride probes, such as anthracene, naphalimide, pyrene, BODIPY, fluorescein, rhodamine, resorufin, coumarin, cyanine, and near-infrared (NIR) dyes. We divide the fluorescent probes for fluoride in cellular application systems into nine groups, for example, type of hydrogen bonds, type of cleavage of Si-O bonds, type of Si-O bond cleavage and cylization reactions, etc. We also review the recent reported carriers in the delivery of fluorescent fluoride probes. Seventy-four typical fluorescent fluoride probes are listed and compared in detail, including quantum yield, reaction medium, excitation and emission wavelengths, linear detection range, selectivity for F-, mechanism, and analytical applications. Finally, we discuss the future challenges of the application of fluorescent fluoride probes in cellular system and in vivo. We wish that more and more excellent fluorescent fluoride probes will be developed

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy

PubMed Central

Butte, Pramod V.; Fang, Qiyin; Jo, Javier A.; Yong, William H.; Pikul, Brian K.; Black, Keith L.; Marcu, Laura

2010-01-01

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337nm, 700ps), and the intensity decay profiles were recorded in the 360-to550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390nm(lifetime=1.8±0.3ns) and 460nm(lifetime=0.8±0.1ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1ns) and reduced in high-grade glioma (N=9; lifetime=1.7±0.4ns). The emission characteristics at 460nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440to460nm; lifetime: 0.8to1.0ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens. PMID:20459282

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

PubMed

Vasconcelos, Luís; Lehto, Tõnis; Madani, Fatemeh; Radoi, Vlad; Hällbrink, Mattias; Vukojević, Vladana; Langel, Ülo

2018-02-01

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2010-01-01

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

Spectroscopic Characterization of Streptavidin Functionalized Quantum dots1

PubMed Central

Wu, Yang; Lopez, Gabriel P.; Sklar, Larry A.; Buranda, Tione

2007-01-01

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot#1005-0045 and QD585, Lot#0905-0031 from Invitrogen). This is the first step in the development of calibration beads, to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited-state lifetime measurements to characterize their spectroscopic behavior, concentrating on the 400-500nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480nm excitation up to 0.4 at 450nm and down again to 0.15 at 350nm. For QD585, QY values were constant at 0.2 between 500nm and 400nm, but dropped to 0.1 at 350nm. We attribute the wavelength dependences to heterogeneity in size and surface defects in the QD525, consistent with characteristics previously described in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots, and the quantitative needs of assay development in biological applications. PMID:17368555

Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling

NASA Astrophysics Data System (ADS)

Song, Wei; Yu, Zehua; Hu, Xinxin; Liu, Rutao

2015-02-01

Studies on the effects of environmental pollutants to protein in vitro has become a global attention. Hydrogen peroxide (H2O2) is used as an effective food preservative and bleacher in industrial production. The toxicity of H2O2 to trypsin was investigated by multiple spectroscopic techniques and the molecular docking method at the molecular level. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the trypsin-H2O2 complex on account of the negative ΔH0 and ΔS0. The binding of H2O2 changed the conformational structures and internal microenvironment of trypsin illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) results. However, the binding site was far away from the active site of trypsin and the trypsin activity was only slightly affected by H2O2, which was further explained by molecular docking investigations.

THE YOUNG SOLAR ANALOGS PROJECT. I. SPECTROSCOPIC AND PHOTOMETRIC METHODS AND MULTI-YEAR TIMESCALE SPECTROSCOPIC RESULTS

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

Gray, R. O.; Briley, M. M.; Lambert, R. A.

2015-12-15

This is the first in a series of papers presenting methods and results from the Young Solar Analogs Project, which began in 2007. This project monitors both spectroscopically and photometrically a set of 31 young (300–1500 Myr) solar-type stars with the goal of gaining insight into the space environment of the Earth during the period when life first appeared. From our spectroscopic observations we derive the Mount Wilson S chromospheric activity index (S{sub MW}), and describe the method we use to transform our instrumental indices to S{sub MW} without the need for a color term. We introduce three photospheric indicesmore » based on strong absorption features in the blue-violet spectrum—the G-band, the Ca i resonance line, and the Hydrogen-γ line—with the expectation that these indices might prove to be useful in detecting variations in the surface temperatures of active solar-type stars. We also describe our photometric program, and in particular our “Superstar technique” for differential photometry which, instead of relying on a handful of comparison stars, uses the photon flux in the entire star field in the CCD image to derive the program star magnitude. This enables photometric errors on the order of 0.005–0.007 magnitude. We present time series plots of our spectroscopic data for all four indices, and carry out extensive statistical tests on those time series demonstrating the reality of variations on timescales of years in all four indices. We also statistically test for and discover correlations and anti-correlations between the four indices. We discuss the physical basis of those correlations. As it turns out, the “photospheric” indices appear to be most strongly affected by emission in the Paschen continuum. We thus anticipate that these indices may prove to be useful proxies for monitoring emission in the ultraviolet Balmer continuum. Future papers in this series will discuss variability of the program stars on medium (days�

Modified Facile Synthesis for Quantitatively Fluorescent Carbon Dots.

PubMed

Hou, Xiaofang; Hu, Yin; Wang, Ping; Yang, Liju; Al Awak, Mohamad M; Tang, Yongan; Twara, Fridah K; Qian, Haijun; Sun, Ya-Ping

2017-10-01

A simple yet consequential modification was made to the popular carbonization processing of citric acid - polyethylenimine precursor mixtures to produce carbon dots (CDots). The modification was primarily on pushing the carbonization processing a little harder at a higher temperature, such as the hydrothermal processing condition of around 330 °C for 6 hours. The CDots thus produced are comparable in spectroscopic and other properties to those obtained in other more controlled syntheses including the deliberate chemical functionalization of preprocessed and selected small carbon nanoparticles, demonstrating the consistency in CDots and reaffirming their general definition as carbon nanoparticles with surface passivation by organic or other species. Equally significant is the finding that the modified processing of citric acid - polyethylenimine precursor mixtures could yield CDots of record-setting fluorescence performance, approaching the upper limit of being quantitatively fluorescent. Thus, the reported work serves as a demonstration on not only the need in selecting the right processing conditions and its associated opportunities in one-pot syntheses of CDots, but also the feasibility in pursuing the preparation of quantitatively fluorescent CDots, which represents an important milestone in the development and understanding of these fluorescent carbon nanomaterials.

Photophysics of α-furil at room temperature and 77 K: Spectroscopic and quantum chemical studies

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

Kundu, Pronab; Chattopadhyay, Nitin, E-mail: nitin.chattopadhyay@yahoo.com

2016-06-21

Steady state and time resolved spectroscopic measurements have been exploited to assign the emissions from different conformations of α-furil (2, 2′-furil) in solution phase at room temperature as well as cryogen (liquid nitrogen, LN{sub 2}) frozen matrices of ethanol and methylcyclohexane. Room temperature studies reveal a single fluorescence from the trans-planar conformer of the fluorophore or two fluorescence bands coming from the trans-planar and the relaxed skew forms depending on excitation at the nπ{sup ∗} or the ππ{sup ∗} absorption band, respectively. Together with the fluorescence bands, the LN{sub 2} studies in both the solvents unambiguously ascertain two phosphorescence emissionsmore » with lifetimes 5 ± 0.3 ms (trans-planar triplet) and 81 ± 3 ms (relaxed skew triplet). Quantum chemical calculations have been performed using density functional theory at CAM-B3LYP/6-311++G{sup ∗∗} level to prop up the spectroscopic surveillance. The simulated potential energy curves (PECs) illustrate that α-furil is capable of giving two emissions from each of the S{sub 1} and the T{sub 1} states—one corresponding to the trans-planar and the other to the relaxed skew conformation. Contrary to the other 1,2-dicarbonyl molecular systems like benzil and α-naphthil, α-furil does not exhibit any fluorescence from its second excited singlet (S{sub 2}) state. This is ascribed to the proximity of the minimum of the PEC of the S{sub 2} state and the hill-top of the PEC of the S{sub 1} state.« less

Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies.

PubMed

Naz, Huma; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2016-06-01

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca(2+), which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca(2+) concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV.

Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Reina, Francesco; Galiani, Silvia; Shrestha, Dilip; Sezgin, Erdinc; de Wit, Gabrielle; Cole, Daniel; Lagerholm, B. Christoffer; Kukura, Philipp; Eggeling, Christian

2018-06-01

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag–gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50  ⩽  t  ⩽  100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag–gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2–3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

Quantification of zinc-porphyrin in dry-cured ham products by spectroscopic methods Comparison of absorption, fluorescence and X-ray fluorescence spectroscopy.

PubMed

Laursen, Kristoffer; Adamsen, Christina E; Laursen, Jens; Olsen, Karsten; Møller, Jens K S

2008-03-01

Zinc-protoporphyrin (Zn-pp), which has been identified as the major pigment in certain dry-cured meat products, was extracted with acetone/water (75%) and isolated from the following meat products: Parma ham, Iberian ham and dry-cured ham with added nitrite. The quantification of Zn-pp by electron absorption, fluorescence and X-ray fluorescence (XRF) spectroscopy was compared (concentration range used [Zn-pp]=0.8-9.7μM). All three hams were found to contain Zn-pp, and the results show no significant difference among the content of Zn-pp quantified by fluorescence, absorbance and X-ray fluorescence spectroscopy for Parma ham and Iberian ham. All three methods can be used for quantification of Zn-pp in acetone/water extracts of different ham types if the content is higher than 1.0ppm. For dry-cured ham with added nitrite, XRF was not applicable due to the low content of Zn-pp (<0.1ppm). In addition, XRF spectroscopy provides further information regarding other trace elements and can therefore be advantageous in this aspect. This study also focused on XRF determination of Fe in the extracts and as no detectable Fe was found in the three types of ham extracts investigated (limit of detection; Fe⩽1.8ppm), it allows the conclusion that iron containing pigments, e.g., heme, do not contribute to the noticeable red colour observed in some of the extracts.

Rotationally resolved fluorescence spectroscopy of molecular iodine

NASA Astrophysics Data System (ADS)

Lemon, Christopher; Canagaratna, Sebastian; Gray, Jeffrey

2008-03-01

Vibration-electronic spectroscopy of I2 vapor is a common, important experiment in physical chemistry lab courses. We use narrow bandwidth diode-pumped solid state (DPSS) lasers to excite specific rotational levels; these lasers are surprisingly stable and are now available at low cost. We also use efficient miniature fiber-optic spectrometers to resolve rotational fluorescence patterns in a vibrational progression. The resolution enables thorough and accurate analysis of spectroscopic constants for the ground electronic state. The high signal-to-noise ratio, which is easily achieved, also enables students to precisely measure fluorescence band intensities, providing further insight into vibrational wavefunctions and the molecular potential function. We will provide a detailed list of parts for the apparatus as well as modeling algorithms with statistical evaluation to facilitate widespread adoption of these experimental improvements by instructors of intermediate and advanced lab courses.

Hydrogen-Bond and Supramolecular-Contact Mediated Fluorescence Enhancement of Electrochromic Azomethines.

PubMed

Wałęsa-Chorab, Monika; Tremblay, Marie-Hélène; Skene, William G

2016-08-01

An electronic push-pull fluorophore consisting of an intrinsically fluorescent central fluorene capped with two diaminophenyl groups was prepared. An aminothiophene was conjugated to the two flanking diphenylamines through a fluorescent quenching azomethine bond. X-ray crystallographic analysis confirmed that the fluorophore formed multiple intermolecular supramolecular bonds. It formed two hydrogen bonds involving a terminal amine, resulting in an antiparallel supramolecular dimer. Hydrogen bonding was also confirmed by FTIR and NMR spectroscopic analyses, and further validated theoretically by DFT calculations. Intrinsic fluorescence quenching modes could be reduced by intermolecular supramolecular contacts. These contacts could be engaged at high concentrations and in thin films, resulting in fluorescence enhancement. The fluorescence of the fluorophore could also be restored to an intensity similar to its azomethine-free counterpart with the addition of water in >50 % v/v in tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and acetonitrile. The fluorophore also exhibited reversible oxidation and its color could be switched between yellow and blue when oxidized. Reversible electrochemically mediated fluorescence turn-off on turn-on was also possible. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations.

PubMed

Wenger, Jérôme; Gérard, Davy; Lenne, Pierre-François; Rigneault, Hervé; Dintinger, José; Ebbesen, Thomas W; Boned, Annie; Conchonaud, Fabien; Marguet, Didier

2006-12-11

Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence cross-correlation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 muM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoaper-tures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.

Spectroscopic thermoacoustic imaging of water and fat composition

NASA Astrophysics Data System (ADS)

Bauer, Daniel R.; Wang, Xiong; Vollin, Jeff; Xin, Hao; Witte, Russell S.

2012-07-01

During clinical studies, thermoacoustic imaging (TAI) failed to reliably identify malignant breast tissue. To increase detection capability, we propose spectroscopic TAI to differentiate samples based on the slope of their dielectric absorption. Phantoms composed of different ratios of water and fat were imaged using excitation frequencies between 2.7 and 3.1 GHz. The frequency-dependent slope of the TA signal was highly correlated with that of its absorption coefficient (R2 = 0.98 and p < 0.01), indicating spectroscopic TAI can distinguish materials based on their intrinsic dielectric properties. This approach potentially enhances cancer detection due to the increased water content of many tumors.

Use of different spectroscopic techniques in the analysis of Roman age wall paintings.

PubMed

Agnoli, Francesca; Calliari, Irene; Mazzocchin, Gian-Antonio

2007-01-01

In this paper the analysis of samples of Roman age wall paintings coming from: Pordenone, Vicenza and Verona is carried out by using three different techniques: energy dispersive x-rays spectroscopy (EDS), x-rays fluorescence (XRF) and proton induced x-rays emission (PIXE). The features of the three spectroscopic techniques in the analysis of samples of archaeological interest are discussed. The studied pigments were: cinnabar, yellow ochre, green earth, Egyptian blue and carbon black.

Spectroscopic Chemical Analysis Methods and Apparatus

NASA Technical Reports Server (NTRS)

Hug, William F.; Reid, Ray D.

2012-01-01

This invention relates to non-contact spectroscopic methods and apparatus for performing chemical analysis and the ideal wavelengths and sources needed for this analysis. It employs deep ultraviolet (200- to 300-nm spectral range) electron-beam-pumped wide bandgap semiconductor lasers, incoherent wide bandgap semiconductor lightemitting devices, and hollow cathode metal ion lasers. Three achieved goals for this innovation are to reduce the size (under 20 L), reduce the weight [under 100 lb (.45 kg)], and reduce the power consumption (under 100 W). This method can be used in microscope or macroscope to provide measurement of Raman and/or native fluorescence emission spectra either by point-by-point measurement, or by global imaging of emissions within specific ultraviolet spectral bands. In other embodiments, the method can be used in analytical instruments such as capillary electrophoresis, capillary electro-chromatography, high-performance liquid chromatography, flow cytometry, and related instruments for detection and identification of unknown analytes using a combination of native fluorescence and/or Raman spectroscopic methods. This design provides an electron-beampumped semiconductor radiation-producing method, or source, that can emit at a wavelength (or wavelengths) below 300 nm, e.g. in the deep ultraviolet between about 200 and 300 nm, and more preferably less than 260 nm. In some variations, the method is to produce incoherent radiation, while in other implementations it produces laser radiation. In some variations, this object is achieved by using an AlGaN emission medium, while in other implementations a diamond emission medium may be used. This instrument irradiates a sample with deep UV radiation, and then uses an improved filter for separating wavelengths to be detected. This provides a multi-stage analysis of the sample. To avoid the difficulties related to producing deep UV semiconductor sources, a pumping approach has been developed that uses

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

PubMed

Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

2017-05-15

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. 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

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

PubMed

Yapici, Ipek; Lee, Kin Sing Stephen; Berbasova, Tetyana; Nosrati, Meisam; Jia, Xiaofei; Vasileiou, Chrysoula; Wang, Wenjing; Santos, Elizabeth M; Geiger, James H; Borhan, Babak

2015-01-28

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2-11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

Fluorescence of molecular hydrogen excited by solar extreme-ultraviolet radiation

NASA Technical Reports Server (NTRS)

Feldman, P. D.; Fastie, W. G.

1973-01-01

During trans-earth coast, the Apollo 17 ultraviolet spectrometer was scheduled to make observations of the far ultraviolet background in selected regions of the sky. In the course of one of these observations, the spacecraft fuel cells were routinely purged of excess hydrogen and water vapor. The ultraviolet fluorescence spectrum of the purged molecular hydrogen excited by solar extreme ultraviolet radiation is interpreted by absorption of solar L-beta and L-gamma radiation in the nearly resonant (6, 0) and (11, 0) Lyman bands. The results are deemed significant for ultraviolet spectroscopic investigations of the atmospheres of the moon and planets since Lyman-band fluorescence provides an unambiguous means of identification of molecular hydrogen in upper atmospheres.

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

PubMed Central

Lubbeck, Jennifer L.; Dean, Kevin M.; Ma, Hairong; Palmer, Amy E.; Jimenez, Ralph

2012-01-01

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts, however the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, non-temporally-coincident excitation beams. Here, we characterize the design and operation of a cytometer with a 3-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW – 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multi-channel fluorescence cytometry, and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

Encapsulation of serotonin in β-cyclodextrin nano-cavities: Fluorescence spectroscopic and molecular modeling studies

NASA Astrophysics Data System (ADS)

Chaudhuri, Sudip; Chakraborty, Sandipan; Sengupta, Pradeep K.

2010-06-01

Serotonin is a physiologically important biogenic amine, deficiency of which leads to mental disorders such as Alzheimer's disease, schizophrenia, infantile autism, and depression. Both β-cyclodextrin (β-CD) and its chemically substituted synthetic varieties (often possessing enhanced aqueous solubility and improved drug complexing abilities) are finding wide applications as drug delivery vehicles. Here we have studied the encapsulation of serotonin in β-CD and succinyl-2-hydroxypropyl β-cyclodextrin (SHP-β-CD) by exploiting the intrinsic serotonin fluorescence. Enhanced fluorescence emission intensity (which increases by ˜18% and 34% in β-CD and SHPβ-CD respectively) and anisotropy ( r) ( r = 0.075 and 0.1 in β-CD and SHPβ-CD respectively) are observed in presence of the cyclodextrins. From the fluorescence data host-guest interaction with 1:1 stoichiometry is evident, the association constants ( K) being 126.06 M -1 and 461.62 M -1 for β-CD and SHPβ-CD respectively. Additionally, molecular docking and semiempirical calculations have been carried out which provide, for the first time, detailed insights regarding the encapsulation process. In particular, it is evident that the indole ring is inserted within the β-CD cavity with the aliphatic amine side chain protruding towards the primary rim of the β-CD cavity. Docking calculations reveal that hydrogen bonding interactions are involved in the formation of the inclusion complex. Semiempirical calculations indicate that formation of the 1:1 inclusion complex is energetically favorable which is consistent with the fluorescence data.

[Retrospective, descriptive, observational study of treatment of multiple actinic keratoses with topical methyl aminolevulinate and red light: results in clinical practice and correlation with fluorescence imaging].

PubMed

Fernández-Guarino, M; Harto, A; Sánchez-Ronco, M; Pérez-García, B; Marquet, A; Jaén, P

2008-12-01

Actinic keratosis (AK) is one of the most common skin diseases seen in clinical practice. In the last 5 years, several studies assessing the efficacy of photodynamic therapy in the treatment of multiple AKs have been published. We aimed to assess the clinical outcomes of photodynamic therapy in patients with multiple AKs and the correlation of those outcomes with fluorescence imaging. In this retrospective, descriptive, observational study of 57 patients treated in our hospital with photodynamic therapy for multiple AKs, we recorded age, sex, and lesion site (face, scalp, and dorsum of the hands). All patients were treated in the same way: methyl aminolevulinic acid (Metvix) was applied for 3 hours and the skin then irradiated with red light at 630 nm, 37 J/cm(2), for 7.5 minutes (Aktilite). The response, remission duration, tolerance, number of sessions, and fluorescence images were recorded by site. The chi(2) test was used to assess between-site differences and the correlation between fluorescence imaging and clinical response. The greatest improvements were obtained for facial lesions; these required fewer sessions and remission lasted longer than lesions at other sites. The treatment was best tolerated on the dorsum of the hands. The fluorescence area and the reduction in intensity on applying treatment were found to be strongly and significantly correlated with the extent of clinical response. Overall, the outcomes of treatment of multiple AKs with photodynamic therapy are better for the face than for the scalp and dorsum of the hands. Fluorescence imaging may be an effective tool for predicting response to treatment.

Glucose Oxidase Adsorption on Sequential Adsorbed Polyelectrolyte Films Studied by Spectroscopic Techniques

NASA Astrophysics Data System (ADS)

Tristán, Ferdinando; Solís, Araceli; Palestino, Gabriela; Gergely, Csilla; Cuisinier, Frédéric; Pérez, Elías

2005-04-01

The adsorption of Glucose Oxidase (GOX) on layers of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) deposited on Sequentially Adsorbed Polyelectrolyte Films (SAPFs) were studied by three different spectroscopic techniques. These techniques are: Optical Wave Light Spectroscopy (OWLS) to measure surface density; Fluorescence Resonance Energy Transfer (FRET) to verify the adsorption of GOX on the surface; and Fourier Transform Infrared Spectroscopy in Attenuated Total Reflection mode (FTIR-HATR) to inspect local structure of polyelectrolytes and GOX. Two positive and two negative polyelectrolytes are used: Cationic poly(ethyleneimine) (PEI) and poly(allylamine hydrochloride) (PAH) and anionic poly(sodium 4-styrene sulfonate) (PSS) and poly(acrylic acid) (PAA). These spectroscopic techniques do not require any labeling for GOX or SAPFs, specifically GOX and PSS are naturally fluorescent and are used as a couple donor-acceptor for the FRET technique. The SAPFs are formed by a (PEI)-(PSS/PAH)2 film followed by (PAA/PAH)n bilayers. GOX is finally deposited on top of SAPFs at different values of n (n=1..5). Our results show that GOX is adsorbed on positive ended SAPFs forming a monolayer. Contrary, GOX adsorption is not observed on negative ended film polyelectrolyte. GOX stability was tested adding a positive and a negative polyelectrolyte after GOX adsorption. Protein is partially removed by PAH and PAA, with lesser force by PAA.

Fluorescence spectroscopy as a specific tool for the interaction study of two surfactants with natural and synthetic organic compounds

NASA Astrophysics Data System (ADS)

Jung, Aude-Valérie; Frochot, Céline; Bersillon, Jean-Luc

2016-04-01

Four different techniques were used to study the binding of cationic cetyltrimethylammonium bromide (CTAB) and non-ionic nonylphenylethoxyl (NPE) surfactants to three synthetic organic components that mimic humic-like aggregates and to two natural aggregated humic substances (HS) extracted from aquatic suspended matter. The composition of synthetic organic components were chosen to be similar to high molecular weight highly processed terrigenous HS and low and high molecular weight less processed terrigenous (or aquatic terrigenous) HS. The natural HS were extracted under two different meteorological conditions (rainy and dry periods). No significant interaction between the non-ionic surfactant and any of the studied compounds was found. Concerning CTAB; pH, conductivity and turbidity measurements, along with fluorescence spectroscopy were combined to provide a better understanding of interactions between organic aggregates and the surfactant. The spectroscopic data show that a "highly processed terrigenous HS" fluorophore interacts in a different way with the cationic surfactant than an "aquatic terrigenous (or less processed terrigenous) HS" fluorophore does. Under similar conditions, some spectral changes in the fluorescence signal are correlated to changes in non-specific physical-chemical parameters (pH, turbidity, conductivity) for the organic compounds tested. The complexation mechanism is essentially governed by charge neutralization, which can be monitored specifically by the fluorescence of the organic moieties.

Direct fluorescence characterisation of a picosecond seeded optical parametric amplifier

NASA Astrophysics Data System (ADS)

Stuart, N. H.; Bigourd, D.; Hill, R. W.; Robinson, T. S.; Mecseki, K.; Patankar, S.; New, G. H. C.; Smith, R. A.

2015-02-01

The temporal intensity contrast of high-power lasers based on optical parametric amplification (OPA) can be limited by parametric fluorescence from the non-linear gain stages. Here we present a spectroscopic method for direct measurement of unwanted parametric fluorescence widely applicable from unseeded to fully seeded and saturated OPA operation. Our technique employs simultaneous spectroscopy of fluorescence photons slightly outside the seed bandwidth and strongly attenuated light at the seed central wavelength. To demonstrate its applicability we have characterised the performance of a two-stage picosecond OPA pre-amplifier with 2.8×105 gain, delivering 335 μJ pulses at 1054 nm. We show that fluorescence from a strongly seeded OPA is reduced by ~500× from the undepleted to full pump depletion regimes. We also determine the vacuum fluctuation driven noise term seeding this OPA fluorescence to be 0.7±0.4 photons ps-1 nm-1 bandwidth. The resulting shot-to-shot statistics highlights a 1.5% probability of a five-fold and 0.3% probability of a ten-fold increase of fluorescence above the average value. Finally, we show that OPA fluorescence can be limited to a few-ps pedestal with 3×10-9 temporal intensity contrast 1.3 ps ahead of an intense laser pulse, a level highly attractive for large scale chirped-pulse OPA laser systems.

Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs

PubMed Central

Elmehriki, Adam AH; Suchý, Mojmír; Chicas, Kirby J; Wojciechowski, Filip; Hudson, Robert HE

2014-01-01

Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2’-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2’-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2’-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior. PMID:25483932

Ultraviolet-visible and fluorescence spectroscopy can be used as a diagnostic tool for gamma irradiation detection in vivo.

PubMed

K-Abdelhalim, Mohamed Anwar; Moussa, Sherif A-Abdelmottaleb

2016-09-01

The spectroscopic properties can indicate important features about the nature and severity of the disease. However, no earlier studies have been used the spectroscopic properties as a diagnostic tool for radiation detection. This study was aimed to use ultraviolet-visible and fluorescence spectroscopy as a diagnostic tool for gamma irradiation detection in rats in vivo. Adult male rats were exposed to 25, 50, 75 and 100 Gray as single dose, using Cobalt-60 (Co-60) source with a dose rate of 0.883 centi Gray/sec (cGy/s). Ultraviolet and fluorescence spectroscopy of rat's blood serum were measured. After gamma irradiation of rats in vivo, the blood serum absorbance peaks for 25, 50, 75 and 100 Gray (Gy) decreased and shifted towards the ultra violet wavelength. A maximal change in fluorescence intensity of blood serum at 350 nm was obtained when exciting light at 194 nm after irradiation. The fluorescence intensity also decreased with the dose. The highest radiation gamma dose might be accompanied with the highest oxidative stress. This study suggests that at the above mentioned gamma radiation doses, the blood is highly fragmented; with low aggregation at 25 Gy and with high aggregation at 50-100 Gy.

Instrumentation of Molecular Imaging on Site-Specific Targeting Fluorescent Peptide for Early Detection of Breast Cancer

NASA Astrophysics Data System (ADS)

Yu, Ping; Ma, Lixin

2012-02-01

In this work we developed two biomedical imaging techniques for early detection of breast cancer. Both image modalities provide molecular imaging capability to probe site-specific targeting dyes. The first technique, heterodyne CCD fluorescence mediated tomography, is a non-invasive biomedical imaging that uses fluorescent photons from the targeted dye on the tumor cells inside human breast tissue. The technique detects a large volume of tissue (20 cm) with a moderate resolution (1 mm) and provides the high sensitivity. The second technique, dual-band spectral-domain optical coherence tomography, is a high-resolution tissue imaging modality. It uses a low coherence interferometer to detect coherent photons hidden in the incoherent background. Due to the coherence detection, a high resolution (20 microns) is possible. We have finished prototype imaging systems for the development of both image modalities and performed imaging experiments on tumor tissues. The spectroscopic/tomographic images show contrasts of dense tumor tissues and tumor necrotic regions. In order to correlate the findings from our results, a diffusion-weighted magnetic resonance imaging (MRI) of the tumors was performed using a small animal 7-Telsa MRI and demonstrated excellent agreement.

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

PubMed

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

Note: a 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy.

PubMed

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A M

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Note: A 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A. M.

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

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

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

Synthesis of fluorescent carbon dots by a microwave heating process: structural characterization and cell imaging applications

NASA Astrophysics Data System (ADS)

Stefanakis, Dimitrios; Philippidis, Aggelos; Sygellou, Labrini; Filippidis, George; Ghanotakis, Demetrios; Anglos, Demetrios

2014-10-01

Two types of highly fluorescent carbon dots (C-dots) were prepared by a single-step procedure based on microwave heating citric acid and 6-aminocaproic acid or citric acid and urea in an aqueous solution. The small size of the isolated carbon dots along with their strong absorption in the UV and their excitation wavelength-dependent fluorescence render them ideal nanomaterials for biomedical applications (imaging and sensing). The structure and properties of the two types of C-dot materials were studied using a series of spectroscopic techniques. The ability of the C-dots to be internalized by HeLa cells was demonstrated via 3-photon fluorescence microscopy imaging.

Hindered Diffusion in Polymeric Solutions Studied by Fluorescence Correlation Spectroscopy

PubMed Central

Zustiak, Silviya P.; Nossal, Ralph; Sackett, Dan L.

2011-01-01

Diffusion of molecules in the crowded and charged interior of the cell has long been of interest for understanding cellular processes. Here, we introduce a model system of hindered diffusion that includes both crowding and binding. In particular, we obtained the diffusivity of the positively charged protein, ribonuclease A (RNase), in solutions of dextrans of various charges (binding) and concentrations (crowding), as well as combinations of both, in a buffer of physiological ionic strength. Using fluorescence correlation spectroscopy, we observed that the diffusivity of RNase was unaffected by the presence of positively charged or neutral dextrans in the dilute regime but was affected by crowding at higher polymer concentrations. Conversely, protein diffusivity was significantly reduced by negatively charged dextrans, even at 0.4 μM (0.02% w/v) dextran. The diffusivity of RNase decreased with increasing concentrations of negative dextran, and the amount of bound RNase increased until it reached a plateau of ∼80% bound RNase. High salt concentrations were used to establish the electrostatic nature of the binding. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration. PMID:21723836

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Spectroscopic studies on the interaction of cysteine capped CuS nanoparticles with tyrosine

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

Prasanth, S.; Raj, D. Rithesh; Kumar, T. V. Vineesh

2015-06-24

Biocompatible cysteine coated CuS nanoparticles were synthesized by a simple aqueous solution method. Hexagonal phase of the samples were confirmed from X-ray diffraction and particle size found to be 9 nm. The possible interaction between the bioactive cysteine capped CuS nanoparticles and tyrosine were investigated using spectroscopic techniques such as UV-Visible absorption and fluorescence spectroscopy. It is observed that the luminescence intensity of tyrosine molecule enhanced by the addition CuS nanoparticles.

Progress Towards Spectroscopic Diagnostics of Plasma Parameters and Neutral Dynamics in Helicon Plasmas

NASA Astrophysics Data System (ADS)

Green, Jonathan; Schmitz, Oliver; Severn, Greg; van Ruremonde, Lars; Winters, Victoria

2017-10-01

The MARIA device at the UW-Madison is used primarily to investigate the dynamics and fueling of neutral particles in helicon discharges. A new systematic method is in development to measure key plasma and neutral particle parameters by spectroscopic methods. The setup relies on spectroscopic line ratios for investigating basic plasma parameters and extrapolation to other states using a collisional radiative model. Active pumping using a Nd:YAG pumped dye laser is used to benchmark and correct the underlying atomic data for the collisional radiative model. First results show a matching linear dependence between electron density and laser induced fluorescence on the magnetic field above 500G. This linear dependence agrees with the helicon dispersion relation and implies MARIA can reliably support the helicon mode and support future measurements. This work was funded by the NSF CAREER award PHY-1455210.

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

PubMed

Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

2015-01-01

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Aerogel volatiles concentrator and analyzer (AVCA) - Collection and concentration of trace volatile organics in aerogel for spectroscopic detection

NASA Astrophysics Data System (ADS)

Tsapin, A.; Jones, S.; Petkov, M.; Borchardt, D.; Anderson, M.

2017-03-01

A study was conducted to determine the efficacy of using silica aerogel to collect and concentrate ambient trace organics for spectroscopic analysis. Silica aerogel was exposed to atmospheres containing trace amounts of polycyclic aromatic and aliphatic hydrocarbons. The organics present were concentrated in the aerogels by factors varying from 10 to more than 1000 over the levels found in the atmospheres, depending on the specific organic present. Since silica aerogel is transparent over a wide range of optical and near infrared wavelengths, UV-induced fluorescence, Raman and infrared spectroscopies were used to detect and identify the organics collected by the aerogel. Measurements were conducted to determine the sensitivity of these spectroscopic methods for determining organics concentrated by aerogels and the effectiveness of this method for identifying systems containing multiple organic species. Polycyclic aromatic hydrocarbons (PAHs) were added to simulated Mars regolith and then vaporized by modest heating in the presence of aerogel. The aerogels adsorbed and concentrated the PAHs, which were detected by induced fluorescence and Raman and FTIR spectroscopies.

Electric Dipole Transition Moments and Solvent-Dependent Interactions of Fluorescent Boron-Nitrogen Substituted Indole Derivatives.

PubMed

Saif, Mari; Widom, Julia R; Xu, Senmiao; Abbey, Eric R; Liu, Shih-Yuan; Marcus, Andrew H

2015-06-25

Fluorescent analogues of the indole side chain of tryptophan can be useful spectroscopic probes of protein-protein and protein-DNA interactions. Here we present linear dichroism and solvent-dependent spectroscopic studies of two fluorescent analogues of indole, in which the organic C═C unit is substituted with the isosteric inorganic B-N unit. We studied the so-called "external" BN indole, which has C2v symmetry, and the "fused" BN indole with Cs symmetry. We performed a combination of absorption and fluorescence spectroscopy, ultraviolet linear dichroism (UV-LD) in stretched poly(ethylene) (PE) films, and quantum chemical calculations on both BN indole compounds. Our measurements allowed us to characterize the degree of alignment for both molecules in stretched PE films. We thus determined the orientations and magnitudes of the two lowest energy electric dipole transition moments (EDTMs) for external BN indole, and the two lowest energy EDTMs for fused BN indole within the 30 000-45 000 cm(-1) spectral range. We compared our experimental results to those of quantum chemical calculations using standard density functional theory (DFT). Our theoretical predictions for the low-energy EDTMs are in good agreement with our experimental data. The absorption and fluorescence spectra of the external and the fused BN indoles are sensitive to solvent polarity. Our results indicate that the fused BN indole experiences much greater solvation interactions with polar solvents than does the external BN indole.

A study of the interaction between malachite green and lysozyme by steady-state fluorescence.

PubMed

Ding, Fei; Liu, Wei; Liu, Feng; Li, Zhi-Yuan; Sun, Ying

2009-09-01

The interaction of a N-methylated diaminotriphenylmethane dye, malachite green, with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results revealed that malachite green caused the fluorescence quenching of lysozyme through a static quenching procedure. The thermodynamic parameters like DeltaH and DeltaS were calculated to be -15.33 kJ mol(-1) and 19.47 J mol(-1) K(-1) according to van't Hoff equation, respectively, which proves main interaction between malachite green and lysozyme is hydrophobic forces and hydrogen bond contact. The distance r between donor (lysozyme) and acceptor (malachite green) was obtained to be 3.82 nm according to Frster's theory. The results of synchronous fluorescence, UV/vis and three-dimensional fluorescence spectra showed that binding of malachite green with lysozyme can induce conformational changes in lysozyme. In addition, the effects of common ions on the constants of lysozyme-malachite green complex were also discussed.

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo*

PubMed Central

Beam, Monica; Silva, M. Catarina; Morimoto, Richard I.

2012-01-01

Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity. PMID:22669943

Enumerating virus-like particles in an optically concentrated suspension by fluorescence correlation spectroscopy.

PubMed

Hu, Yi; Cheng, Xuanhong; Daniel Ou-Yang, H

2013-01-01

Fluorescence correlation spectroscopy (FCS) is one of the most sensitive methods for enumerating low concentration nanoparticles in a suspension. However, biological nanoparticles such as viruses often exist at a concentration much lower than the FCS detection limit. While optically generated trapping potentials are shown to effectively enhance the concentration of nanoparticles, feasibility of FCS for enumerating field-enriched nanoparticles requires understanding of the nanoparticle behavior in the external field. This paper reports an experimental study that combines optical trapping and FCS to examine existing theoretical predictions of particle concentration. Colloidal suspensions of polystyrene (PS) nanospheres and HIV-1 virus-like particles are used as model systems. Optical trapping energies and statistical analysis are used to discuss the applicability of FCS for enumerating nanoparticles in a potential well produced by a force field.

Simple and cost-effective fluorescent labeling of 5-hydroxymethylcytosine

NASA Astrophysics Data System (ADS)

Shahal, Tamar; Green, Ori; Hananel, Uri; Michaeli, Yael; Shabat, Doron; Ebenstein, Yuval

2016-12-01

The nucleobase 5-hydroxymethylcytosine (5-hmC), a modified form of cytosine, is an important epigenetic mark related to regulation of gene expression. 5-hmC levels are highly dynamic during early development and are modulated during the progression of neurodegenerative disease and cancer. We describe a spectroscopic method for the global quantification of 5-hmC in genomic DNA. This method relies on the enzymatic glucosylation of 5-hmC, followed by a glucose oxidation step that results in the formation of aldehyde moieties that are covalently linked to a fluorescent reporter by oxime ligation. The fluorescence intensity of the labeled sample is directly proportional to its 5-hmC content. We show that this simple and cost-effective technique is suitable for quantification of 5-hmC content in different mouse tissues.

Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

NASA Astrophysics Data System (ADS)

Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

2005-08-01

The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

Absorption and Luminescence Studies of Some Highly Fluorescent Derivatives of Vitamin B1; Solvent and pH Effects

NASA Astrophysics Data System (ADS)

Marciniak, B.; Koput, J.; Kozubek, H.

1990-08-01

The influence of solvent on the UV-visible absorption and luminescence spectra of some highly fluorescent vitamin B1 derivatives, the products of the reaction of N-methylated vitamin B1 with cytidine (I), adenosine (II) and 2-amino-4-methylpyridine (III) is studied. Spectroscopic manifestations of protonation of I and II are also investigated using a semiempirical INDO/S CI method. Singlet and triplet energy levels of the free ion and several protonated species are calculated, and transition energies and oscillator strengths are compared with the experimental spectra. Calculated charge densities on heteroatoms in the ground and excited singlet and triplet states are correlated with changes of the experimental pKa values with excitation. The results for I and II are compared with those for the trimethylated pyrichrominium ion (III) previously studied

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Elucidating the Structure-Reactivity Correlations of Phenothiazine-Based Fluorescent Probes toward ClO.

PubMed

Wang, Shichao; Zhang, Boyu; Wang, Wenjing; Feng, Gang; Yuan, Daqiang; Zhang, Xuanjun

2018-06-07

In this work, with the aim of developing effective molecular probes and investigating the structure-reactivity correlation, a short series of phenothiazine-based fluorescent probes are designed for the detection of ClO - with differing electron push-pull groups. Sensing experiment results and single-crystal X-ray analysis with the aid of time-dependent DFT (TD-DFT) calculations reveal that substituting groups with increasing electron-withdrawing ability can increase the dihedral angle of the phenothiazine moiety and reduce the gap energy of the probes, leading to enhanced reactivity toward ClO - . Both PT1 and PT2 show two-color switching upon detection of ClO - . PT1, with the strong electron-donating group thiophene, shows a fluorescence color switch from salmon to blue. PT2, with a medium electron-donating/accepting group benzothiazole, shows a fluorescence color switch from red to green. However, both PT1 and PT2 show almost no response to ONOO - . Through the introduction of strong electron-withdrawing ketone combined with a cyano group, PT3 shows a cyan emission upon detection of ClO - and weak red emission upon detection of ONOO - . HRMS and 1 H NMR results confirm that PT1 and PT2 have the same sensing mode, in which the divalent sulfur of phenothiazine can be oxidized to sulfoxide by ClO - . Upon reaction with ClO - , PT3 experiences two-step reactions. It is first oxidized into the sulfone structure by ClO - , and then transformed into sulfoxide phenothiazine aldehyde. Upon encountering ONOO - , PT3 changes into an aldehyde structure and some nonfluorescent byproducts. Owing to their special selectivity and high sensitivity, PT1 and PT2 are applied to image the endogenous ClO - in macrophage cells and zebrafish larvae. This study is expected to provide useful guidelines for probe design for various applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Temperature measurements of micro-droplets using pulsed 2-color laser-induced fluorescence with MDR-enhanced energy transfer

NASA Astrophysics Data System (ADS)

Palmer, Johannes; Reddemann, Manuel A.; Kirsch, Valeri; Kneer, Reinhold

2016-12-01

In this work, a new measurement system is presented for studying temperature of micro-droplets by pulsed 2-color laser-induced fluorescence. Pulsed fluorescence excitation allows motion blur suppression and thus simultaneous measurements of droplet size, velocity and temperature. However, high excitation intensities of pulsed lasers lead to morphology-dependent resonances inside micro-droplets, which are accompanied by disruptive stimulated emission. Investigations showed that stimulated emission can be avoided by enhanced energy transfer via an additional dye. The suitability and accuracy of the new pulsed method are verified on the basis of a spectroscopic analysis and comparison to continuously excited 2-color laser-induced fluorescence.

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

NASA Astrophysics Data System (ADS)

Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

Laser-induced fluorescence in the detection of esophageal carcinoma

NASA Astrophysics Data System (ADS)

Wang, Kenneth K.; Gutta, Kumar; Laukka, Mark A.; Densmore, John

1995-01-01

Laser induced fluorescence (LIF) is a technique which can perform an 'optical biopsy' of gastrointestinal mucosa. LIF was performed in resected specimens using a pulsed N2-laser coupled fiberoptically to a probe. Fluorescence was measured using a 0.2 meter spectroscope with an intensified photodiode array. Measurements were made on fresh (<30 minutes after resection) esophageal specimens containing normal mucosa, Barrett's esophagus, and adenocarcinoma. Each tissue section was examined using an optical probe consisting of a central fiber for delivering the excitation energy and a 6 fiber bundle surrounding the central fiber for detection of the fluorescence. An excitation wavelength of 337 nm was used which generated 3-ns pulses while fluorescence intensities were acquired from 300-800 nm. Spectra were obtained from each section in a standardized fashion and background spectra subtracted. Fluorescence readings were taken from 54 normal esophageal sections and 32 sections of adenocarcinoma. A fluorescence index obtained from the tumor sections was 0.68+/- 0.01 compared with 0.51+/- 0.01 for the normal sections (p<0.001). Using a discriminant value of 0.65, this technique had a sensitivity of 81% and a specificity of 100% for detection of malignant tissue. The positive predictive value was 100% and the negative predictive value was 90% for an overall accuracy of 93%. LIF is a promising technique which has the capability of distinguishing normal versus malignant tissue in the esophagus with good accuracy.

Modeling Carbon Dioxide Vibrational Frequencies in Ionic Liquids: II. Spectroscopic Map.

PubMed

Daly, Clyde A; Berquist, Eric J; Brinzer, Thomas; Garrett-Roe, Sean; Lambrecht, Daniel S; Corcelli, Steven A

2016-12-15

The primary challenge for connecting molecular dynamics (MD) simulations to linear and two-dimensional infrared measurements is the calculation of the vibrational frequency for the chromophore of interest. Computing the vibrational frequency at each time step of the simulation with a quantum mechanical method like density functional theory (DFT) is generally prohibitively expensive. One approach to circumnavigate this problem is the use of spectroscopic maps. Spectroscopic maps are empirical relationships that correlate the frequency of interest to properties of the surrounding solvent that are readily accessible in the MD simulation. Here, we develop a spectroscopic map for the asymmetric stretch of CO 2 in the 1-butyl-3-methylimidazolium hexafluorophosphate ([C 4 C 1 im][PF 6 ]) ionic liquid (IL). DFT is used to compute the vibrational frequency of 500 statistically independent CO 2 -[C 4 C 1 im][PF 6 ] clusters extracted from an MD simulation. When the map was tested on 500 different CO 2 -[C 4 C 1 im][PF 6 ] clusters, the correlation coefficient between the benchmark frequencies and the predicted frequencies was R = 0.94, and the root-mean-square error was 2.7 cm -1 . The calculated distribution of frequencies also agrees well with experiment. The spectroscopic map required information about the CO 2 angle, the electrostatics of the surrounding solvent, and the Lennard-Jones interaction between the CO 2 and the IL. The contribution of each term in the map was investigated using symmetry-adapted perturbation theory calculations.

Automatic classification of fluorescence and optical diffusion spectroscopy data in neuro-oncology

NASA Astrophysics Data System (ADS)

Savelieva, T. A.; Loshchenov, V. B.; Goryajnov, S. A.; Potapov, A. A.

2018-04-01

The complexity of the biological tissue spectroscopic analysis due to the overlap of biological molecules' absorption spectra, multiple scattering effect, as well as measurement geometry in vivo has caused the relevance of this work. In the neurooncology the problem of tumor boundaries delineation is especially acute and requires the development of new methods of intraoperative diagnosis. Methods of optical spectroscopy allow detecting various diagnostically significant parameters non-invasively. 5-ALA induced protoporphyrin IX is frequently used as fluorescent tumor marker in neurooncology. At the same time analysis of the concentration and the oxygenation level of haemoglobin and significant changes of light scattering in tumor tissues have a high diagnostic value. This paper presents an original method for the simultaneous registration of backward diffuse reflectance and fluorescence spectra, which allows defining all the parameters listed above simultaneously. The clinical studies involving 47 patients with intracranial glial tumors of II-IV Grades were carried out in N.N. Burdenko National Medical Research Center of Neurosurgery. To register the spectral dependences the spectroscopic system LESA- 01-BIOSPEC was used with specially developed w-shaped diagnostic fiber optic probe. The original algorithm of combined spectroscopic signal processing was developed. We have created a software and hardware, which allowed (as compared with the methods currently used in neurosurgical practice) to increase the sensitivity of intraoperative demarcation of intracranial tumors from 78% to 96%, specificity of 60% to 82%. The result of analysis of different techniques of automatic classification shows that in our case the most appropriate is the k Nearest Neighbors algorithm with cubic metrics.

White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX

NASA Astrophysics Data System (ADS)

Flynn, Brendan P.; DSouza, Alisha V.; Kanick, Stephen C.; Davis, Scott C.; Pogue, Brian W.

2013-04-01

Subsurface fluorescence imaging is desirable for medical applications, including protoporphyrin-IX (PpIX)-based skin tumor diagnosis, surgical guidance, and dosimetry in photodynamic therapy. While tissue optical properties and heterogeneities make true subsurface fluorescence mapping an ill-posed problem, ultrasound-guided fluorescence-tomography (USFT) provides regional fluorescence mapping. Here USFT is implemented with spectroscopic decoupling of fluorescence signals (auto-fluorescence, PpIX, photoproducts), and white light spectroscopy-determined bulk optical properties. Segmented US images provide a priori spatial information for fluorescence reconstruction using region-based, diffuse FT. The method was tested in simulations, tissue homogeneous and inclusion phantoms, and an injected-inclusion animal model. Reconstructed fluorescence yield was linear with PpIX concentration, including the lowest concentration used, 0.025 μg/ml. White light spectroscopy informed optical properties, which improved fluorescence reconstruction accuracy compared to the use of fixed, literature-based optical properties, reduced reconstruction error and reconstructed fluorescence standard deviation by factors of 8.9 and 2.0, respectively. Recovered contrast-to-background error was 25% and 74% for inclusion phantoms without and with a 2-mm skin-like layer, respectively. Preliminary mouse-model imaging demonstrated system feasibility for subsurface fluorescence measurement in vivo. These data suggest that this implementation of USFT is capable of regional PpIX mapping in human skin tumors during photodynamic therapy, to be used in dosimetric evaluations.

Dynamics of hybrid amoeba proteus containing zoochlorellae studied using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Liu, C.-H.; Fong, B. A.; Alfano, S. A., Jr.; Rakhlin, I.; Wang, W. B.; Ni, X. H.; Yang, Y. L.; Zhou, F.; Zuzolo, R. C.; Alfano, R. R.

2011-03-01

The microinjection of organelles, plants, particles or chemical solutions into Amoeba proteus coupled with spectroscopic analysis and observed for a period of time provides a unique new model for cancer treatment and studies. The amoeba is a eukaryote having many similar features of mammalian cells. The amoeba biochemical functions monitored spectroscopically can provide time sequence in vivo information about many metabolic transitions and metabolic exchanges between cellar organelles and substances microinjected into the amoeba. It is possible to microinject algae, plant mitochondria, drugs or carcinogenic solutions followed by recording the native fluorescence spectra of these composites. This model can be used to spectroscopically monitor the pre-metabolic transitions in developing diseased cells such as a cancer. Knowing specific metabolic transitions could offer solutions to inhibit cancer or reverse it as well as many other diseases. In the present study a simple experiment was designed to test the feasibility of this unique new model by injecting algae and chloroplasts into amoeba. The nonradiative dynamics found from these composites are evidence in terms of the emission ratios between the intensities at 337nm and 419nm; and 684nm bands. There were reductions in the metabolic and photosynthetic processes in amoebae that were microinjected with chloroplasts and zoochlorellae as well of those amoebae that ingested the algae and chloroplasts. The changes in the intensity of the emissions of the peaks indicate that the zoochlorellae lived in the amoebae for ten days. Spectral changes in intensity under the UV and 633nm wavelength excitation are from the energy transfer of DNA and RNA, protein-bound chromophores and chlorophylls present in zoochlorellae undergoing photosynthesis. The fluorescence spectroscopic probes established the biochemical interplay between the cell organelles and the algae present in the cell cytoplasm. This hybrid state is indicative

Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants.

PubMed

Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

2004-12-01

It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a nonfluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a nonfluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

Raman spectroscopic signature of vaginal fluid and its potential application in forensic body fluid identification.

PubMed

Sikirzhytskaya, Aliaksandra; Sikirzhytski, Vitali; Lednev, Igor K

2012-03-10

Traces of human body fluids, such as blood, saliva, sweat, semen and vaginal fluid, play an increasingly important role in forensic investigations. However, a nondestructive, easy and rapid identification of body fluid traces at the scene of a crime has not yet been developed. The obstacles have recently been addressed in our studies, which demonstrated the considerable potential of Raman spectroscopy. In this study, we continued to build a full library of body fluid spectroscopic signatures. The problems concerning vaginal fluid stain identification were addressed using Raman spectroscopy coupled with advanced statistical analysis. Calculated characteristic Raman and fluorescent spectral components were used to build a multidimensional spectroscopic signature of vaginal fluid, which demonstrated good specificity and was able to handle heterogeneous samples from different donors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

Glutamate receptors as seen by light: Spectroscopic studies of structure-function relationships

PubMed Central

Mankiewicz, Kimberly A.; Jayaraman, Vasanthi

2010-01-01

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand binding domain and subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction of the insight gained from X-ray crystallography and nuclear magnetic resonance (NMR) investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer (FRET) to study the behavior of the isolated ligand binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation. PMID:17934637

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

PubMed Central

Dedecker, Peter

2017-01-01

Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. PMID:28930199

Biophysical influence of isocarbophos on bovine serum albumin: Spectroscopic probing

NASA Astrophysics Data System (ADS)

Zhang, Hua-xin; Zhou, Ying; Liu, E.

Isocarbophos (ICP) is a phosphorous pesticide with high toxicity. It has been detected in several kinds of food and therefore can enter human body. In this paper, spectroscopic approaches including three-dimensional fluorescence (3D-FL) spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy were employed to explore the binding of ICP to bovine serum albumin (BSA) at simulated physiological conditions. It was found that the fluorescence quenching of BSA was caused by the formation of ICP-BSA complex at ground state and belonged to static quenching mechanism. The binding constants, the number of binding sites, enthalpy change (ΔHθ), Gibbs free energy change (ΔGθ) and entropy change (ΔSθ) were calculated at four different temperatures according to Scatchard model and thermodynamic equations. To identify the binding location, fluorescence probe techniques were used. The results showed that warfarin, an acknowledged site marker for BSA, could be partially replaced by ICP when ICP was added to warfarin-BSA systems, which demonstrated that ICP primarily bound on Sudlow's site I in domain IIA of BSA molecule. The distance r (3.06 nm) between donor (Trp-212) and acceptor (ICP) was obtained based on Förster's non-radiation fluorescence resonance energy transfer (FRET) theory. Furthermore, the CD spectral results indicated that the secondary structure of BSA was changed in presence of ICP. The study is helpful to evaluating the toxicology of ICP and understanding its effects on the function of protein during the blood transportation process.

Further Insights into Metal-DOM Interaction: Consideration of Both Fluorescent and Non-Fluorescent Substances

PubMed Central

Xu, Huacheng; Zhong, Jicheng; Yu, Guanghui; Wu, Jun; Jiang, Helong; Yang, Liuyan

2014-01-01

Information on metal binding with fluorescent substances has been widely studied. By contrast, information on metal binding with non-fluorescent substances remains lacking despite the dominance of these substances in aquatic systems. In this study, the metal binding properties of both fluorescent and non-fluorescent substances were investigated by using metal titration combined with two-dimensional correlation spectroscopy (2D–COS) analysis. The organic matters in the eutrophic algae-rich lake, including natural organic matters (NOM) and algae-induced extracellular polymeric substances (EPS), both contained fluorescent and non-fluorescent substances. The peaks in the one-dimensional spectra strongly overlapped, while 2D–COS can decompose the overlapped peaks and thus enhanced the spectral resolution. Moreover, 2D FTIR COS demonstrated that the binding susceptibility of organic ligands in both NOM and algal EPS matrices followed the order: 3400>1380>1650 cm−1, indicative the significant contribution of non-fluorescent ligands in metal binding. The modified Stern-Volmer equation also revealed a substantial metal binding potential for the non-fluorescent substances (logKM: 3.57∼4.92). As for the effects of organic ligands on metal binding, EPS was characterized with higher binding ability than NOM for both fluorescent and non-fluorescent ligands. Algae-induced EPS and the non-fluorescent substances in eutrophic algae-rich lakes should not be overlooked because of their high metal binding potential. PMID:25380246

Fluorescence lifetime imaging ophthalmoscopy.

PubMed

Dysli, Chantal; Wolf, Sebastian; Berezin, Mikhail Y; Sauer, Lydia; Hammer, Martin; Zinkernagel, Martin S

2017-09-01

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Recognition of Mg²⁺ by a new fluorescent "turn-on" chemosensor based on pyridyl-hydrazono-coumarin.

PubMed

Orrego-Hernández, Jessica; Nuñez-Dallos, Nelson; Portilla, Jaime

2016-05-15

A new fluoroionophore PyHC bearing 2-pyridylhydrazone and 7-hydroxycoumarin moieties for selective detection of Mg(2+) was synthesized and characterized. This chemosensor exhibited "turn-on" fluorescence behavior and was sensitive to Mg(2+) concentrations as low as 105 nmol L(-1) in ethanol-water solution. Detailed spectroscopic studies revealed the binding mode of a 1:1 complex between PyHC and Mg(2+) that leads to a fluorescence enhancement. Copyright © 2016 Elsevier B.V. All rights reserved.

25 ns software correlator for photon and fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Magatti, Davide; Ferri, Fabio

2003-02-01

A 25 ns time resolution, multi-tau software correlator developed in LABVIEW based on the use of a standard photon counting unit, a fast timer/counter board (6602-PCI National Instrument) and a personal computer (PC) (1.5 GHz Pentium 4) is presented and quantitatively discussed. The correlator works by processing the stream of incoming data in parallel according to two different algorithms: For large lag times (τ⩾100 μs), a classical time-mode (TM) scheme, based on the measure of the number of pulses per time interval, is used; differently, for τ⩽100 μs a photon-mode (PM) scheme is adopted and the time sequence of the arrival times of the photon pulses is measured. By combining the two methods, we developed a system capable of working out correlation functions on line, in full real time for the TM correlator and partially in batch processing for the PM correlator. For the latter one, the duty cycle depends on the count rate of the incoming pulses, being ˜100% for count rates ⩽3×104 Hz, ˜15% at 105 Hz, and ˜1% at 106 Hz. For limitations imposed by the fairly small first-in, first-out (FIFO) buffer available on the counter board, the maximum count rate permissible for a proper functioning of the PM correlator is limited to ˜105 Hz. However, this limit can be removed by using a board with a deeper FIFO. Similarly, the 25 ns time resolution is only limited by maximum clock frequency available on the 6602-PCI and can be easily improved by using a faster clock. When tested on dilute solutions of calibrated latex spheres, the overall performances of the correlator appear to be comparable with those of commercial hardware correlators, but with several nontrivial advantages related to its flexibility, low cost, and easy adaptability to future developments of PC and data acquisition technology.

Plant stress detection by remote measurement of fluorescence

USGS Publications Warehouse

McFarlane, J. C.; Watson, Robert D.; Theisen, Arnold F.; Jackson, R. D.; Ehrler, W. L.; Pinter, P. J.; Idso, S. B.; Reginato, R. J.

1980-01-01

Chlorophyll fluorescence of mature lemon trees was measured with a Fraunhofer line discriminator (FLD). An increase in fluorescence was correlated with plant water stress as measured by stomatal resistance and twig water potential.

Pathological changes in Alzheimer"s brain evaluated with fluorescence emission analysis (FEA)

NASA Astrophysics Data System (ADS)

Christov, Alexander; Ottman, Todd; Grammas, Paula

2004-07-01

Development of AD is associated with cerebrovascular deposition of amyloid beta (Aβ) as well as a progressive increase in vasular collagen content. Both AΒ and collagen are naturally fluorescent compounds when exposed to UV light. We analyzed autofluorescence emitted from brain tissue samples and isolated brain resistance vessels harvested postmortem from patients with Alzheimer's disease (AD) and age-matched controls. Fluorescence emission, excited at 355 nm with an Nd:YAG laser, was measured using a fiber-optic based fluorescence spectroscopic system for tissue analysis. Significantly higher values of fluorescence emission intensity (P<0.001) in the spectral region from 465 to 490 nm were detected in brain resistance vessel samples from AD patients compared to the normal individuals. Results from western blot analysis showed elevated levels of type I and type III collagen, and reduced levels of type IV collagen in resistance vessels from AD patients, compared to control samples. In addition, using direct scanning of the cortical suface for fluoresxcence emission by the laser-induced fluorescence spectroscopy system we detected a significantly (P<0.05) higher level of apoptosis in AD brain tissue compared to age-matched controls. Fluorescence emission analysis (FEA) appears to be a sensitive technique for detecting structural changes in AD brain tissue.

Assessment of the unidentified organic matter fraction in fogwater using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Valsaraj, K.; Birdwell, J.

2010-07-01

Dissolved organic matter (DOM) in fogwaters from southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix (EEM) fluorescence spectroscopy. The results demonstrate that fluorescence spectroscopy can be used to obtain a qualitative assessment of the large fraction of fogwater organic carbon (~40 - 80% by weight) that cannot be identified in terms of specific chemical compounds. The method has the principle advantage that it can be applied at natural abundance concentrations, thus eliminating the need for large sample volumes required to isolate DOM for characterization by other spectroscopic (NMR, FTIR) and chemical (elemental) analyses. It was anticipated that the fogwater organic matter fluorescence spectra would resemble those of surface and rain waters, containing peaks indicative of both humic substances and fluorescent amino acids. Humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices had values comparable to other natural waters. Biological character (intensity of tyrosine and tryptophan peaks) was found to increase with organic carbon concentration. Fogwater organic matter appears to contain a mixture of terrestrially- and microbially-derived material. The fluorescence results show that most of the unidentified fogwater organic carbon can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems.

Studies of interaction of emodin and DNA in the presence of ethidium bromide by spectroscopic method

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Zhang, Hanqi; Qiao, Chunyu; Sun, Ying; Liu, Chunming

2008-01-01

Emodin interacting with deoxyribonucleic acid (DNA) has been studied by different spectroscopic techniques, such as fluorescence, ultraviolet and visible (UV-vis), and fourier transform infared (FT-IR) spectroscopies, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of emodin shows that the fluorescence quenching of DNA-EB complex by emodin occurs. The binding constants of emodin with DNA in the presence of EB are 6.02 × 10 4, 9.20 × 10 4 and 1.17 × 10 5 L mol -1 at 20, 35 and 50 °C, respectively. FT-IR spectrum further suggests that both the phosphate groups and the bases of DNA react with emodin. The reaction of DNA with emodin in the presence of EB is affected by ionic strength and temperature. The values of melting temperature ( Tm) of DNA-EB complex and emodin-DNA-EB complexes were determined, respectively. From the experiment evidences, the major binding mode of emodin with DNA should be the groove binding.

Surface Transient Binding-Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of the Time Window to Seconds.

PubMed

Peng, Sijia; Wang, Wenjuan; Chen, Chunlai

2018-05-10

Fluorescence correlation spectroscopy is a powerful single-molecule tool that is able to capture kinetic processes occurring at the nanosecond time scale. However, the upper limit of its time window is restricted by the dwell time of the molecule of interest in the confocal detection volume, which is usually around submilliseconds for a freely diffusing biomolecule. Here, we present a simple and easy-to-implement method, named surface transient binding-based fluorescence correlation spectroscopy (STB-FCS), which extends the upper limit of the time window to seconds. We further demonstrated that STB-FCS enables capture of both intramolecular and intermolecular kinetic processes whose time scales cross several orders of magnitude.

Time-resolved resonance fluorescence spectroscopy for study of chemical reactions in laser-induced plasmas.

PubMed

Liu, Lei; Deng, Leimin; Fan, Lisha; Huang, Xi; Lu, Yao; Shen, Xiaokang; Jiang, Lan; Silvain, Jean-François; Lu, Yongfeng

2017-10-30

Identification of chemical intermediates and study of chemical reaction pathways and mechanisms in laser-induced plasmas are important for laser-ablated applications. Laser-induced breakdown spectroscopy (LIBS), as a promising spectroscopic technique, is efficient for elemental analyses but can only provide limited information about chemical products in laser-induced plasmas. In this work, time-resolved resonance fluorescence spectroscopy was studied as a promising tool for the study of chemical reactions in laser-induced plasmas. Resonance fluorescence excitation of diatomic aluminum monoxide (AlO) and triatomic dialuminum monoxide (Al 2 O) was used to identify these chemical intermediates. Time-resolved fluorescence spectra of AlO and Al 2 O were used to observe the temporal evolution in laser-induced Al plasmas and to study their formation in the Al-O 2 chemistry in air.

“Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

PubMed Central

2015-01-01

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2–11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues. PMID:25534273

“Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

DOE PAGES

Yapici, Ipek; Lee, Kin Sing Stephen; Berbasova, Tetyana; ...

2014-12-22

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2$-$11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observedmore » spectroscopic behavior that results from single mutation of key residues.« less

A detailed spectroscopic study on the interaction of Rhodamine 6G with human hemoglobin.

PubMed

Mandal, Paulami; Bardhan, Munmun; Ganguly, Tapan

2010-05-03

UV-vis, time-resolved fluorescence and circular dichroism spectroscopic investigations have been made to reveal the nature of the interactions between xanthene dye Rhodamine 6G and the well known protein hemoglobin. From the analysis of the steady-state and time-resolved fluorescence quenching of Rhodamine 6G in aqueous solutions in presence of hemoglobin, it is revealed that the quenching is static in nature. The primary binding pattern between Rhodamine and hemoglobin has been interpreted as combined effect of hydrophobic association and electrostatic interaction. The binding constants, number of binding sites and thermodynamic parameters at various pH of the environment have been computed. The binding average distance between the energy donor Rhodamine and acceptor hemoglobin has been determined from the Forster's theory. Copyright 2010 Elsevier B.V. All rights reserved.

Updating a synchronous fluorescence spectroscopic virgin olive oil adulteration calibration to a new geographical region.

PubMed

Kunz, Matthew Ross; Ottaway, Joshua; Kalivas, John H; Georgiou, Constantinos A; Mousdis, George A

2011-02-23

Detecting and quantifying extra virgin olive adulteration is of great importance to the olive oil industry. Many spectroscopic methods in conjunction with multivariate analysis have been used to solve these issues. However, successes to date are limited as calibration models are built to a specific set of geographical regions, growing seasons, cultivars, and oil extraction methods (the composite primary condition). Samples from new geographical regions, growing seasons, etc. (secondary conditions) are not always correctly predicted by the primary model due to different olive oil and/or adulterant compositions stemming from secondary conditions not matching the primary conditions. Three Tikhonov regularization (TR) variants are used in this paper to allow adulterant (sunflower oil) concentration predictions in samples from geographical regions not part of the original primary calibration domain. Of the three TR variants, ridge regression with an additional 2-norm penalty provides the smallest validation sample prediction errors. Although the paper reports on using TR for model updating to predict adulterant oil concentration, the methods should also be applicable to updating models distinguishing adulterated samples from pure extra virgin olive oil. Additionally, the approaches are general and can be used with other spectroscopic methods and adulterants as well as with other agriculture products.

VizieR Online Data Catalog: Spectroscopic analysis of 348 red giants (Zielinski+, 2012)

NASA Astrophysics Data System (ADS)

Zielinski, P.; Niedzielski, A.; Wolszczan, A.; Adamow, M.; Nowak, G.

2012-10-01

The atmospheric parameters were derived using a strictly spectroscopic method based on the LTE analysis of equivalent widths of FeI and FeII lines. With existing photometric data and the Hipparcos parallaxes, we estimated stellar masses and ages via evolutionary tracks fitting. The stellar radii were calculated from either estimated masses and the spectroscopic logg or from the spectroscopic Teff and estimated luminosities. The absolute radial velocities were obtained by cross-correlating spectra with a numerical template. Our high-quality, high-resolution optical spectra have been collected since 2004 with the Hobby-Eberly Telescope (HET), located in the McDonald Observatory. The telescope was equipped with the High Resolution Spectrograph (HRS; R~60000 resolution). (2 data files).

Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange

NASA Astrophysics Data System (ADS)

Hansda, Chaitali; Chakraborty, Utsav; Hussain, Syed Arshad; Bhattacharjee, Debajyoti; Paul, Pabitra Kumar

2016-03-01

Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π-π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV-Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe.

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Fluorescence Spectrum and Decay Measurement for Hsil VS Normal Cytology Differentiation in Liquid Pap Smear Supernatant

NASA Astrophysics Data System (ADS)

Vaitkuviene, A.; Gegzna, V.; Juodkazis, S.; Jursenas, S.; Miasojedovas, S.; Kurtinaitiene, R.; Rimiene, J.; Vaitkus, J.

2009-06-01

Cervical smear material contains endo and exocervical cells, mucus and inflammative, immune cells in cases of pathology. Just not destroyed keratinocytes lay on the glass for microscopy. Liquid cytology supernatant apart other diagnostics could be used for photodiagnostic. The spectroscopic parameters suitable for Normal and HSIL cytology groups supernatant differentiation are demonstrated. The dried liquid PAP supernatant fractions—sediment and liquid were investigated. Excitation and emission matrices (EEM), supernatant fluorescence decay measured under 280 nm diode short pulse excitation and fluorescence spectroscopy by excitation with 355 nm laser light were analyzed. The differences between Normal and HSIL groups were statistically proven in the certain spectral regions. Fluorescence decay peculiarities show spectral regions consisting of few fluorophores. Obtained results on fluorescence differences in Normal and HSIL groups' supernatant shows the potency of photodiagnosis application in cervical screening.

Analytical Raman spectroscopic study for discriminant analysis of different animal-derived feedstuff: Understanding the high correlation between Raman spectroscopy and lipid characteristics.

PubMed

Gao, Fei; Xu, Lingzhi; Zhang, Yuejing; Yang, Zengling; Han, Lujia; Liu, Xian

2018-02-01

The objectives of the current study were to explore the correlation between Raman spectroscopy and lipid characteristics and to assess the potential of Raman spectroscopic methods for distinguishing the different sources of animal-originated feed based on lipid characteristics. A total of 105 lipid samples derived from five animal species have been analyzed by gas chromatography (GC) and FT-Raman spectroscopy. High correlations (r 2 >0.94) were found between the characteristic peak ratio of the Raman spectra (1654/1748 and 1654/1445) and the degree of unsaturation of the animal lipids. The results of FT-Raman data combined with chemometrics showed that the fishmeal, poultry, porcine and ruminant (bovine and ovine) MBMs could be well separated based on their lipid spectral characteristics. This study demonstrated that FT-Raman spectroscopy can mostly exhibit the lipid structure specificity of different species of animal-originated feed and can be used to discriminate different animal-originated feed samples. Copyright © 2017. Published by Elsevier Ltd.

Light emitting diode excitation emission matrix fluorescence spectroscopy.

PubMed

Hart, Sean J; JiJi, Renée D

2002-12-01

An excitation emission matrix (EEM) fluorescence instrument has been developed using a linear array of light emitting diodes (LED). The wavelengths covered extend from the upper UV through the visible spectrum: 370-640 nm. Using an LED array to excite fluorescence emission at multiple excitation wavelengths is a low-cost alternative to an expensive high power lamp and imaging spectrograph. The LED-EEM system is a departure from other EEM spectroscopy systems in that LEDs often have broad excitation ranges which may overlap with neighboring channels. The LED array can be considered a hybrid between a spectroscopic and sensor system, as the broad LED excitation range produces a partially selective optical measurement. The instrument has been tested and characterized using fluorescent dyes: limits of detection (LOD) for 9,10-bis(phenylethynyl)-anthracene and rhodamine B were in the mid parts-per-trillion range; detection limits for the other compounds were in the low parts-per-billion range (< 5 ppb). The LED-EEMs were analyzed using parallel factor analysis (PARAFAC), which allowed the mathematical resolution of the individual contributions of the mono- and dianion fluorescein tautomers a priori. Correct identification and quantitation of six fluorescent dyes in two to six component mixtures (concentrations between 12.5 and 500 ppb) has been achieved with root mean squared errors of prediction (RMSEP) of less than 4.0 ppb for all components.

Optical fluorescence biosensor for plant water stress detection

NASA Astrophysics Data System (ADS)

Chong, Jenny P. C.; Liew, O. W.; Li, B. Q.; Asundi, A. K.

2007-05-01

Precision farming in arable agriculture and horticulture allows conservative use of resources that are applied according to plant needs. The growing concern for sustainability in crop production has accentuated the significance of our work to develop a rapid, sensitive and non-destructive spectroscopic method for real-time monitoring of plant water stress. Elucidation of crop water status before the onset of irreversible cellular damage is critical for effective water management to ensure maximum crop yield and profit margin. A two-component bio-sensing system comprising transgenic 'Indicator Plants' and a spectrometer-linked stereoscopic microscope was developed to detect early signs of water stress before the permanent wilting point is reached. The 'Indicator Plants' are transgenic Petunia hybrida genetically engineered with a drought-responsive promoter-linked enhanced green fluorescent protein marker gene (EGFP). No EGFP fluorescence was detected prior to induction of dehydration stress. Fluorescence emission intensity increased with dehydration period and was found mainly in the stems, leaf veins and leaf tips. While fluorescence emission above endogenous background was detectable after 2 hours of water stress treatment, the plants reached permanent wilting point after 6 hours, showing that our system was able to detect water stress prior to plant entry into the stage of irreversible damage. Future work will be geared towards overcoming biological and instrument-related difficulties encountered in our initial detection system.

Synthesis, characterization and spectroscopic behavior of novel 2-oxo-1,4-disubstituted-1,2,5,6-tetrahydrobenzo[h]quinoline-3-carbonitrile dyes.

PubMed

Khan, Salman A; Asiri, Abdullah M; Al-Thaqafy, Saad H; Faidallah, Hassan M; El-Daly, Samy A

2014-12-10

Two synthetic pathways were adopted to synthesize the target 2-oxo-1,4-disubstituted-1,2,5,6-tetrahydro-benzo[h]quinoline-3-carbonitriles. Structure of the synthesized compounds has been characterized based on FT-IR, (1)H NMR, (13)C NMR and elemental analyses. UV-Vis and fluorescence spectroscopy measurements provided that all compounds are good absorbent and fluorescent. Fluorescence polarity study demonstrated that these compounds were sensitive to the polarity of the microenvironment provided by different solvents. In addition, spectroscopic and physicochemical parameters, including singlet absorption, extinction coefficient, Stokes shift, oscillator strength and dipole moment were investigated in order to explore the analytical potential of synthesized compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

PubMed Central

Vogt, Alexander; D'Angelo, Cecilia; Oswald, Franz; Denzel, Andrea; Mazel, Charles H.; Matz, Mikhail V.; Ivanchenko, Sergey; Nienhaus, G. Ulrich; Wiedenmann, Jörg

2008-01-01

A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ∼15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins. PMID:19018285

Differences in fluorescence profiles from breast cancer tissues due to changes in relative tryptophan content via energy transfer: tryptophan content correlates with histologic grade and tumor size but not with lymph node metastases

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, Robert R.

2014-12-01

The correlation between histologic grade, an increasingly important measure of prognosis for patients with breast cancer, and tryptophan levels from tissues of 15 breast carcinoma patients was investigated. Changes in the relative content of key native organic biomolecule tryptophan were seen from the fluorescence spectra of cancerous and paired normal tissues with excitation wavelengths of 280 and 300 nm. Due to a large spectral overlap and matching excitation-emission spectra, fluorescence resonance energy transfer from tryptophan-donor to reduced nicotinamide adenine dinucleotides-acceptor was noted. We used the ratios of fluorescence intensities at their spectral emission peaks, or spectral fingerprint peaks, at 340, 440, and 460 nm. Higher ratios correlated strongly with high histologic grade, while lower-grade tumors had low ratios. Large tumor size also correlated with high ratios, while the number of lymph node metastases, a major factor in staging, was not correlated with tryptophan levels. High histologic grade correlates strongly with increased content of tryptophan in breast cancer tissues and suggests that measurement of tryptophan content may be useful as a part of the evaluation of these patients.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.

PubMed

Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

2014-10-10

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. Copyright © 2014 Elsevier Inc. All rights reserved.

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bavari, Sina; Kaplan, Gerardo

2018-04-01

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials. Published by Elsevier B.V.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy.

PubMed

Sun, Yang; Park, Jesung; Stephens, Douglas N; Jo, Javier A; Sun, Lei; Cannata, Jonathan M; Saroufeem, Ramez M G; Shung, K Kirk; Marcu, Laura

2009-06-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy

PubMed Central

Sun, Yang; Park, Jesung; Stephens, Douglas N.; Jo, Javier A.; Sun, Lei; Cannata, Jonathan M.; Saroufeem, Ramez M. G.; Shung, K. Kirk; Marcu, Laura

2009-01-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 μm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque. PMID:19566223

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy

NASA Astrophysics Data System (ADS)

Sun, Yang; Park, Jesung; Stephens, Douglas N.; Jo, Javier A.; Sun, Lei; Cannata, Jonathan M.; Saroufeem, Ramez M. G.; Shung, K. Kirk; Marcu, Laura

2009-06-01

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 μm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

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.

Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

NASA Astrophysics Data System (ADS)

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

2016-02-01

Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

NASA Astrophysics Data System (ADS)

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

2015-12-01

Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

Application of correlation constrained multivariate curve resolution alternating least-squares methods for determination of compounds of interest in biodiesel blends using NIR and UV-visible spectroscopic data.

PubMed

de Oliveira, Rodrigo Rocha; de Lima, Kássio Michell Gomes; Tauler, Romà; de Juan, Anna

2014-07-01

This study describes two applications of a variant of the multivariate curve resolution alternating least squares (MCR-ALS) method with a correlation constraint. The first application describes the use of MCR-ALS for the determination of biodiesel concentrations in biodiesel blends using near infrared (NIR) spectroscopic data. In the second application, the proposed method allowed the determination of the synthetic antioxidant N,N'-Di-sec-butyl-p-phenylenediamine (PDA) present in biodiesel mixtures from different vegetable sources using UV-visible spectroscopy. Well established multivariate regression algorithm, partial least squares (PLS), were calculated for comparison of the quantification performance in the models developed in both applications. The correlation constraint has been adapted to handle the presence of batch-to-batch matrix effects due to ageing effects, which might occur when different groups of samples were used to build a calibration model in the first application. Different data set configurations and diverse modes of application of the correlation constraint are explored and guidelines are given to cope with different type of analytical problems, such as the correction of matrix effects among biodiesel samples, where MCR-ALS outperformed PLS reducing the relative error of prediction RE (%) from 9.82% to 4.85% in the first application, or the determination of minor compound with overlapped weak spectroscopic signals, where MCR-ALS gave higher (RE (%)=3.16%) for prediction of PDA compared to PLS (RE (%)=1.99%), but with the advantage of recovering the related pure spectral profile of analytes and interferences. The obtained results show the potential of the MCR-ALS method with correlation constraint to be adapted to diverse data set configurations and analytical problems related to the determination of biodiesel mixtures and added compounds therein. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Calibration method for spectroscopic systems

DOEpatents

Sandison, David R.

1998-01-01

Calibration spots of optically-characterized material placed in the field of view of a spectroscopic system allow calibration of the spectroscopic system. Response from the calibration spots is measured and used to calibrate for varying spectroscopic system operating parameters. The accurate calibration achieved allows quantitative spectroscopic analysis of responses taken at different times, different excitation conditions, and of different targets.

Calibration method for spectroscopic systems

DOEpatents

Sandison, D.R.

1998-11-17

Calibration spots of optically-characterized material placed in the field of view of a spectroscopic system allow calibration of the spectroscopic system. Response from the calibration spots is measured and used to calibrate for varying spectroscopic system operating parameters. The accurate calibration achieved allows quantitative spectroscopic analysis of responses taken at different times, different excitation conditions, and of different targets. 3 figs.

Real-time autocorrelator for fluorescence correlation spectroscopy based on graphical-processor-unit architecture: method, implementation, and comparative studies

NASA Astrophysics Data System (ADS)

Laracuente, Nicholas; Grossman, Carl

2013-03-01

We developed an algorithm and software to calculate autocorrelation functions from real-time photon-counting data using the fast, parallel capabilities of graphical processor units (GPUs). Recent developments in hardware and software have allowed for general purpose computing with inexpensive GPU hardware. These devices are more suited for emulating hardware autocorrelators than traditional CPU-based software applications by emphasizing parallel throughput over sequential speed. Incoming data are binned in a standard multi-tau scheme with configurable points-per-bin size and are mapped into a GPU memory pattern to reduce time-expensive memory access. Applications include dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) experiments. We ran the software on a 64-core graphics pci card in a 3.2 GHz Intel i5 CPU based computer running Linux. FCS measurements were made on Alexa-546 and Texas Red dyes in a standard buffer (PBS). Software correlations were compared to hardware correlator measurements on the same signals. Supported by HHMI and Swarthmore College

A versatile in situ spectroscopic cell for fluorescence/transmission EXAFS and X-ray diffraction of heterogeneous catalysts in gas and liquid phase.

PubMed

Hannemann, Stefan; Casapu, Maria; Grunwaldt, Jan Dierk; Haider, Peter; Trüssel, Philippe; Baiker, Alfons; Welter, Edmund

2007-07-01

A new spectroscopic cell suitable for the analysis of heterogeneous catalysts by fluorescence EXAFS (extended X-ray absorption fine structure), transmission EXAFS and X-ray diffraction during in situ treatments and during catalysis is described. Both gas-phase and liquid-phase reactions can be investigated combined with on-line product analysis performed either by mass spectrometry or infrared spectroscopy. The set-up allows measurements from liquid-nitrogen temperature to 973 K. The catalysts are loaded preferentially as powders, but also as self-supporting wafers. The reaction cell was tested in various case studies demonstrating its flexibility and its wide applicability from model studies at liquid-nitrogen temperature to operando studies under realistic reaction conditions. Examples include structural studies during (i) the reduction of alumina-supported noble metal particles prepared by flame-spray pyrolysis and analysis of alloying in bimetallic noble metal particles (0.1%Pt-0.1%Pd/Al(2)O(3), 0.1%Pt-0.1%Ru/Al(2)O(3), 0.1%Pt-0.1%Rh/Al(2)O(3), 0.1%Au-0.1%Pd/Al(2)O(3)), (ii) reactivation of aged 0.8%Pt-16%BaO-CeO(2) NO(x) storage-reduction catalysts including the NO(x) storage/reduction cycle, and (iii) alcohol oxidation over gold catalysts (0.6%Au-20%CuO-CeO(2)).

Improved murine glioma detection following modified diet and photobleaching of skin PpIX fluorescence

NASA Astrophysics Data System (ADS)

Gibbs, Summer L.; O'Hara, Julia A.; Hoopes, P. Jack; Pogue, Brian W.

2007-02-01

The Aminolevulinic Acid (ALA) - Protoporphyrin IX (PpIX) system is unique in the world of photosensitizers in that the prodrug ALA is enzymatically transformed via the tissue of interest into fluorescently detectable levels of PpIX. This system can be used to monitor cellular metabolism of tumor tissue for applications such as therapy monitoring. Detecting PpIX fluorescence noninvasively has proven difficult due to the high levels of PpIX produced in the skin compared to other tissue both with and without ALA administration. In the current study, methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration have been examined. Use of a purified diet is found to decrease both skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration, while addition of a broad spectrum antibiotic to the water shows little effect. Following ALA administration, improved brain tumor detection is seen when skin PpIX fluorescence is photobleached via blue light prior to transmission spectroscopic measurements of tumor bearing and control animals. Both of these methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration are shown to have a large effect on the ability to detect tumor tissue PpIX fluorescence noninvasively in vivo.

In-vivo kinetics of ALA-induced fluorescence in the canine oral cavity: influence of drug dose and tissue type

NASA Astrophysics Data System (ADS)

Vaidyanathan, Vijay; Rastegar, Sohi; Fossum, Theresa W.; Flores, P.; van der Breggen, E. W. J.; Egger, N. G.; Jacques, Steven L.; Motamedi, Massoud

1997-06-01

Fluorescence spectroscopic detection and photodynamic therapy may provide an effective approach for early detection and treatment of oral cancer. Thus the development of a safe photosensitizer that could enhance the spectroscopic contrast between normal and neoplastic tissue, while allowing for selective photosensitization and treatment of pre-malignant and malignant lesions in the oral cavity, is highly desired. In this study, the pharmacokinetics and a safety of 5-aminolevulinic acid (ALA) that could induce an endogenous precursor of protoporphyrin IX and heme in the biosynthetic pathway was investigated. Two doses of ALA:25 and 75 mg/kg were administered intravenously to 4 and 3 dogs, respectively. A 'wash-out' period of 1 week between administration of each does was allowed to ensure against PpIX build-up. Using an optical multichannel analyzer, the fluorescence from the oral cavity was recorded at 3 sites: buccal mucosa, gums, and the tongue, and also from a remote site, the skin. A fiber optic probe was used to deliver excitation and collect the emitted fluorescence. Results showed that the ALA-induced fluorescence reached a peak at 2-4 hours, and returned to baseline in 24-31 hours. The dogs were stable during the course of the study, minimal vomiting was noted. In conclusion, the study showed that higher doses result in a higher peak at a later time.It was observed that different tissues have different pharmacokinetic response, the tongue and the gums have the highest peak fluorescence values, followed by the buccal mucosa and skin.

Uranium(VI) Binding Forms in Selected Human Body Fluids: Thermodynamic Calculations versus Spectroscopic Measurements.

PubMed

Osman, Alfatih A A; Geipel, Gerhard; Barkleit, Astrid; Bernhard, Gert

2015-02-16

Human exposure to uranium increasingly becomes a subject of interest in many scientific disciplines such as environmental medicine, toxicology, and radiation protection. Knowledge about uranium chemical binding forms(speciation) in human body fluids can be of great importance to understand not only its biokinetics but also its relevance in risk assessment and in designing decorporation therapy in the case of accidental overexposure. In this study, thermodynamic calculations of uranium speciation in relevant simulated and original body fluids were compared with spectroscopic data after ex-situ uranium addition. For the first time, experimental data on U(VI) speciation in body fluids (saliva, sweat, urine) was obtained by means of cryogenic time-resolved laser-induced fluorescence spectroscopy (cryo-TRLFS) at 153 K. By using the time dependency of fluorescence decay and the band positions of the emission spectra, various uranyl complexes were demonstrated in the studied samples. The variations of the body fluids in terms of chemical composition, pH, and ionic strength resulted in different binding forms of U(VI). The speciation of U(VI) in saliva and in urine was affected by the presence of bioorganic ligands, whereas in sweat, the distribution depends mainly on inorganic ligands. We also elucidated the role of biological buffers, i.e., phosphate (H(2)PO(4−)/HPO(4)(2−)) on U(VI) distribution, and the system Ca(2+)/UO(2)(2+)/PO(4)(3−) was discussed in detail in both saliva and urine. The theoretical speciation calculations of the main U(VI) species in the investigated body fluids were significantly consistent with the spectroscopic data. Laser fluorescence spectroscopy showed success and reliability for direct determination of U(VI) in such biological matrices with the possibility for further improvement.

Biophysical influence of isocarbophos on bovine serum albumin: spectroscopic probing.

PubMed

Zhang, Hua-xin; Zhou, Ying; Liu, E

2012-06-15

Isocarbophos (ICP) is a phosphorous pesticide with high toxicity. It has been detected in several kinds of food and therefore can enter human body. In this paper, spectroscopic approaches including three-dimensional fluorescence (3D-FL) spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy were employed to explore the binding of ICP to bovine serum albumin (BSA) at simulated physiological conditions. It was found that the fluorescence quenching of BSA was caused by the formation of ICP-BSA complex at ground state and belonged to static quenching mechanism. The binding constants, the number of binding sites, enthalpy change (ΔH(θ)), Gibbs free energy change (ΔG(θ)) and entropy change (ΔS(θ)) were calculated at four different temperatures according to Scatchard model and thermodynamic equations. To identify the binding location, fluorescence probe techniques were used. The results showed that warfarin, an acknowledged site marker for BSA, could be partially replaced by ICP when ICP was added to warfarin-BSA systems, which demonstrated that ICP primarily bound on Sudlow's site I in domain IIA of BSA molecule. The distance r (3.06 nm) between donor (Trp-212) and acceptor (ICP) was obtained based on Förster's non-radiation fluorescence resonance energy transfer (FRET) theory. Furthermore, the CD spectral results indicated that the secondary structure of BSA was changed in presence of ICP. The study is helpful to evaluating the toxicology of ICP and understanding its effects on the function of protein during the blood transportation process. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis, characterization and bioimaging of fluorescent labeled polyoxometalates.

PubMed

Geisberger, Georg; Gyenge, Emina Besic; Hinger, Doris; Bösiger, Peter; Maake, Caroline; Patzke, Greta R

2013-07-21

A fluorescent labeled Wells-Dawson type POM ({P2W17O61Fluo}) was newly synthesized and characterized by a wide range of analytical methods. {P2W17O61Fluo} was functionalized with fluorescein amine through a stable amide bond, and its long time stability was verified by UV/vis spectroscopic techniques at physiologically relevant pH values. No significant impact on the cell viability or morphology of HeLa cells was observed for POM concentrations up to 100 μg mL(-1). Cellular uptake of fluorescent {P2W17O61Fluo} was monitored by confocal laser scanning microscopy. POM uptake occurs mainly after prolonged incubation times of 24 h resulting in different intracellular patterns, i.e. randomly distributed over the entire cytoplasm, or aggregated in larger clusters. This direct monitoring strategy for the interaction of POMs with cells opens up new pathways for elucidating their unknown mode of action on the way to POM-based drug development.

Guided fluorescence diagnosis of childhood caries: preliminary measures correlate with depth of carious decay

NASA Astrophysics Data System (ADS)

Timoshchuk, Mari-Alina; Zhang, Liang; Dickinson, Brian A.; Ridge, Jeremy S.; Kim, Amy S.; Baltuck, Camille T.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

2014-02-01

The current rise in childhood caries worldwide has increased the demand for portable technologies that can quickly and accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype for detecting and diagnosing occlusal caries demineralization in vivo has been developed and pilot tested. The device uses a 405-nm laser as a scanned illumination source to obtain high resolution and high surface contrast reflectance images, which allows the user to quickly image and screen for any signs of demineralized enamel. When a suspicious region is located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitations. These spectra are used to compute an auto-fluorescence (AF) ratio of the suspicious region and the percent difference of AF ratios from a healthy region of the same tooth. The device was tested on 7 children's teeth in vivo with clinically diagnosed carious lesions. Lesion depth was then visually estimated from the video image using the 405-nm scanned light source, and within a month the maximum drill depth was assessed by a clinician. The researcher and clinicians were masked from previous measurements in a blinded study protocol. Preliminary results show that the ratiometric percent difference measurement of the AF spectrum of the tooth correlates with the severity of the demineralization as assessed by the clinician after drilling.

Noninvasive determination of cell nucleoplasmic viscosity by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Liang, Lifang; Wang, Xichao; Xing, Da; Chen, Tongsheng; Chen, Wei R.

2009-03-01

Noninvasive and reliable quantification of rheological characteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study examines the use of fluorescence correlation spectroscopy (FCS) to noninvasively determine nucleoplasmic viscosity (ɛnu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that ɛnu of lung adenocarcinoma (ASTC-a-1) and HeLa cells are 1.77+/-0.42 cP and 1.40+/-0.27 cP, respectively, about three to four times larger than the water viscosity at 37 °C. ɛnu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24 °C. In addition, we found that ɛnu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of ɛnu between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of ɛnu in living cells synchronized in the G1, S, and G2 phases.

Novel tissue phantom for testing a dual-modality diagnostic system: time-resolved fluorescence spectroscopy and high frequency ultrasound

NASA Astrophysics Data System (ADS)

Sun, Yang; Liao, Kuo-Chih; Sun, Yinghua; Park, Jesung; Marcu, Laura

2008-02-01

A unique tissue phantom is reported here that mimics the optical and acoustical properties of biological tissue and enables testing and validation of a dual-modality clinical diagnostic system combining time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasound backscatter microscopy (UBM). The phantom consisted of contrast agents including silicon dioxide particles with a range of diameters from 0.5 to 10 μm acting as optical and acoustical scatterers, and FITC-conjugated dextran mimicking the endogenous fluorophore in tissue. The agents were encapsulated in a polymer bead attached to the end of an optical fiber with a 200 μm diameter using a UV-induced polymerization technique. A set of beads with fibers were then implanted into a gel-based matrix with controlled patterns including a design with lateral distribution and a design with successively changing depth. The configuration presented here allowed the validation of the hybrid fluorescence spectroscopic and ultrasonic system by detecting the lateral and depth distribution of the contrast agents, as well as for coregistration of the ultrasonic image with spectroscopic data. In addition, the depth of the beads in the gel matrix was changed to explore the effect of different concentration ratio of the mixture on the fluorescence signal emitted.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Living Supramolecular Polymerization of a Perylene Bisimide Dye into Fluorescent J-Aggregates.

PubMed

Wagner, Wolfgang; Wehner, Marius; Stepanenko, Vladimir; Ogi, Soichiro; Würthner, Frank

2017-12-11

The self-assembly of a new perylene bisimide (PBI) organogelator with 1,7-dimethoxy substituents in the bay position affords non-fluorescent H-aggregates at high cooling rates and fluorescent J-aggregates at low cooling rates. Under properly adjusted conditions, the kinetically trapped "off-pathway" H-aggregates are transformed into the thermodynamically favored J-aggregates, a process that can be accelerated by the addition of J-aggregate seeds. Spectroscopic studies revealed a subtle interplay of π-π interactions and intra- and intermolecular hydrogen bonding for monomeric, H-, and J-aggregated PBIs. Multiple polymerization cycles initiated from the seed termini demonstrate the living character of this chain-growth supramolecular polymerization process. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spectroscopic analysis on the resveratrol-DNA binding interactions at physiological pH

NASA Astrophysics Data System (ADS)

Zhang, Shufang; Sun, Xuejun; Jing, Zhihong; Qu, Fengli

2011-11-01

The interaction of resveratrol with calf thymus deoxyribonucleic acid (ctDNA) under physiological conditions (Tris-HCl buffer solutions, pH 7.4) was studied by spectroscopy, fluorescence spectroscopy and viscosity measurement method, respectively. Results indicated that a complex of resveratrol with ctDNA was formed with a binding constant of K17 °C = 5.49 × 10 3 L mol -1 and K37 °C = 1.90 × 10 4 L mol -1. The fluorescence quenching mechanism of acridine orange (AO)-ctDNA by resveratrol was shown to be a static quenching type. The thermodynamic parameters of the complex were calculated by a double reciprocal method: ΔHms=4.64×10 J mol, ΔSms=231.8 J K mol and ΔGms=-2.54×10 J mol (37 °C). Spectroscopic techniques together with viscosity determination provided evidences of intercalation mode of binding for the interaction between resveratrol and ctDNA.

Convective-diffusion-based fluorescence correlation spectroscopy for detection of a trace amount of E. coli in water.

PubMed

Qing, De-Kui; Mengüç, M Pinar; Payne, Fred A; Danao, Mary-Grace C

2003-06-01

Fluorescence correlation spectroscopy (FCS) is adapted for a new procedure to detect trace amounts of Escherichia coli in water. The present concept is based on convective diffusion rather than Brownian diffusion and employs confocal microscopy as in traditional FCS. With this system it is possible to detect concentrations as small as 1.5 x 10(5) E. coli per milliliter (2.5 x 10(-16) M). This concentration corresponds to an approximately 1.0-nM level of Rhodamine 6G dyes. A detailed analysis of the optical system is presented, and further improvements for the procedure are discussed.

Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

NASA Astrophysics Data System (ADS)

Novikov, Eugene G.; Skakun, Victor V.; Borst, Jan Willem; Visser, Antonie J. W. G.

2018-01-01

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

Fluorescence spectroscopy of Rhodamine 6G: concentration and solvent effects.

PubMed

Zehentbauer, Florian M; Moretto, Claudia; Stephen, Ryan; Thevar, Thangavel; Gilchrist, John R; Pokrajac, Dubravka; Richard, Katherine L; Kiefer, Johannes

2014-01-01

Rhodamine 6G (R6G), also known as Rhodamine 590, is one of the most frequently used dyes for application in dye lasers and as a fluorescence tracer, e.g., in the area of environmental hydraulics. Knowing the spectroscopic characteristics of the optical emission is key to obtaining high conversion efficiency and measurement accuracy, respectively. In this work, solvent and concentration effects are studied. A series of eight different organic solvents (methanol, ethanol, n-propanol, iso-propanol, n-butanol, n-pentanol, acetone, and dimethyl sulfoxide (DMSO)) are investigated at constant dye concentration. Relatively small changes of the fluorescence spectrum are observed for the different solvents; the highest fluorescence intensity is observed for methanol and lowest for DMSO. The shortest peak wavelength is found in methanol (568 nm) and the longest in DMSO (579 nm). Concentration effects in aqueous R6G solutions are studied over the full concentration range from the solubility limit to highly dilute states. Changing the dye concentration provides tunability between ∼550 nm in the dilute case and ∼620 nm at high concentration, at which point the fluorescence spectrum indicates the formation of R6G aggregates. Copyright © 2013 Elsevier B.V. All rights reserved.

Raman microscopy of bladder cancer cells expressing green fluorescent protein

NASA Astrophysics Data System (ADS)

Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

2016-11-01

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange.

PubMed

Hansda, Chaitali; Chakraborty, Utsav; Hussain, Syed Arshad; Bhattacharjee, Debajyoti; Paul, Pabitra Kumar

2016-03-15

Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π-π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV-Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe. Copyright © 2015 Elsevier B.V. All rights reserved.

Measuring the diffusion coefficient of ganglioside on cell membrane by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Dong, Shiqing; You, Minghai; Chen, Jianling; Zhou, Jie; Xie, Shusen; Yang, Hongqin

2017-06-01

The fluidity of proteins and lipids on cell membrane plays an important role in cell’s physiological functions. Fluorescence correlation spectroscopy (FCS) is an effective technique to detect the rapid dynamic behaviors of proteins and/or lipids in living cells. In this study, we used the rhodamine6G solution to optimize the FCS system. And, cholera toxin B subunit (CT-B) was used to label ganglioside on living Hela cell membranes. The diffusion time and coefficients of ganglioside can be obtained through fitting the autocorrelation curve based on the model of two-dimensional cell membrane. The results showed that the diffusion coefficients of ganglioside distributed within a wide range. It revealed the lateral diffusion of lipids on cell membrane was inhomogeneous, which was due to different microstructures of cytoplasmic membrane. The study provides a helpful method for further studying the dynamic characteristics of proteins and lipids molecules on living cell membrane.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

phyA-GFP is spectroscopically and photochemically similar to phyA and comprises both its native types, phyA' and phyA''.

PubMed

Sineshchekov, Vitaly; Sudnitsin, Artem; Ádám, Éva; Schäfer, Eberhard; Viczián, András

2014-12-01

Low-temperature fluorescence investigations of phyA-GFP used in experiments on its nuclear-cytoplasmic partitioning were carried out. In etiolated hypocotyls of phyA-deficient Arabidopsis thaliana expressing phyA-GFP, it was found that it is similar to phyA in spectroscopic parameters with both its native types, phyA' and phyA'', present and their ratio shifted towards phyA'. In transgenic tobacco hypocotyls, native phyA and rice phyA-GFP were also identical to phyA in the wild type whereas phyA-GFP belonged primarily to the phyA' type. Finally, truncated oat Δ6-12 phyA-GFP expressed in phyA-deficient Arabidopsis was represented by the phyA' type in contrast to full-length oat phyA-GFP with an approximately equal proportion of the two phyA types. This correlates with a previous observation that Δ6-12 phyA-GFP can form only numerous tiny subnuclear speckles while its wild-type counterpart can also localize into bigger and fewer subnuclear protein complexes. Thus, phyA-GFP is spectroscopically and photochemically similar or identical to the native phyA, suggesting that the GFP tag does not affect the chromophore. phyA-GFP comprises phyA'-GFP and phyA''-GFP, suggesting that both of them are potential participants in nuclear-cytoplasmic partitioning, which may contribute to its complexity.

Characterization of binding site heterogeneity for copper within dissolved organic matter fractions using two-dimensional correlation fluorescence spectroscopy.

PubMed

Hur, Jin; Lee, Bo-Mi

2011-06-01

The heterogeneity of copper binding characteristics for dissolved organic matter (DOM) fractions was investigated based on the fluorescence quenching of the synchronous fluorescence spectra upon the addition of copper and two-dimensional correlation spectroscopy (2D-COS). Hydrophobic acid (HoA) and hydrophilic (Hi) fractions of two different DOM (algal and leaf litter DOM) were used for this study. For both DOM, fluorescence quenching occurred at a wider range of wavelengths for the HoA fractions compared to the Hi fractions. The combined information of the synchronous and asynchronous maps derived from 2D-COS provided a clear picture of the heterogeneous distribution of the copper binding sites within each DOM fraction, which was not readily recognized by a simple comparison of the changes in the synchronous fluorescence spectra upon the addition of copper. For the algal DOM, higher stability constants were exhibited for the HoA versus the Hi fractions. The logarithms of the stability constants ranged from 4.8 to 6.1 and from 4.5 to 5.0 for the HoA and the Hi fractions of the algal DOM, respectively, depending on the associated wavelength and the fitted models. In contrast, no distinctive difference in the binding characteristics was found between the two fractions of the leaf litter DOM. This suggests that influences of the structural and chemical properties of DOM on copper binding may differ for DOM from different sources. The relative difference of the calculated stability constants within the DOM fractions were consistent with the sequential orders interpreted from the asynchronous 2D-COS. It is expected that 2D-COS will be widely applied to other DOM studies requiring detailed information on the heterogeneous nature and subsequent effects under a range of environmental conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection.

PubMed

Roberts, David W; Olson, Jonathan D; Evans, Linton T; Kolste, Kolbein K; Kanick, Stephen C; Fan, Xiaoyao; Bravo, Jaime J; Wilson, Brian C; Leblond, Frederic; Marois, Mikael; Paulsen, Keith D

2018-06-01

OBJECTIVE The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination. METHODS To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620-640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis. RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery. CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation. Clinical trial registration no.: NCT02191488 (clinicaltrials.gov).

The effect of intermolecular hydrogen bonding on the fluorescence of a bimetallic platinum complex.

PubMed

Zhao, Guang-Jiu; Northrop, Brian H; Han, Ke-Li; Stang, Peter J

2010-09-02

The bimetallic platinum complexes are known as unique building blocks and arewidely utilized in the coordination-driven self-assembly of functionalized supramolecular metallacycles. Hence, photophysical study of the bimetallic platinum complexes will be very helpful for the understanding on the optical properties and further applications of coordination-driven self-assembled supramolecular metallacycles. Herein, we report steady-state and time-resolved spectroscopic experiments as well as quantum chemistry calculations to investigate the significant intermolecular hydrogen bonding effects on the intramolecular charge transfer (ICT) fluorescence of a bimetallic platinum compound 4,4'-bis(trans-Pt(PEt(3))(2)OTf)benzophenone 3 in solution. We demonstrated that the fluorescent state of compound 3 can be assigned as a metal-to-ligand charge transfer (MLCT) state. Moreover, it was observed that the formation of intermolecular hydrogen bonds can effectively lengthen the fluorescence lifetime of 3 in alcoholic solvents compared with that in hexane solvent. At the same time, the electronically excited states of 3 in solution are definitely changed by intermolecular hydrogen bonding interactions. As a consequence, we propose a new fluorescence modulation mechanism by hydrogen bonding to explain different fluorescence emissions of 3 in hydrogen-bonding solvents and nonhydrogen-bonding solvents.

Spectroscopic and theoretical studies of charge-transfer interaction of 1-(2-pyridylazo)-2-napthol with nitroaromatics

NASA Astrophysics Data System (ADS)

Karmakar, Animesh; Singh, Bula

2017-05-01

1-(2-Pyridylazo)-2-napthol (hereafter 1Q) is widely used as a chelating ligand applied in chelatometric, spectrophotometric analysis of metal ions. It appeared from the literature survey that no inclusion complex of 1Q was reported with nitroaromatics. The formation of charge-transfer complex gives an opportunity to improve the physico-chemical properties of different donors. So the complex of 1Q with 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP), picric acid (PA), and 3,5-dinitrosalicylic acid (3,5-DNSA) was described in this work in methanol medium. The ground and excited state binding constants and other spectroscopic data have been determined using UV-vis and fluorescence spectroscopic studies. All the complexes have been synthesized and characterized using FT-IR, 1H NMR, and elemental analysis. Spectroscopic data reveal that 1Q joins by a N+sbnd Hsbnd O- type hydrogen bond with nitroaromatics. Job's plot of the continuous variation of absorbance indicates that stoichiometry of CT-complex was 1:1. Thermal stability of the synthesized complex has determined by TGA-DTA analysis. Energy-minimization DFT calculation further supported the formation of the H-bonded charge-transfer adduct.

Jet-Cooled Laser-Induced Fluorescence Spectroscopy of T-Butoxy

NASA Astrophysics Data System (ADS)

Reilly, Neil J.; Cheng, Lan; Stanton, John F.; Miller, Terry A.; Liu, Jinjun

2015-06-01

The vibrational structures of the tilde A ^2A_1 and tilde X ^2E states of t-butoxy were obtained in jet-cooled laser-induced fluorescence (LIF) and dispersed fluorescence (DF) spectroscopic measurements. The observed transitions are assigned based on vibrational frequencies calculated using Complete Active Space Self-Consistent Field (CASSCF) method and the predicted Franck-Condon factors. The spin-orbit (SO) splitting was measured to be 35(5) cm-1 for the lowest vibrational level of the ground (tilde X ^2E) state and increases with increasing vibrational quantum number of the CO stretch mode. Vibronic analysis of the DF spectra suggests that Jahn-Teller (JT)-active modes of the ground-state t-butoxy radical are similar to those of methoxy and would be the same if methyl groups were replaced by hydrogen atoms. Coupled-cluster calculations show that electron delocalization, introduced by the substitution of hydrogens with methyl groups, reduces the electronic contribution of the SO splittings by only around ten percent, and a calculation on the vibronic levels based on quasidiabatic model Hamiltonian clearly attributes the relatively small SO splitting of the tilde X ^2E state of t-butoxy mainly to stronger reduction of orbital angular momentum by the JT-active modes when compared to methoxy. The rotational and fine structure of the LIF transition to the first CO stretch overtone level of the tilde A^2A_1 state has been simulated using a spectroscopic model first proposed for methoxy, yielding an accurate determination of the rotational constants of both tilde A and tilde X states.

Rapid screening test for porphyria diagnosis using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Lang, A.; Stepp, H.; Homann, C.; Hennig, G.; Brittenham, G. M.; Vogeser, M.

2015-07-01

Porphyrias are rare genetic metabolic disorders, which result from deficiencies of enzymes in the heme biosynthesis pathway. Depending on the enzyme defect, different types of porphyrins and heme precursors accumulate for the different porphyria diseases in erythrocytes, liver, blood plasma, urine and stool. Patients with acute hepatic porphyrias can suffer from acute neuropathic attacks, which can lead to death when undiagnosed, but show only unspecific clinical symptoms such as abdominal pain. Therefore, in addition to chromatographic methods, a rapid screening test is required to allow for immediate identification and treatment of these patients. In this study, fluorescence spectroscopic measurements were conducted on blood plasma and phantom material, mimicking the composition of blood plasma of porphyria patients. Hydrochloric acid was used to differentiate the occurring porphyrins (uroporphyrin-III and coproporphyrin-III) spectroscopically despite their initially overlapping excitation spectra. Plasma phantom mixtures were measured using dual wavelength excitation and the corresponding concentrations of uroporphyrin-III and coproporphyrin-III were determined. Additionally, three plasma samples of porphyria patients were examined and traces of coproporphyrin-III and uroporphyrin-III were identified. This study may therefore help to establish a rapid screening test method with spectroscopic differentiation of the occurring porphyrins, which consequently allows for the distinction of different porphyrias. This may be a valuable tool for clinical porphyria diagnosis and rapid or immediate treatment.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Williams, D. L.

1986-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, Emmett W.; Williams, Darrel L.

1987-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Optical spectroscopic characteristics of lactate and mitochondrion as new biomarkers in cancer diagnosis: understanding Warburg effect

NASA Astrophysics Data System (ADS)

Liu, C.-H.; Ni, X. H.; Pu, Yang; Yang, Y. L.; Zhou, F.; Zuzolo, R.; Wang, W. B.; Masilamani, V.; Rizwan, A.; Alfano, R. R.

2012-01-01

Cancer cells display high rates of glycolysis even under normoxia and mostly under hypoxia. Warburg proposed this effect of altered metabolism in cells more than 80 years ago. It is considered as a hallmark of cancer. Optical spectroscopy can be used to explore this effect. Pathophysiological studies indicate that mitochondria of cancer cells are enlarged and increased in number. Warburg observed that cancer cells tend to convert most glucose to lactate regardless of the presence of oxygen. Previous observations show increased lactate in breast cancer lines. The focus of this study is to investigate the relative content changes of lactate and mitochondria in human cancerous and normal breast tissue samples using optical spectroscopic techniques. The optical spectra were obtained from 30 cancerous and 25 normal breast tissue samples and five model components (Tryptophan, fat, collagen, lactate and mitochondrion) using fluorescence, Stokes shift and Raman spectroscopy. The basic biochemical component analysis model (BBCA) and a set of algorithm were used to analyze the spectra. Our analyses of fluorescence spectra showed a 14 percent increase in lactate content and 2.5 times increase in mitochondria number in cancerous breast tissue as compared with normal tissue. Our findings indicate that optical spectroscopic techniques may be used to understand Warburg effect. Lactate and mitochondrion content changes in tumors examined using optical spectroscopy may be used as a prognostic molecular marker in clinic applications.

Spectroscopic and thermodynamic studies on ferulic acid - Alpha-2-macroglobulin interaction

NASA Astrophysics Data System (ADS)

Rehman, Ahmed Abdur; Sarwar, Tarique; Arif, Hussain; Ali, Syed Saqib; Ahsan, Haseeb; Tabish, Mohammad; Khan, Fahim Halim

2017-09-01

Ferulic acid is a major phenolic acid found in numerous plant species in conjugated form. It binds to enzymes and oligomeric proteins and modifies their structure and function. This study was designed to examine the interaction of ferulic acid, an active ingredient of some important medicines, with α2M, a key serum proteinase, under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques such as, UV-visible absorption, fluorescence spectroscopy, circular dichroism along with isothermal titration calorimetry. Fluorescence quenching of α2M by ferulic acid demonstrated the formation of α2M-ferulic acid complex by static quenching mechanism. Binding parameters calculated by Stern-Volmer method showed that ferulic acid binds to α2M with moderate affinity of the order of ∼104 M-1. The thermodynamic signatures reveal that binding was enthalpy driven and hydrogen bonding played a major role in ferulic acid-α2M binding. CD spectra analysis suggests very little conformational changes in α2M on ferulic acid binding.

Detection of proximal caries using quantitative light-induced fluorescence-digital and laser fluorescence: a comparative study.

PubMed

Yoon, Hyung-In; Yoo, Min-Jeong; Park, Eun-Jin

2017-12-01

The purpose of this study was to evaluate the in vitro validity of quantitative light-induced fluorescence-digital (QLF-D) and laser fluorescence (DIAGNOdent) for assessing proximal caries in extracted premolars, using digital radiography as reference method. A total of 102 extracted premolars with similar lengths and shapes were used. A single operator conducted all the examinations using three different detection methods (bitewing radiography, QLF-D, and DIAGNOdent). The bitewing x-ray scale, QLF-D fluorescence loss (ΔF), and DIAGNOdent peak readings were compared and statistically analyzed. Each method showed an excellent reliability. The correlation coefficient between bitewing radiography and QLF-D, DIAGNOdent were -0.644 and 0.448, respectively, while the value between QLF-D and DIAGNOdent was -0.382. The kappa statistics for bitewing radiography and QLF-D had a higher diagnosis consensus than those for bitewing radiography and DIAGNOdent. The QLF-D was moderately to highly accurate (AUC = 0.753 - 0.908), while DIAGNOdent was moderately to less accurate (AUC = 0.622 - 0.784). All detection methods showed statistically significant correlation and high correlation between the bitewing radiography and QLF-D. QLF-D was found to be a valid and reliable alternative diagnostic method to digital bitewing radiography for in vitro detection of proximal caries.

Connecting active to passive fluorescence with photosynthesis: a method for evaluating remote sensing measurements of Chl fluorescence.

PubMed

Magney, Troy S; Frankenberg, Christian; Fisher, Joshua B; Sun, Ying; North, Gretchen B; Davis, Thomas S; Kornfeld, Ari; Siebke, Katharina

2017-09-01

Recent advances in the retrieval of Chl fluorescence from space using passive methods (solar-induced Chl fluorescence, SIF) promise improved mapping of plant photosynthesis globally. However, unresolved issues related to the spatial, spectral, and temporal dynamics of vegetation fluorescence complicate our ability to interpret SIF measurements. We developed an instrument to measure leaf-level gas exchange simultaneously with pulse-amplitude modulation (PAM) and spectrally resolved fluorescence over the same field of view - allowing us to investigate the relationships between active and passive fluorescence with photosynthesis. Strongly correlated, slope-dependent relationships were observed between measured spectra across all wavelengths (F λ , 670-850 nm) and PAM fluorescence parameters under a range of actinic light intensities (steady-state fluorescence yields, F t ) and saturation pulses (maximal fluorescence yields, F m ). Our results suggest that this method can accurately reproduce the full Chl emission spectra - capturing the spectral dynamics associated with changes in the yields of fluorescence, photochemical (ΦPSII), and nonphotochemical quenching (NPQ). We discuss how this method may establish a link between photosynthetic capacity and the mechanistic drivers of wavelength-specific fluorescence emission during changes in environmental conditions (light, temperature, humidity). Our emphasis is on future research directions linking spectral fluorescence to photosynthesis, ΦPSII, and NPQ. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid-Base Equilibrium of Two Cis Configurations.

PubMed

Higashino, Asuka; Mizuno, Misao; Mizutani, Yasuhisa

2016-04-07

Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5.2 ± 0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.

Depth-resolved fluorescence of biological tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-06-01

The depth-resolved autofluorescence ofrabbit oral tissue, normal and dysplastic human ectocervical tissue within l20μm depth were investigated utilizing a confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of oral and ectocervical tissue, strong keratin fluorescence with the spectral characteristics similar to collagen was observed. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. Furthermore, NADH and FADfluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

A comparative study of gallstones from children and adults using FTIR spectroscopy and fluorescence microscopy

PubMed Central

Kleiner, Oleg; Ramesh, Jagannathan; Huleihel, Mahmoud; Cohen, Beny; Kantarovich, Keren; Levi, Chen; Polyak, Boris; Marks, Robert S; Mordehai, Jacov; Cohen, Zahavi; Mordechai, Shaul

2002-01-01

Background Cholelithiasis is the gallstone disease (GSD) where stones are formed in the gallbladder. The main function of the gallbladder is to concentrate bile by the absorption of water and sodium. GSD has high prevalence among elderly adults. There are three major types of gallstones found in patients, White, Black and Brown. The major chemical component of white stones is cholesterol. Black and brown stones contain different proportions of cholesterol and bilirubin. The pathogenesis of gallstones is not clearly understood. Analysis of the chemical composition of gallstones using various spectroscopic techniques offers clues to the pathogenesis of gallstones. Recent years has seen an increasing trend in the number of cases involving children. The focus of this study is on the analysis of the chemical composition of gallstones from child and adult patients using spectroscopic methods. Methods In this report, we present FTIR spectroscopic studies and fluorescence microscopic analysis of gallstones obtained from 67 adult and 21 child patients. The gallstones were removed during surgical operations at Soroka University Medical Center. Results Our results show that black stones from adults and children are rich in bilirubin. Brown stones are composed of varying amounts of bilirubin and cholesterol. Green stones removed from an adult, which is rare, was found to be composed mainly of cholesterol. Our results also indicated that cholesterol and bilirubin could be the risk factors for gallstone formation in adults and children respectively. Fluorescence micrographs showed that the Ca-bilirubinate was present in all stones in different quantities and however, Cu-bilirubinate was present only in the mixed and black stones. Conclusions Analysis based on FTIR suggest that the composition of black and brown stones from both children and adults are similar. Various layers of the brown stone from adults differ by having varying quantities of cholesterol and calcium carbonate

A new screening method for flunitrazepam in vodka and tequila by fluorescence spectroscopy.

PubMed

Leesakul, Nararak; Pongampai, Sirintip; Kanatharana, Proespichaya; Sudkeaw, Pravit; Tantirungrotechai, Yuthana; Buranachai, Chittanon

2013-01-01

A new screening method for flunitrazepam in colourless alcoholic beverages based on a spectroscopic technique is proposed. Absorption and steady-state fluorescence of flunitrazepam and its protonated form with various acids were investigated. The redshift of the wavelength of maximum absorption was distinctively observed in protonated flunitrazepam. An emissive fluorescence at 472 nm was detected in colourless spirits (vodka and tequila) at room temperature. 2-M perchloric acid was the most appropriated proton source. By using electron ionization mass spectrometry and time-dependent density functional theory calculations, the possible structure of protonated flunitrazepam was identified to be 2-nitro-N-methylacridone, an acridone derivative as opposed to 2-methylamino-5-nitro-2'-fluorobenzophenone, a benzophenone derivative. Copyright © 2012 John Wiley & Sons, Ltd.

Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.

PubMed

Stepanenko, Olesya V; Verkhusha, Vladislav V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, K K

2008-08-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

PubMed Central

Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, K.K.

2010-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology. PMID:18691124

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

NASA Astrophysics Data System (ADS)

Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

2015-05-01

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

λ-Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides

PubMed Central

Samiee, K. T.; Foquet, M.; Guo, L.; Cox, E. C.; Craighead, H. G.

2005-01-01

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage λ repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding. PMID:15613638

Simultaneous characterization of lateral lipid and prothrombin diffusion coefficients by z-scan fluorescence correlation spectroscopy.

PubMed

Stefl, Martin; Kułakowska, Anna; Hof, Martin

2009-08-05

A new (to our knowledge) robust approach for the determination of lateral diffusion coefficients of weakly bound proteins is applied for the phosphatidylserine specific membrane interaction of bovine prothrombin. It is shown that z-scan fluorescence correlation spectroscopy in combination with pulsed interleaved dual excitation allows simultaneous monitoring of the lateral diffusion of labeled protein and phospholipids. Moreover, from the dependencies of the particle numbers on the axial sample positions at different protein concentrations phosphatidylserine-dependent equilibrium dissociation constants are derived confirming literature values. Increasing the amount of membrane-bound prothrombin retards the lateral protein and lipid diffusion, indicating coupling of both processes. The lateral diffusion coefficients of labeled lipids are considerably larger than the simultaneously determined lateral diffusion coefficients of prothrombin, which contradicts findings reported for the isolated N-terminus of prothrombin.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

Optical Antenna-Based Fluorescence Correlation Spectroscopy to Probe the Nanoscale Dynamics of Biological Membranes.

PubMed

Winkler, Pamina M; Regmi, Raju; Flauraud, Valentin; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F

2018-01-04

The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.

Chapter 7: Total internal reflection fluorescence microscopy.

PubMed

Axelrod, Daniel

2008-01-01

Total internal reflection fluorescence microscopy (TIRFM), also known as evanescent wave microscopy, is used in a wide range of applications, particularly to view single molecules attached to planar surfaces and to study the position and dynamics of molecules and organelles in living culture cells near the contact regions with the glass coverslip. TIRFM selectively illuminates fluorophores only in a very thin (less than 100 nm deep) layer near the substrate, thereby avoiding excitation of fluorophores outside this subresolution optical section. This chapter reviews the history, current applications in cell biology and biochemistry, basic optical theory, combinations with numerous other optical and spectroscopic approaches, and a range of setup methods, both commercial and custom.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane.

PubMed

Stanca, Sarmiza E; Matthäus, Christian; Neugebauer, Ute; Nietzsche, Sandor; Fritzsche, Wolfgang; Dellith, Jan; Heintzmann, Rainer; Weber, Karina; Deckert, Volker; Krafft, Christoph; Popp, Jürgen

2015-10-01

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954 cm(-1) which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. This method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering. Copyright © 2015 Elsevier Inc. All rights reserved.

Quenching interaction of BSA with DTAB is dynamic in nature: A spectroscopic insight

NASA Astrophysics Data System (ADS)

Das, Nirmal Kumar; Pawar, Lavanya; Kumar, Naveen; Mukherjee, Saptarshi

2015-08-01

The role of electrostatic interactions between the protein, Bovine Serum Albumin (BSA) and the cationic surfactant, dodecyltrimethylammonium bromide (DTAB) has been substantiated using spectroscopic approaches. The primary mechanism of fluorescence quenching of the tryptophan of BSA is most probably dynamic in nature as the complex formation resulting in a protein-surfactant assembly is not very spontaneous. The weak interaction buries the tryptophan amino acid residue inside the protein scaffolds which have been quantitatively proved by our acrylamide quenching studies. The loss in the secondary structure of the protein as a result of interaction with DTAB has been elucidated by CD spectroscopy.

Optimization of spectroscopic surveys for testing non-Gaussianity